TRANSLATION STALLING COMPOSITIONS AND METHODS OF USE THEREOF

CROSS -REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/572,244, filed October 13, 2017, which application is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. P50-GM 102706 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

[0003] A Sequence Listing is provided herewith as a text file, "BERK-

369WO_SEQ_LISTING_ST25.txt" created on October 3, 2018 and having a size of 95 KB. The contents of the text file are incorporated by reference herein in their entirety.

INTRODUCTION

[0004] Proteins are major products of the genome known to express phenotypes in biology. In humans, many proteins cause disease due to their overexpression or due to mutations in the sequences encoding them. It has proven difficult to find chemical inhibitors of many such disease- associated proteins, and it would be desirable to prevent their synthesis in the first place.

Additionally, it is desirable to identify proteins with different or improved functions in all areas of biotechnology; thus, methods for molecular evolution of proteins to modify their function have been developed, and include technologies such as ribosome display.

[0005] There is a need in the art for methods and compositions for stalling translation of nascent

proteins on a ribosome. Such methods and compositions can be used for identifying new chemical entities that stall translation of target proteins of interest, and for the identification and/or directed evolution of proteins with new functions. SUMMARY

[0006] The present disclosure provides ribonucleic acids that provide for translation stalling. The

present disclosure provides ribosome/nucleic acid complexes comprising the ribonucleic acids, as well as ribosome/nucleic acid/nascent chain polypeptide complexes comprising the ribonucleic acids. The present disclosure provides methods of identifying a translation stalling agent. The present disclosure provides libraries of ribosome/nucleic acid/nascent chain polypeptide complexes, and methods of using such libraries.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A and IB provide a diagram (FIG. 1A) of a construct designed to express mRNA

transcripts encoding a ribosome stalling polypeptide (comprising a CDHl stalling signal protector polypeptide and a CDHl ribosome stalling signal), which transcripts were translated in in vitro cell extracts in the absence or presence of the translation stalling agent Compound 1 ; and data (FIG. IB) showing that translation of the lucif erase reporter gene was reduced in the presence of Compound 1.

[0008] FIG. 2A-2C provide a diagram (FIG. 2A) of a construct designed to express mRNA transcripts encoding another Compound 1-sensitive ribosome stalling polypeptide, comprising a CDHl stalling signal protector polypeptide and a PCSK9 ribosome stalling signal; and data (FIG. 2B and 2C) showing that translation of FLAG was reduced in the presence of Compound 1.

[0009] FIG. 3 A and 3B provide an amino acid sequence of CDHl, with the CDHl stalling signal protector polypeptide (residues 586-696) underlined and the CDHl ribosome stalling signal double underlined (FIG. 3A); and an amino acid sequence of a CDHl stalling signal protector polypeptide (FIG. 3B).

[0010] FIG. 4 provides Compound 1-sensitive ribosome stalling signals.

[0011] FIG. 5A-5D provide diagrams of the PF846 stalled ribosome nascent chains (RNCs) including a schematic representation of the DNA construct used to prepare PF846 stalled RNCs (FIG. 5A); stalling of the chimeric PCSK9 nascent chain in in vitro translation reactions in the absence or presence of PF846 (FIG. 5B); cryo-electron microscopy (cryo-EM) structure of the stalled CDH1-RNC (FIG. 5C); and the structure of the stalled CDH1-RNC in the rotated state (FIG. 5D).

[0012] FIG. 6A-6B provide diagrams of the PF846-mediated stalling of RNCs including in vitro

translation assay results of PCSK9 and CDH1 nascent chains with different affinity tags in the absence or presence of PF846 (FIG. 6A); and western blot results with anti-FLAG® antibody of affinity purified stalled PCSK9-RNCs after elution and pelleting (FIG. 6B).

[0013] FIG. 7A-7C provide diagrams of the cryo-EM data processing and model validation of PF846 stalled CDHl-RNCs including EM micrographs for classification and refinement of selected subsets (FIG. 7A); final Fourier shell correlation (FSC) curves of CDHl-RNCs (FIG. 7B); and model to map correlation at FSC value of 0.5.

[0014] FIG. 8A-8E provide diagrams of the cryo-electron data processing of PF846 stalled PCSK9-

RNCs including the processing steps for stalled PCSK9-RNCs as described in FIG. 7A-7C (FIG. 8A); final FSC curves of the CDHl-RNCs (FIG. 8B); model to map correlation at FSC value of 0.5 (FIG. 8C); data processing steps for a sample prepared with a short incubation time (FIG. 8D); and final FSC curves of the RNCs (FIG. 8E).

[0015] FIG. 9 provides a table of data collection, structure model refinement, and validation statistics of the CDHl-RNCs.

[0016] FIG. lOA-lOC provide diagrams of the cryo-EM reconstructions of PF846-stalled PCSK9 RNCs including a PCSK9-RNC in the rotated state with A/P and P/E tRNA (FIG. 10A); with A/A and P/E tRNA (FIG. 10B); and PCSK9-RNC in the non-rotated state (FIG. IOC).

[0017] FIG. 11A-11F provide diagrams of the interactions of A/P tRNAs with mRNA in PF846 stalled CDHl-RNCs including observed densities for representative A-U (FIG. 11 A), G-U (FIG. 11B), and G-C (FIG. 11C) base pairs in 18S rRNA; and observed densities for tRNA anticodon-mRNA codon base pairs at the 3rd position of the codon in the A and P sites, of two G-U (FIG. 11D- 1 IE) base pairs and a G-C base pair (FIG. 1 IF).

[0018] FIG. 12A-12I provide diagrams of the difference density maps defining purine and pyrimidine bases including representative negative difference densities for an incorrect G-C base pair (FIG. 12A); incorrect U-A base pair (FIG. 12B); incorrectly placed G instead of U in a base pair (FIG. 12C); incorrect G-C pair (FIG. 12D); and negative difference density maps of tRNA anticodon- mRNA codon base pairs at the 3rd position of the codon in the A and P sites (FIG. 12E-12I). [0019] FIG. 13A-13H provide diagrams of the interactions of PF846 with the human ribosome including phylogenetic analysis of the 28S rRNA binding pocket in eukaryotes (FIG. 13 A) and bacteria (FIG. 13B); PF846 chemical (FIG. 13C) and three-dimensional structures (FIG. 13D); 28S rRNA residues with direct interactions with PF846 (FIG. 13E); interactions of PF846 and nucleotides from 28S rRNA (FIG. 13F-13G); and interactions of PF846 with U4517, with van der Waals surfaces as in FIG. 13E.

[0020] FIG. 14A-14F provide diagrams of the docking of different conformations of PF846 into

densities extracted from the CDH1-RNC cryo-EM maps, including X-ray structures of PF846 (FIG. 14A-14B) and low energy conformations of PF846 (FIG. 14C-14F).

[0021] FIG. 15 provides a table of the calculated energies of the various conformations of PF846 as shown in FIG. 14A-14F.

[0022] FIG 16A-16E provide diagrams of the PF846 in the predicted binding pocket of E. coli

ribosome rRNA including a cartoon representation of the conserved small molecule binding loop from E. coli and human ribosomes (FIG. 16A); residues that contribute to the binding of PF846 (FIG. 16B) and 16S rRNA residues from E. coli that have direct interactions with PF846 (FIG. 16C-16E).

[0023] FIG. 17A-17D provide diagrams of the CDHl nascent chains and comparisons with other stalled peptides in the ribosome exit tunnel including a molecular model of CDHl nascent chain (FIG.

17A); and superpositions of hCMV and SRP nascent chain within the exit tunnel showing the predicted steric clashes with PF846 (FIG. 17B-17C). FIG. 17D shows stalling of the libraries induced by PF-06446846, reported in relative luciferase units.

[0024] FIG. 18A-18B provide diagrams of the cryo-EM densities of PF846 stalled nascent chains including densities extracted from maps of the CDH1-RNC (FIG. 18 A) and PCSK9-RNC (FIG.

18B) in the rotated state with A/P nascent chain-tRNA (NC-tRNA), shown in the same orientation.

[0025] FIG. 19A-19B provide diagrams of the interactions between CDHl (FIG. 19A) and PCSK9 (FIG. 19B) nascent chains, and the ribosome exit tunnel.

[0026] FIG. 20A-20C provide diagrams of the mechanism of PF846-induced stalling including

diagrams of the poor base pairing of the A/P NC-tRNA 3' -CCA end in the ribosomal P-loop (FIG. 20A-20B) and a schematic diagram of the PF846 binding to the exit tunnel (FIG. 20C). [0027] FIG. 21A-21B provide diagrams of the poor base pairing of the 3 'CCA end of A/P NC-tRNA in PCSK9-stalled RNCs including interactions between the 3'CCA end of A/P tRNA and 28S rRNA nucleotides in the P-loop (FIG. 21 A), where C75 was flipped without pairing with the P- loop (FIG. 21B).

DEFINITIONS

[0028] The term "polypeptide -ribosome complex" or "ribosome -bound nascent chain" or "RNC" as used herein refers to a complex comprising a ribosome attached to a polypeptide being synthesized on the ribosome. The polypeptide of an RNC can be: i) non-naturally occurring; ii) naturally- occurring; iii) a fusion polypeptide; or iii) a variant of a wild-type polypeptide; and the like.

[0029] A "stalled" ribosome refers to a ribosome that, in the presence of a stalling agent, is unable to or has a reduced capacity for performing translation of an mRNA into a polypeptide, compared to the level of translation in the absence of the stalling agent.

[0030] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the

product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms. Alternatively, DNA sequences encoding RNA that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring ("wild type") or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a

"recombinant" polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring ("wild type") or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence.

[0031] The term "naturally-occurring" or "unmodified" or "native" as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring. Accordingly, a biomolecule or organism may be "unnatural" or "non-naturally-occurring", e.g., where a biomolecule, e.g., a nucleic acid, a polypeptide, e.g., or a cell, or an organism, is intentionally modified by a human, e.g., in the laboratory.

[0032] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as "oligomers" or "oligos" and may be isolated from genes, or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0033] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0034] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine -leucine - isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0035] A polynucleotide or polypeptide has a certain percent "sequence identity" to another

polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences.

Sequence identity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi(dot)nlm(dot)nili(dot)gov/BLAST, ebi(dot)ac(dot)uk/Tools/msa/tcoffee/,

ebi(dot)ac(dot)uk/Tools/msa/muscle/, mafft(dot)cbrc(dot)jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0036] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0037] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0039] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an mRNA" includes a plurality of such mRNAs and reference to "the transcript" includes reference to one or more transcripts and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0040] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0042] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a ribosome stalling polypeptide and a nucleotide sequence encoding a heterologous polypeptide of interest. The present disclosure provides a complex comprising the nucleic acid and a ribosome. The present disclosure provides a cell comprising a nucleic acid of the present disclosure or a complex of the present disclosure. Methods of identifying translation-stalling agents, and methods of generating a stalled ribosome nascent chain complex, are also provided. The present disclosure provides a method of identifying a polypeptide of interest, involving

NUCLEIC ACID ENCODING A RIBOSOME STALLING POLYPEPTIDE

[0043] The present disclosure provides a nucleic acid (e.g., a ribonucleic acid, or a deoxyribonucleic acid comprising a nucleotide sequence encoding the ribonucleic acid) comprising a nucleotide sequence encoding a ribosome stalling polypeptide and a nucleotide sequence encoding a heterologous polypeptide of interest.

[0044] The present disclosure provides a nucleic acid (e.g., a ribonucleic acid, or a deoxyribonucleic acid comprising a nucleotide sequence encoding the ribonucleic acid) comprising: a) a nucleotide sequence encoding a ribosome stalling polypeptide, wherein the ribosome stalling polypeptide comprises: i) a stalling signal protector polypeptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 2B; and ii) a ribosome stalling signal; and b) i) a nucleotide sequence encoding a heterologous polypeptide of interest; or ii) an insertion site for a nucleotide sequence encoding a heterologous polypeptide of interest.

[0045] In some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a heterologous polypeptide -encoding nucleotide sequence; and b) a ribosome stalling polypeptide- encoding nucleotide sequence. In other cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a ribosome stalling polypeptide-encoding nucleotide sequence; and b) a heterologous polypeptide-encoding nucleotide sequence.

[0046] In some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) an internal ribosome entry site (IRES); b) a heterologous polypeptide-encoding nucleotide sequence; and c) a ribosome stalling polypeptide-encoding nucleotide sequence. In other cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) an IRES; b) a ribosome stalling polypeptide-encoding nucleotide sequence; and c) a heterologous polypeptide- encoding nucleotide sequence.

[0047] A ribosome stalling polypeptide comprises: a) a stalling signal protector polypeptide; and b) a ribosome stalling signal. In some cases, a ribosome stalling polypeptide comprises two ribosome stalling signals. In some cases, a ribosome stalling polypeptide comprises more than two ribosome stalling signals. Where a ribosome stalling polypeptide comprises two ribosome stalling signals, in some cases, the two ribosome stalling signals comprise the same amino acid sequence. In some cases, a ribosome stalling polypeptide comprises a first ribosome stalling signal and a second ribosome stalling signal, where the first ribosome stalling signal and the second ribosome stalling signal have different amino acid sequences. For example, in some cases, a ribosome stalling polypeptide comprises a first ribosome stalling signal and a second ribosome stalling signal, where the first ribosome stalling signal is a CDH1 ribosome stalling signal and the second ribosome stalling signal is a PCSK9 ribosome stalling signal. In some other cases, a ribosome stalling polypeptide comprises a PF-06446846 (PF846) ribosome stalling signal. A PF846 ribosome stalling signal is also referred to herein as "Compound 1".

[0048] In some cases, compound 1 stalls translation of the ribosome polypeptide in the rotated state. In some cases, compound 1 inhibits translocation of specific nascent chains by preventing peptidyl- tRNA from binding stably in the hybride A/P site. In some cases, the peptidyl-tRNA requires tRNA 3' CCA end to properly base pair with 28S rRNA nucleotides in a P-loop. In some cases, a non-rotated ribosome nascent chain is a transient state during Compound 1 mediated translation stalling. In some cases, Compound 1 stalls ribosome nascent chains in the rotated state, and the non-rotated state with a P/P-site NC-tRNA is resolved during translation in the presence of Compound 1. [0049] In some cases, a CDH1 ribosome stalling signal is stalled near its C-terminus. In some cases, a CDH1 ribosome stalling signal is stalled near its C-terminus by Compound 1. In some cases, the CDH1 ribosome stalling signal stalled near its C-terminus by Compound 1 provides for the extension for the peptide N-terminus beyond the confines of the ribosomal exit tunnel.

Stalling signal protector polypeptide

[0050] A stalling signal protector polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCR (SEQ ID NO: l). In some cases, one or more of the cysteine residues is substituted with an amino acid other than cysteine.

[0051] A stalling signal protector polypeptide can have a length of from about 95 amino acids to about 120 amino acids, e.g., from about 95 amino acids (aa) to about 100 amino acids, from about 100 amino acids to about 105 amino acids, from about 105 amino acids to about 110 amino acids, from about 110 amino acids to about 115 amino acids, or from about 115 amino acids to about 120 amino acids.

Ribosome stalling signals

[0052] A suitable ribosome stalling signal induces, in the presence of a stalling agent, a permanent or transient translational arrest of a ribosome on a nucleic acid.

[0053] A ribosome stalling signal can have a length of from about 5 amino acids to about 75 amino acids, e.g., from about 5 amino acids (aa) to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa to about 75 aa. In some cases, a ribosome stalling signal has a length of 8 amino acids. In some cases, a ribosome stalling signal has a length of from 10 amino acids to 20 amino acids. In some cases, a ribosome stalling signal has a length of from 20 amino acids to 30 amino acids. In some cases, a ribosome stalling signal has a length of from 30 amino acids to 40 amino acids. In some cases, a ribosome stalling signal has a length of from 40 amino acids to 50 amino acids. In some cases, a ribosome stalling signal has a length of from 50 amino acids to 60 amino acids.

[0054] In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a small amino acid, or a polar amino acid. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome- stalling signal is a Gly or Ala. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Gly. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Ala. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a Thr, Asp, Leu, Arg, or Glu. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Thr. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Asp. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Leu. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Arg. In some cases, the amino acid that is 12 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Glu.

[0055] In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a small amino acid, or a polar amino acid. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome- stalling signal is a Gly or Ala. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Gly. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Ala. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a Thr, Asp, Leu, Arg, or Glu. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Thr. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Asp. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Leu. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Arg. In some cases, the amino acid that is 24 amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Glu.

[0056] In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a small amino acid, or a polar amino acid. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome- stalling signal is a Gly or Ala. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome- stalling signal is Gly. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Ala. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is a Thr, Asp, Leu, Arg, or Glu. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome- stalling signal is Thr. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Asp. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Leu. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N-terminal to the C-terminal amino acid of the ribosome-stalling signal is Arg. In some cases, the amino acid that is 20 or more, 40 or more, 80 or more, or 100 or more amino acids N- terminal to the C-terminal amino acid of the ribosome-stalling signal is Glu.

[0057] A suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences:

[0058] i) EGAAGVCRKAQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO:2) [CDH1];

[0059] ii) MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDED (SEQ ID NO: 3) [PCSK9];

[0060] iii) MQHRGFLLLTLLALLALTSAV (SEQ ID NO:4) [MDK];

[0061] iv) MKFLLDILLLLPLLIVC (SEQ ID NO:5) [HSD17B11];

[0062] v) MGLWWVTVQPPARRMGWLPLLLLLTQCLGVPGQRSPLNDF (SEQ ID NO:6) [MSTl]; [0063] vi) MGKFMKPGKVVLVLAGRYSGRKAVIVK (SEQ ID NO:7) [RPL27];

[0064] vii) MDAGVTESGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO: 8) [PCBP1];

[0065] viii) MSLQWTAVATFLYAEVFVVLLLCIP (SEQ ID NO:9) [BCAP31];

[0066] ix) MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO: 10) [PODXL2];

[0067] x) MGLHLRPYRVGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO: 11)

[PLA2G15];

[0068] xi) RMKPWFEVGDENSGWSAQKVTNLHLMLQLVRVLVSPTNPPGA (SEQ ID NO: 12)

[USOl];

[0069] xii) MAAPALGLVCGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO: 13) [SUMF1];

[0070] xiii) MAAALWGFFPVLLLLLLS (SEQ ID NO: 14) [EMC7];

[0071] xiv) MARGAALALLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO: 15) [CD99];

[0072] xv) MRAHAQRGRGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO: 16)

[SUMF2];

[0073] xvi) MDTGVIEGGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO: 17) [PCBP2];

[0074] xvii) MGARASGGPLARAGLLLLLL (SEQ ID NO: 18) [BRI3BP]; and

[0075] xviii) GPLKKSNAPLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO: 19)

[IFI30];

[0076] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

EGAAGVCRKAQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO:2). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0077] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

CRKAQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO: 140). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal. [0078] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDED (SEQ ID NO:3). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0079] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: WWPLPLLLLLLLLLGPAGARAQED (SEQ ID NO: 141). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0080] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MQHRGFLLLTLLALLALTSAV (SEQ ID NO:4). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0081] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MKFLLDILLLLPLLIVC (SEQ ID NO:5). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0082] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGLWWVTVQPPARRMGWLPLLLLLTQCLGVPGQRSPLNDF (SEQ ID NO:6). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0083] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MGKFMKPGKVVLVLAGRYSGRKAVIVK (SEQ ID NO:7). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal.

[0084] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MDAGVTESGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO: 8). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0085] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MSLQWTAVATFLYAEVFVVLLLCIP (SEQ ID NO:9). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0086] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO: 10). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0087] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGLHLRPYRVGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO: 11). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0088] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

RMKPWFEVGDENSGWSAQKVTNLHLMLQLVRVLVSPTNPPGA (SEQ ID NO: 12). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal. [0089] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MAAPALGLVCGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO: 13). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0090] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MAAALWGFFPVLLLLLLS (SEQ ID NO: 14).

In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0091] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MARGAALALLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO: 15). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0092] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MRAHAQRGRGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO:16). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0093] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MDTGVIEGGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO: 17). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal. [0094] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MGARASGGPLARAGLLLLLL (SEQ ID NO: 18). In some cases, such a ribosome stalling signal is a Compound 1 -sensitive signal.

[0095] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

GPLKKSNAPLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO: 19). In some cases, such a ribosome stalling signal is a Compound 1-sensitive signal. In some cases, such a ribosome stalling signal is a Compound 3-sensitive signal.

[0096] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCREGAAGVCRK AOPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO:20), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCREGAAGVCR K

AQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO:20); and has a length of from 150 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREGAAGVCR K

AOPVEAGLOIPAILGILGGILALLILILLLLLF (SEQ ID NO:20); and has a length of 153 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ

ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREGAAGVCR K AOPVEAGLOIPAILGILGGILALLILILLLLLF (SEQ ID NO:20); and has a length of 153 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[0097] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGTVSSRRS WWPLPLLLLLLLLLGPAGARAOEDED (SEQ ID NO:21), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGTVSSRRS WWPLPLLLLLLLLLGPAGARAOEDED (SEQ ID NO:21); and has a length of from 145 amino acids to 155 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGTVSSRRS WWPLPLLLLLLLLLGPAGARAOEDED (SEQ ID NO:21); and has a length of 146 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGTVSSRRS WWPLPLLLLLLLLLGPAGARAOEDED (SEQ ID NO:21); and has a length of 146 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[0098] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMOHRGFLLL TLLALLALTSAV (SEQ ID NO:22), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMOHRGFLLL TLLALLALTSAV (SEQ ID NO:22); and has a length of from 130 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMQHRGFLLL TLLALLALTSAV (SEQ ID NO:22); and has a length of 132 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMQHRGFLLL TLLALLALTSAV (SEQ ID NO:22); and has a length of 132 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3 -sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMKFLLDILLL LPLLIVC (SEQ ID NO:23), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMKFLLDILLL LPLLIVC (SEQ ID NO:23); and has a length of from 126 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMKFLLDILLL LPLLIVC (SEQ ID NO:23); and has a length of 128 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMKFLLDILLL LPLLIVC (SEQ ID NO:23); and has a length of 128 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3 -sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLWWVTV QPPARRMGWLPLLLLLTQCLGVPGQRSPLNDF (SEQ ID NO:24), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGLWWVTV OPPARRMGWLPLLLLLTOCLGVPGORSPLNDF (SEQ ID NO:24); and has a length of from 150 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLWWVTV QPPARRMGWLPLLLLLTQCLGVPGQRSPLNDF (SEQ ID NO:24); and has a length of 151 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGLWWVTV OPPARRMGWLPLLLLLTOCLGVPGORSPLNDF (SEQ ID NO:24); and has a length of 151 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[00101] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGKFMKPG KVVLVLAGRYSGRKAVIVK (SEQ ID NO:25), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGKFMKPG KVVLVLAGRYSGRKAVIVK (SEQ ID NO:25); and has a length of from 136 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGKFMKPG KVVLVLAGRYSGRKAVIVK (SEQ ID NO:25); and has a length of 138 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGKFMKPG

KVVLVLAGRYSGRKAVIVK (SEQ ID NO:25); and has a length of 138 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive.

[00102] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMDAGVTESG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:26), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMDAGVTESG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:26); and has a length of from 142 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMDAGVTESG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:26); and has a length of 144 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMDAGVTESG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:26); and has a length of 144 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMSLOWTAV ATFLYAEVFVVLLLCIP (SEQ ID NO:27), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMSLOWTAV ATFLYAEVFVVLLLCIP (SEQ ID NO:27); and has a length of from 134 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMSLOWTAV ATFLYAEVFVVLLLCIP (SEQ ID NO:27); and has a length of 136 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMSLQWTAV ATFLYAEVFVVLLLCIP (SEQ ID NO:27); and has a length of 136 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of from 140 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of 143 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of 143 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGLHLRPYR VGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO:29), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLHLRPYR VGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO:29); and has a length of from 148 amino acids to 155 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGLHLRPYR VGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO:29); and has a length of 150 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLHLRPYR VGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO:29); and has a length of 150 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[00106] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRMKPWFEV GDENSGWSAOKVTNLHLMLOLVRVLVSPTNPPGA (SEQ ID NO:30), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRRMKPWFEV GDENSGWSAOKVTNLHLMLQLVRVLVSPTNPPGA (SEQ ID NO:30); and has a length of from 151 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRRMKPWFEV GDENSGWSAOKVTNLHLMLOLVRVLVSPTNPPGA (SEQ ID NO:30); and has a length of 153 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRMKPWFEV GDENSGWSAOKVTNLHLMLOLVRVLVSPTNPPGA (SEQ ID NO:30); and has a length of 153 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[00107] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAAPALGLV CGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO:31), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAAPALGLV CGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO:31); and has a length of from 141 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMAAPALGLV CGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO:31); and has a length of 143 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMAAPALGLV CGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO:31); and has a length of 143 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAAALWGF FPVLLLLLLS (SEQ ID NO:32), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMAAALWGF FPVLLLLLLS (SEQ ID NO:32); and has a length of from 127 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAAALWGF FPVLLLLLLS (SEQ ID NO:32); and has a length of 129 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMAAALWGF FPVLLLLLLS (SEQ ID NO:32); and has a length of 129 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3 -sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMARGAALA LLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO:33), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMARGAALA LLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO:33); and has a length of from 140 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMARGAALA LLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO:33); and has a length of 142 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMARGAALA LLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO:33); and has a length of 142 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMRAHAQRG RGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO:34), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMRAHAQRG RGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO:34); and has a length of from 150 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMRAHAORG RGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO:34); and has a length of 153 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMRAHAORG RGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID NO:34); and has a length of 153 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive. [00111] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMDTGVIEGG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:35), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMDTGVIEGG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:35); and has a length of from 142 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMDTGVIEGG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:35); and has a length of 144 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMDTGVIEGG LNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:35); and has a length of 144 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[00112] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGARASGGP LARAGLLLLLL (SEQ ID NO:36), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGARASGGP LARAGLLLLLL (SEQ ID NO:36); and has a length of from 129 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGARASGGP LARAGLLLLLL (SEQ ID NO:36); and has a length of 131 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGARASGGP LARAGLLLLLL (SEQ ID NO:36); and has a length of 131 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3 -sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRGPLKKSNAP LVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO:37), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRGPLKKSNAP LVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO:37); and has a length of from 150 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRGPLKKSNAP LVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO:37); and has a length of 154 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRGPLKKSNAP LVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID NO:37); and has a length of 154 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 1 sensitive. In some cases, such a ribosome stalling polypeptide is Compound 3-sensitive.

[00114] A suitable ribosome stalling signal comprises an amino acid sequence having at least

85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences:

[00115] i) NSVGEACTDMKREYDQCFNRWFAEK [TR1AP1] (SEQ ID NO:38);

[00116] ii) SHIQIPPGLTELLQGYTVEVLRQQPP [PRKAR2A] (SEQ ID NO:39);

[00117] iii) EWWASSPLRLWLLLRLLP [PLOGLUT1] (SEQ ID NO:40);

[00118] iv) NRVLCAPAAGAVRALRLIGWASRSLHP [C16ofr91[ (SEQ ID NO:41);

[00119] v) FSSSKANPHRWSVGHTMGKGHQRPWWKVLPLSCFLVALIIWCY [C16ofr91[

(SEQ ID NO:42);

[00120] vi) SRPQLRRWRLVSSPP [CTBS] (SEQ ID NO:43);

[00121] vii) FEVFVFDVGQKTWKSYDWSQITTVATFGKYDSELMCYAHSKGARVV

[CTBS] (SEQ ID NO:44);

[00122] viii) SASVVSVISRFLEEYLSSTPQRLK [DAD1] (SEQ ID NO:45);

[00123] ix) HLLAILFCALWSAVL [PTX3] (SEQ ID NO:46);

[00124] x) TFLYGTPTMFVDILNQPDFSSYDISTMCGGVIAGSPAPPE [ACSF2] (SEQ ID

NO:47);

[00125] xi) NEEYDVIVLGTGLTECILSGIM [DGI2] (SEQ ID NO:48);

[00126] xii) FPRVSTFLPLRPLSRHPLSSGSPETSAAAIMLLTVRH [MRSP31] (SEQ ID

NO:49);

[00127] xiii) ALFVRLLA [TGFBI] (SEQ ID NO:50); [00128] xiv) RIEKCYFCSGPIYP [RSL24D1] (SEQ ID NO:51);

[00129] xv) AAMASLGALALLLLSSLSRC [SSR4] (SEQ ID NO:52);

[00130] xvi) RSLGALLLLLSACLAVSAGPVPTPPD [AMBP] (SEQ ID NO:53);

[00131] xvii) MLLKTVLLLGHVAQVLMLDNGLLQTPPMGW [NAGA] (SEQ ID NO:54);

[00132] xviii) MGGRVFLVFLAFCVWLTLPGAE [ADEGRE2] (SEQ ID NO:55);

[00133] xix) MGAVWSALLVGGGLAGALFVWLLRGGPG [STBD1] (SEQ ID NO:56);

[00134] xx) MGPAGSLLGSGQMQITLWGSLAAVAIFFVITFLIFLCS [PAG1] (SEQ ID

NO:57);

[00135] xxi) MSRFLNVLRSWLVMVSIIAMGNTLQSFRDHTFLYEKLYTGKPNL [C14orfl]

(SEQ ID NO:58);

[00136] xxii) MGLQACLLGLFALILSGKCSYSPEPD [TPP1] (SEQ ID NO:59);

[00137] xxiii) MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA [PODXL2] (SEQ ID

NO: 10);

[00138] xxiv) MAADLNLEWISLPRSWTYGITRGGRVFFINEEAKS [PLEKHAS] (SEQ ID

NO:60);

[00139] xxv) MCTGGCARCLGGTLIPLAFFGFLANILLFFPGGKVIDDND [TM4SF] (SEQ ID

NO:61); and

[00140] xxvi) MGPVRLGILLFLFL [CNPY4] (SEQ ID NO:62).

[00141] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

NSVGEACTDMKREYDQCFNRWFAEK (SEQ ID NO:38). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00142] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

NSVGEACTDMKREYDQCFNRWFAEK (SEQ ID NO:38). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00143] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SHIQIPPGLTELLQGYTVEVLRQQPP (SEQ ID NO:39). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00144] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EWWASSPLRLWLLLRLLP (SEQ ID NO:40). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00145] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

NRVLCAPAAGAVRALRLIGWASRSLHP (SEQ ID NO:41). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00146] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

FSSSKANPHRWSVGHTMGKGHQRPWWKVLPLSCFLVALIIWCY (SEQ ID NO:42). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00147] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SRPQLRRWRLVSSPP (SEQ ID NO:43). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00148] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

FEVFVFDVGQKTWKSYDWSQITTVATFGKYDSELMCYAHSKGARVV (SEQ ID NO:44). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00149] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SASVVSVISRFLEEYLSSTPQRLK (SEQ ID NO:45). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00150] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: HLLAILFCALWSAVL (SEQ ID NO:46). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00151] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

TFLYGTPTMFVDILNQPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:47). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00152] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: NEEYDVIVLGTGLTECILSGIM (SEQ ID NO:48). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00153] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

FPRVSTFLPLRPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:49). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00154] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: ALFVRLLA (SEQ ID NO:50). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00155] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: RIEKCYFCSGPIYP (SEQ ID NO:51). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal. [00156] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: AAMASLGALALLLLSSLSRC (SEQ ID NO:52). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00157] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

RSLGALLLLLSACLAVSAGPVPTPPD (SEQ ID NO:53). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00158] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MLLKTVLLLGHVAQVLMLDNGLLQTPPMGW (SEQ ID NO:54). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00159] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MGGRVFLVFLAFCVWLTLPGAE (SEQ ID NO:55). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00160] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGAVWSALLVGGGLAGALFVWLLRGGPG (SEQ ID NO:56). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00161] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGPAGSLLGSGQMQITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:57). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal. [00162] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MSRFLNVLRSWLVMVSIIAMGNTLQSFRDHTFLYEKLYTGKPNL (SEQ ID NO:58). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00163] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGLQACLLGLFALILSGKCSYSPEPD (SEQ ID NO:59). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00164] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO: 10). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00165] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MAADLNLEWISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:60). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00166] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MCTGGCARCLGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO:61). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal.

[00167] In some cases, a suitable ribosome stalling signal comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MGPVRLGILLFLFL (SEQ ID NO:62). In some cases, such a ribosome stalling signal is a Compound 2-sensitive signal. [00168] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNSVGEACTD MKREYDOCFNRWFAEK (SEQ ID NO:63), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRNSVGEACTD MKREYDOCFNRWFAEK (SEQ ID NO:63); and has a length of from 133 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRNSVGEACTD MKREYDOCFNRWFAEK (SEQ ID NO:63); and has a length of 136 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNSVGEACTD MKREYDOCFNRWFAEK (SEQ ID NO:63); and has a length of 136 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00169] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSHIOIPPGLTE LLOGYTVEVLROOPP (SEQ ID NO:64), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSHIOIPPGLTE LLOGYTVEVLROOPP (SEQ ID NO:64); and has a length of from 135 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSHIQIPPGLTE LLOGYTVEVLROOPP (SEQ ID NO:64); and has a length of 137 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDOVTTLEVSVCDCEGAAGVCRSHIQIPPGLTE LLOGYTVEVLROOPP (SEQ ID NO:64); and has a length of 137 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREWWASSPLR LWLLLRLLP (SEQ ID NO:65), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREWWASSPLR LWLLLRLLP (SEQ ID NO:65); and has a length of from 127 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREWWASSPLR LWLLLRLLP (SEQ ID NO:65); and has a length of 129 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCREWWASSPLR LWLLLRLLP (SEQ ID NO:65); and has a length of 129 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRNRVLCAPAA GAVRALRLIGWASRSLHP (SEQ ID NO:66), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRNRVLCAPAA GAVRALRLIGWASRSLHP (SEQ ID NO:66); and has a length of from 136 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNRVLCAPAA GAVRALRLIGWASRSLHP (SEQ ID NO:66); and has a length of 138 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNRVLCAPAA GAVRALRLIGWASRSLHP (SEQ ID NO:66); and has a length of 138 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive. [00172] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFSSSKANPH RWSVGHTMGKGHORPWWKVLPLSCFLVALIIWCY (SEQ ID NO:67), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRFSSSKANPH RWSVGHTMGKGHQRPWWKVLPLSCFLVALIIWCY (SEQ ID NO:67); and has a length of from 150 amino acids to 165 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRFSSSKANPH RWSVGHTMGKGHQRPWWKVLPLSCFLVALIIWCY (SEQ ID NO:67); and has a length of 154 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFSSSKANPH RWSVGHTMGKGHORPWWKVLPLSCFLVALIIWCY (SEQ ID NO:67); and has a length of 154 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00173] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSRPOLRRW R

LVSSPP (SEQ ID NO:68), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSRPOLRRWR LVSSPP (SEQ ID NO:68); and has a length of from 124 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRSRPOLRRWR LVSSPP (SEQ ID NO:68); and has a length of 126 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRSRPOLRRWR LVSSPP (SEQ ID NO:68); and has a length of 126 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFEVFVFDVG OKTWKSYDWSOITTVATFGKYDSELMCYAHSKGARVV (SEQ ID NO:69), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFEVFVFDVG OKTWKSYDWSOITTVATFGKYDSELMCYAHSKGARVV (SEQ ID NO:69); and has a length of from 155 amino acids to 170 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFEVFVFDVG OKTWKSYDWSOITTVATFGKYDSELMCYAHSKGARVV (SEQ ID NO:69); and has a length of 157 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFEVFVFDVG QKTWKSYDWSOITTVATFGKYDSELMCYAHSKGARVV (SEQ ID NO:69); and has a length of 157 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRSASVVSVISR FLEE YLS STPQRLK (SEQ ID NO:70), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSASVVSVISR FLEE YLS STPQRLK (SEQ ID NO:70); and has a length of from 133 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSASVVSVISR FLEE YLS STPQRLK (SEQ ID NO:70); and has a length of 135 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRSASVVSVISR FLEE YLS STPORLK (SEQ ID NO:70); and has a length of 135 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00176] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRHLLAILFCAL WSAVL (SEQ ID NO:71), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRHLLAILFCAL WSAVL (SEQ ID NO:71); and has a length of from 123 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRHLLAILFCAL WSAVL (SEQ ID NO:71); and has a length of 126 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRHLLAILFCAL WSAVL (SEQ ID NO:71); and has a length of 126 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00177] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRTFLYGTPT M FVDILNOPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:72), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRTFLYGTPTM FVDILNOPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:72); and has a length of from 148 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRTFLYGTPTM FVDILNOPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:72); and has a length of 151 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRTFLYGTPTM FVDILNQPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:72); and has a length of 151 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNEEYDVIVL GTGLTECILSGIM (SEQ ID NO:73), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRNEEYDVIVL GTGLTECILSGIM (SEQ ID NO:73); and has a length of from 130 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNEEYDVIVL GTGLTECILSGIM (SEQ ID NO:73); and has a length of 133 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRNEEYDVIVL GTGLTECILSGIM (SEQ ID NO:73); and has a length of 133 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRFPRVSJFLPL RPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:74), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFPRVSTFLPL RPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:74); and has a length of from 145 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRFPRVSTFLPL RPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:74); and has a length of 148 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRFPRVSJFLPL RPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:74); and has a length of 148 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00180] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRALFVRLLA

(SEQ ID NO:75), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRALFVRLLA

(SEQ ID NO:75); and has a length of from 117 amino acids to 130 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRALFVRLLA

(SEQ ID NO:75); and has a length of 119 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRALFVRLLA

(SEQ ID NO:75); and has a length of 119 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00181] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRAAMASLGA L

ALLLLSSLSRC (SEQ ID NO:76), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRAAMASLGAL ALLLLSSLSRC (SEQ ID NO:76); and has a length of from 129 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRAAMASLGAL ALLLLSSLSRC (SEQ ID NO:76); and has a length of 131 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRAAMASLGAL ALLLLSSLSRC (SEQ ID NO:76); and has a length of 131 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRIEKCYFCSG PIYP (SEQ ID NO:77), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRIEKCYFCSG PIYP (SEQ ID NO:77); and has a length of from 123 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRIEKCYFCSG PIYP (SEQ ID NO:77); and has a length of 125 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRIEKCYFCSG PIYP (SEQ ID NO:77); and has a length of 125 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRRSLGALLLLL SACLAVSAGPVPTPPD (SEQ ID NO:78), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRSLGALLLLL SACLAVSAGPVPTPPD (SEQ ID NO:78); and has a length of from 135 amino acids to 145 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRRSLGALLLLL SACLAVSAGPVPTPPD (SEQ ID NO:78); and has a length of 137 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRRSLGALLLLL SACLAVSAGPVPTPPD (SEQ ID NO:78); and has a length of 137 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00184] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMLLKTVLLL GHVAOVLMLDNGLLOTPPMGW (SEQ ID NO:79), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMLLKTVLLL GHVAOVLMLDNGLLOTPPMGW (SEQ ID NO:79); and has a length of from 139 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMLLKTVLLL GHVAOVLMLDNGLLOTPPMGW (SEQ ID NO:79); and has a length of 141 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMLLKTVLLL GHVAOVLMLDNGLLOTPPMGW (SEQ ID NO:79); and has a length of 141 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00185] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGGRVFLVF LAFCVWLTLPGAE (SEQ ID NO:80), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGGRVFLVF LAFCVWLTLPGAE (SEQ ID NO:80); and has a length of from 130 amino acids to 140 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGGRVFLVF LAFCVWLTLPGAE (SEQ ID NO: 80); and has a length of 133 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGGRVFLVF LAFCVWLTLPGAE (SEQ ID NO: 80); and has a length of 133 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGAVWSAL LVGGGLAGALFVWLLRGGPG (SEQ ID NO:81), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGAVWSAL LVGGGLAGALFVWLLRGGPG (SEQ ID NO:81); and has a length of from 137 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGAVWSAL LVGGGLAGALFVWLLRGGPG (SEQ ID NO:81); and has a length of 139 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGAVWSAL LVGGGLAGALFVWLLRGGPG (SEQ ID NO:81); and has a length of 139 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMGPAGSLLG SGOMQITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:82), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPAGSLLG SGOMQITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:82); and has a length of from 147 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPAGSLLG SGOMQITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:82); and has a length of 149 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGPAGSLLG SGOMOITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:82); and has a length of 149 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00188] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMSRFLNVLR SWLVMVSIIAMGNTLOSFRDHTFLYEKLYTGKPNL (SEQ ID NO: 83), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDQVTTLEVSVCDCEGAAGVCRMSRFLNVLR SWLVMVSIIAMGNTLQSFRDHTFLYEKLYTGKPNL (SEQ ID NO:83); and has a length of from 153 amino acids to 165 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMSRFLNVLR SWLVMVSIIAMGNTLOSFRDHTFLYEKLYTGKPNL (SEQ ID NO:83); and has a length of 155 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMSRFLNVLR SWLVMVSIIAMGNTLOSFRDHTFLYEKLYTGKPNL (SEQ ID NO:83); and has a length of 155 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00189] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ

ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLQACLL G LFALILSGKCSYSPEPD (SEQ ID NO:84), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGLOACLLG LFALILSGKCSYSPEPD (SEQ ID NO:84); and has a length of from 135 amino acids to 150 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGLQACLLG LFALILSGKCSYSPEPD (SEQ ID NO: 84); and has a length of 137 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGLQACLLG LFALILSGKCSYSPEPD (SEQ ID NO: 84); and has a length of 137 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of from 140 amino acids to 155 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of 143 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGRLLRAAR LPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO:28); and has a length of 143 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMAADLNLE WISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:85), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAADLNLE WISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:85); and has a length of from 144 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQY NDPTQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMAADLNLE WISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:85); and has a length of 146 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMAADLNLE WISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:85); and has a length of 146 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00192] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMCTGGCARC LGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO: 86), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMCTGGCARC LGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO:86); and has a length of from 149 amino acids to 160 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRMCTGGCARC LGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO:86); and has a length of 151 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMCTGGCARC LGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO:86); and has a length of 151 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

[00193] In some cases, a ribosome stalling polypeptide comprises an amino acid sequence

having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPVRLGIL LFLFL (SEQ ID NO:87), where the ribosome stalling signal is underlined. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPVRLGIL LFLFL (SEQ ID NO:87); and has a length of from 123 amino acids to 135 amino acids. In some cases, a ribosome stalling polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPVRLGIL LFLFL (SEQ ID NO:87); and has a length of 125 amino acids. In some cases, a ribosome stalling polypeptide comprises the amino acid sequence

SDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDP TQ ESIILKPKMALEVGDYKINLKLMDNONKDOVTTLEVSVCDCEGAAGVCRMGPVRLGIL LFLFL (SEQ ID NO:87); and has a length of 125 amino acids. In some cases, such a ribosome stalling polypeptide is Compound 2 sensitive.

Linkers

[00194] In some cases, a peptide linker is interposed between a stalling signal protector

polypeptide and a ribosome stalling signal present in a ribosome stalling polypeptide. In some cases, a peptide linker is interposed between a ribosome stalling polypeptide and a heterologous polypeptide.

[00195] Suitable linkers (also referred to as "spacers") can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable linkers can have a length of more than 20 amino acids, e.g., from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, or from 40 amino acids to 50 amino acids. Tags

[00196] In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a tag, e.g., an affinity tag.

[00197] Suitable tags include, e.g., His5 (HHHHH) (SEQ ID NO:88), HisX6 (HHHHHH) (SEQ

ID NO:89), C-myc (EQKLISEEDL) (SEQ ID NO:90), Flag (DYKDDDDK) (SEQ ID NO:91), StrepTag (WSHPQFEK) (SEQ ID NO:92), hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:93), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:94), Phe-His-His-Thr (SEQ ID NO:95), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:96), metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

[00198] In some cases, the tag is selected from an Avi tag (GLNDIFEAQKIEWHE) (SEQ ID

NO:98), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO:99), a FLAG tag (DYKDDDDK) (SEQ ID NO:91), a His-FLAG tag (HHHHHHDYKDHDG) (SEQ ID NO: 142), a 3XFLAG tag (DYKDHDGDYKDHDIDYKDDDDK) (SEQ ID NO: 145), a hemagglutinin tag (YPYDVPDYA) (SEQ ID NO:93), a poly(histidine) tag (HHHHHH) (SEQ ID NO:89), a Myc tag (EQKLISEEDL) (SEQ ID NO:90), an S tag (KETAAAKFERQHMDS) (SEQ ID NO: 100), an SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ GQREP) (SEQ ID NO: 101), a Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 102), a Softag 3 (TQDPSRVG) (SEQ ID NO: 103), a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 104), an Xpress tag (DLYDDDDK) (SEQ ID NO: 105), an Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 106), a SpyTag (AHIVMVDAYKPTK) (SEQ ID NO: 107), a TwinStrep tag

(WSHPQFEKGAMTGWSHPQFEK) (SEQ ID NO: 144) and a streptactin tag (Strep-tag II: WSHPQFEK) (SEQ ID NO:92).

[00199] A nucleotide sequence encoding a tag can be placed in any position in a nucleic acid of the present disclosure. In some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a heterologous polypeptide-encoding nucleotide sequence; b) a tag- encoding nucleotide sequence; and c) a ribosome stalling polypeptide-encoding nucleotide sequence. In other cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a ribosome stalling polypeptide -encoding nucleotide sequence; b) a tag-encoding nucleotide sequence; and c) a heterologous polypeptide-encoding nucleotide sequence.

IRES

[00200] In some cases, a nucleic acid of the present disclosure comprises an internal ribosome entry site (IRES). In some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) an IRES; b) a heterologous polypeptide-encoding nucleotide sequence; and c) a ribosome stalling polypeptide-encoding nucleotide sequence. In other cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) an IRES; b) a ribosome stalling polypeptide-encoding nucleotide sequence; and c) a heterologous polypeptide-encoding nucleotide sequence.

[00201] Suitable IRES include picornavirus IRES (e.g., poliovirus IRES; rhinovirus IRES;

encephalomyocarditis virus IRES); aphto virus IRES; hepatitis A virus IRES; hepatitis C virus IRES; pesti virus IRES; encephalomyocarditis virus IRES; cytomegalovirus IRES; fibroblast growth factor-1 IRES; FGF-2 IRES; platelet-derived growth factor B IRES; vascular endothelial growth factor IRES; insulin-like growth factor-2 IRES; c-myc RES; apoptotic protease activating factor-1 IRES; ΝΚ-κΒ repressing factor IRES; and the like. The nucleotide sequences of numerous IRESs are known in the art, and any such IRES sequence can be used.

Heterologous polypeptides

[00202] A ribonucleic acid of the present disclosure (or a DNA molecule encoding same) can comprise a nucleotide sequence encoding a heterologous polypeptide of interest. In some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a heterologous polypeptide-encoding nucleotide sequence; and b) a ribosome stalling polypeptide-encoding nucleotide sequence. In other cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a ribosome stalling polypeptide-encoding nucleotide sequence; and b) a heterologous polypeptide-encoding nucleotide sequence. In some cases, the heterologous polypeptide of interest produces a detectable signal.

[00203] In some cases, the heterologous polypeptide of interest generates a detectable signal. In some cases, the heterologous polypeptide of interest is a luminescent protein. In some cases, the heterologous polypeptide of interest is a chromogenic protein. In some cases, the heterologous polypeptide of interest is a fluorescent protein. In some cases, the heterologous polypeptide of interest is an enzyme that catalyzes production of a chromophore, luminophore, or other product that produces a detectable signal. In some instances, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a ribosome stalling polypeptide-encoding nucleotide sequence; and b) a heterologous polypeptide-encoding nucleotide sequence, where the heterologous polypeptide produces a detectable signal.

[00204] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein

(GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

[00205] Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, β-glucuronidase, invertase, Xanthine Oxidase, luciferase (e.g., firefly lucif erase), glucose oxidase (GO), and the like.

[00206] In some cases, the heterologous polypeptide comprises one or more amino acid

differences compared to the amino acid sequence of a reference polypeptide. For example, a reference polypeptide can be an antibody, an enzyme, a receptor, or any other polypeptide for which generation of variants is of interest. For example, in some cases, a ribonucleic acid of the present disclosure comprises, in order from 5' to 3' : a) a heterologous polypeptide-encoding nucleotide sequence, where the heterologous polypeptide is a variant of a reference polypeptide; and b) a ribosome stalling polypeptide-encoding nucleotide sequence. [00207] In some cases, the one or more amino acid differences are selected from one or more of an amino acid substitution, an amino acid insertion, and an amino acid deletion. For example, a variant heterologous polypeptide of interest can have a single amino acid substitution compared to the amino acid sequence a reference polypeptide. A variant heterologous polypeptide of interest can have 2, 3, 4 5, 6, 7, 8, 9, 10, or more than 10, amino acid substitutions compared to the amino acid sequence a reference polypeptide.

[00208] A reference polypeptide can be a wild-type polypeptide, or any polypeptide for which variants are to be generated and tested. In some cases, a reference polypeptide is an antigen- binding portion of an antibody. In some cases, a reference polypeptide is an enzyme. For example, the enzyme can be an enzyme of a metabolic pathway (e.g., a mevalonate pathway; a polyketide synthesis pathway; and the like). In some cases, a reference polypeptide is a receptor. In some cases, the reference polypeptide is an antibody or an antigen-binding fragment such as a nanobody, a single-chain Fv, and the like. In some cases, the reference polypeptide is a protease inhibitor. In some cases, the reference polypeptide is a nanobody. In some cases, the reference polypeptide is an Affitin. In some cases, the reference polypeptide is a bacterial periplasmic binding protein. In some cases, the reference polypeptide is a fluorescent protein. In some cases, the reference polypeptide is a T7 phage gene 2 protein. In some cases, the reference polypeptide is an ankyrin repeat protein. In some cases, the reference polypeptide is an affibody. In some cases, the reference polypeptide is a knottin. In some cases, the reference polypeptide is a receptor ligand. In some cases, the reference polypeptide is a viral capsid protein.

Stalling agents

[00209] A nucleic acid of the present disclosure can be an RNA that, when present in a

eukaryotic cell comprising a translation-stalling agent, or when present in a composition such as a cell-free in vitro translation system comprising a translation-stalling agent, is translated, wherein the ribosome stalling polypeptide and the translation-stalling agent together provide for ribosome stalling.

[00210] A suitable stalling agent is a substituted amide compound of Formula I:

[00211] wherein:

[00212] R ¹ is pyrid-2-yl, isoquinolin-l-yl or lH-pyrrolo[2,3-c]pyridin-7-yl;

[00213] R ¹ is optionally mono- or di-substituted with chloro or (Ci-C4)alkyl; X and Y are

independently either N or C(H), provided that at least one of X or Y is C(H);

[00214] R ² is H, fluoro, hydroxyl or methyl;

[00215] R ³ is

[00216] where R ⁶ and R ⁸ re each independently H, methyl, halo or (Ci-C4)alkyloxy, provided that only one of R ⁶ and R ⁸ is halo; wherein R ¹⁰ and R ⁿ are each independently H, (Ci-C4)alkyl or (C3-Cs)cycloalkyl; and wherein R ⁷ is hydroxyl, (Ci-C4)alkyloxy, (Ci-C4)alkoxycarbonyloxy(Ci- C4)alkyloxy, or (Ci-C4)alkylcarbonyloxy(Ci-C4)alkoxy. [00217] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl. In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl and the piperidinyl C* is (R).

[00218] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl; and X and Y are both C(H), R ² is H and R ¹ is optionally mono-substituted with chloro or methyl. In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H and R ¹ is optionally mono-substituted with chloro or methyl.

[00219] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl; and X and Y are both C(H), R ² is H and R ¹ is optionally mono-substituted with chloro or methyl; and where:

[00220] R ³ is:

[00221] R ⁷ hydroxyl, (Ci-C ₂ )alkyloxy or

[00222] R ¹⁰ is methyl; and

[00223] R ⁿ is H.

[00224] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H and R ¹ is optionally mono- substituted with chloro or methyl; and where:

[00225] R ³ is:

[00226] R ⁷ hydroxy!, (Ci-C ₂ )alkyloxy or

[00227] R ¹⁰ is methyl; and

[00228] R ⁿ is H.

[00229] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl; and X and Y are both C(H), R ² is H and R ¹ is optionally mono-substituted with chloro or methyl; and

[00230] R ³ is:

[00231] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H and R ¹ is optionally mono- substituted with chloro or methyl; and

[00232] R ³ is:

[00233] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl; and X and Y are both C(H), R ² is H and R ¹ is optionally mono-substituted with chloro or methyl; and

[00234] R ³ is:

[00235] R ⁶ is H or methyl and R ⁸ is H.

[00236] In some cases, a suitable agent is a compound of Formula I, wherein R ¹ is pyrid-2-yl and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H and R ¹ is optionally mono- substituted with chloro or methyl; and

[00237] R ³ is:

[00238] R ⁶ is H or methyl and R ⁸ is H. [00239] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-1- yl. In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-l-yl; and the piperidinyl C* is (R).

[00240] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-l- yl; X and Y are both C(H), R ² is H, hydroxyl, or methyl; and R ¹ is optionally mono-substituted with chloro or methyl. In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-l-yl; and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H, hydroxyl, or methyl and R ¹ is optionally mono-substituted with chloro or methyl.

[00241] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-l- yl; and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H, hydroxyl, or methyl and R ¹ is optionally mono-substituted with chloro or methyl, where R ³ is:

[00242] R ⁷ is hydroxyl, Ci-C ₂ )alkoxy or

[00243] R ¹⁰ is methyl; and R ⁿ is H.

[00244] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-l- yl; and X and Y are both C(H), R ² is H, hydroxyl, or methyl and R ¹ is optionally mono- substituted with chloro or methyl, where R ³ is:

[00245] R ⁷ is hydroxyl, Ci-C2)alkoxy or

[00246] R ¹⁰ is methyl; and R ⁿ is H.

[00247] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-1- yl; and X and Y are both C(H), R ² is H, hydroxyl, or methyl and R ¹ is optionally mono- substituted with chloro or methyl; where R ³ is:

[00248] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-1- yl; and the piperidinyl C* is (R); and X and Y are both C(H), R ² is H, hydroxyl, or methyl and R ¹ is optionally mono-substituted with chloro or methyl; where R ³ is:

[00249] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-1- yl; where X and Y are both C(H); R ² is H, hydroxyl, or methyl; and R ¹ is optionally mono- substituted with chloro or methyl; and R ³ is:

[00250] R ⁶ is H or methyl; and R ⁸ is H.

[00251] In some cases, a suitable agent is a compound of Formula I, where R ¹ is isoquinolin-1- yl; and the piperidinyl C* is (R); where X and Y are both C(H); R ² is H, hydroxyl, or methyl; and R ¹ is optionally mono-substituted with chloro or methyl; and R ³ is:

[00252] R ⁶ is H or methyl; and R ⁸ is H.

[00253] In some cases, a suitable agent is a compound of Formula I, where R ¹ is lH-pyrrolo[2,3- c]pyridin-7-yl. In some cases, a suitable agent is a compound of Formula I, where R ¹ is 1H- pyrrolo[2,3-c]pyridin-7-yl and the piperidinyl C* is (R). [00254] In some cases, a suitable agent is a compound of Formula I, where R ¹ is lH-pyrrolo[2,3- c]pyridin-7-yl; where X and Y are both C(H); and where R ² is H, hydroxyl, or methyl; and R ¹ is optionally mono-substituted with chloro or methyl. In some cases, a suitable agent is a compound of Formula I, where R ¹ is lH-pyrrolo[2,3-c]pyridin-7-yl and the piperidinyl C* is (R); where X and Y are both C(H); and where R ² is H, hydroxyl, or methyl; and R ¹ is optionally mono-substituted with chloro or methyl.

[00255] In some cases, the ribosome stalling agent is N-(3-chloropyridin-2-yl)-N-[(3R)- piperidin-3-yl]-4-(3H-[l ,2,3]triazolo[4,5-b]pyridine-3-yl)benzamide, referred to herein as "Compound 1."

[00256 following structure:

[00257] In some cases, the ribosome stalling agent is (R)-N-(isoquinolin-l-yl)-N-(piperidine-3- yl)-4-(pyrazolo[l ,5-a]pyrimidin-3-yl)banzamide, referred to herein as "Compound 2."

[00258] 2 has the following structure:

[00259] In some cases, the ribosome stalling agent is N-(3-chloropyridin-2-yl)-5-(6-methyl-3H-

[1 ,2,3] triazolo[4,5-b]pyridine-3-yl)-N-[(3R)-piperidin-3-yl]pyridin e-2-caboxamide, referred to herein as "Compound 3."

[00260] Compound 3 has the following structure:

Nucleic acid encoding a ribonucleic acid; recombinant expression vectors

[00261] The present disclosure also provides a nucleic acid (e.g., a DNA) comprising a

nucleotide sequence encoding an RNA of the present disclosure.

[00262] In some cases, the DNA is present in a recombinant expression vector. In some cases, the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus (AAV) construct, a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc. In some cases, the recombinant expression vector is a plasmid construct. In some cases, a recombinant vector (e.g., a recombinant DNA plasmid; a recombinant viral vector; etc.) of the present disclosure comprises a nucleotide sequence encoding an RNA of the present disclosure.

[00263] In some cases, the nucleotide sequence encoding an RNA of the present disclosure is operably linked to a transcription control element. In some cases, the nucleotide sequence encoding an RNA of the present disclosure is operably linked to a promoter, e.g., a promoter that is functional in a eukaryotic cell. In some cases, the promoter is regulatable. In some cases, the promoter is constitutive. Suitable promoters can be any known promoter and include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc.

[00264] In some cases, a DNA recombinant nucleic acid encoding an RNA of the present

disclosure comprises an amino acid sequence encoding a tag, where suitable tags are describe elsewhere herein. Such tags can facilitate recovery of an RNC of interest. [00265] In some cases, a DNA recombinant nucleic acid encoding an RNA of the present disclosure comprises an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide. For example, an insertion site can be located 5' of the ribosome stalling polypeptide-encoding nucleotide sequence. As another example, an insertion site can be located 3' of the ribosome stalling polypeptide-encoding nucleotide sequence. The insertion site could be within 80 nucleotides (nt), within 60 nt, within 40 nt, within 20 nt, within 15 nt, within 10 nt, within 5 nt, or within 2 nt, of the ribosome stalling polypeptide-encoding nucleotide sequence.

[00266] A DNA of the present disclosure can be introduced into a cell, generating a genetically modified cell.

Nascent polypeptide

[00267] The present disclosure provides a nascent chain polypeptide encoded by a nucleic acid of the present disclosure. The nascent chain polypeptide can have a length of from 1 amino acid to 900 or more amino acids. For example, the nascent chain polypeptide can have a length of from 1 amino acid (aa) to 5 aa, from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa, from 700 aa to 800 aa, or from 800 aa to 900 aa. The RNC polypeptide can have a length of more than 900 aa.

[00268] Because translation is stalled, the nascent chain polypeptide can be attached to the

ribonucleic acid/ribosome complex. Thus, the present disclosure provides a ribosome/ribonucleic acid/nascent chain polypeptide complex.

NUCLEIC ACID-RIBOSOME COMPLEX

[00269] The present disclosure provides a complex comprising: a) a ribonucleic acid of the present disclosure; and b) a ribosome. The present disclosure provides a complex comprising: a) a ribonucleic acid of the present disclosure; b) a ribosome; and c) a nascent chain polypeptide encoded by the ribonucleic acid.

[00270] The result of ribosomal stalling is the formation of a stable arrested

ribosome/RNA/nascent chain polypeptide complex. Such a complex can be used in biophysical analytical methods, and in the identification and characterization of biologically and

pharmacologically important polypeptides.

[00271] A complex of the present disclosure is stable at room temperature for a period of time of from about 15 minutes to 48 hours, e.g., from about 15 minutes to about 30 minutes, from about 30 minutes to about 60 minutes, from about 1 hour to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 48 hours.

[00272] In some cases, the ribosome is a eukaryotic ribosome. In some cases, the ribosome is a mammalian ribosome. The ribosome can be from any of a variety of eukaryotic cells, e.g., a mammalian cell; an insect cell; an arachnid cell; an invertebrate cell; a reptile cell; a yeast cell; an algal cell; and the like.

[00273] In some cases, the ribosome is a prokaryotic ribosome. In some cases, the ribosome is an archaeal ribosome.

Compositions

[00274] The present disclosure provides a composition comprising a complex of the present disclosure. The present disclosure provides a composition comprising: a) a ribonucleic acid of the present disclosure; and b) a ribosome. The present disclosure provides a composition comprising: a) a ribonucleic acid of the present disclosure; b) a ribosome; and c) a nascent chain polypeptide encoded by the ribonucleic acid.

[00275] The composition can comprise a cell lysate. Suitable cell lysates include, but are not limited to, a wheat germ lysate, a reticulocyte lysate, a yeast cell lysate, a mammalian cell lysate, and an insect cell lysate.

[00276] A composition of the present disclosure can comprise a stalling agent. For example, a composition of the present disclosure can comprise a compound of Formula I. As one example, a composition of the present disclosure can comprise Compound 1. As another example, a composition of the present disclosure can comprise Compound 2. As another example, a composition of the present disclosure can comprise Compound 3.

[00277] A composition of the present disclosure can include one or more of: a buffer; a salt; a pH adjusting agent; a reducing agent; adenosine triphosphate (ATP); spermidine; putrescine; amino acids; creatine phosphate; creatine kinase; and an RNAse inhibitor. Libraries

[00278] The present disclosure provides a library comprising a plurality of ribosome -ribonucleic acid complexes of the present disclosure. In some cases, each of the ribonucleic acids comprises a nucleotide sequence (e.g., a nucleotide sequence of from 5 to 30 (e.g., 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30) nucleotides in length) that serves as a barcode. A library of complexes of the present disclosure is useful in methods of identifying a polypeptide of interest (e.g., identifying a variant polypeptide of interest), as described below.

[00279] A library of the present disclosure can comprise a plurality of members, which differ from one another in the nucleotide sequence encoding the heterologous polypeptide of interest. A library of the present disclosure can comprise from 10 to 10 ¹² different members. A library of the present disclosure can comprise from 10 to 10 ² different members, from 10 ² to 10 ³ different members, from 10 ³ to 10 ⁴ different members, from 10 ⁴ to 10 ^s different members, from 10 ^s to 10 ⁶ different members, from 10 ⁶ to 10 ⁷ different members, from 10 ⁷ to 10 ^s different members, from 10 ^s to 10 ⁹ different members, from 10 ⁹ to 10 ¹⁰ different members, from 10 ¹⁰ to 10 ⁿ different members, from 10 ⁿ to 10 ¹² different members, or from 10 ¹² to 10 ¹³ different members. In some cases, a library of the present disclosure comprises more than 10 ¹³ different members.

[00280] A library of the present disclosure can comprise a plurality of ribosome -ribonucleic acid complexes of the present disclosure, comprising nucleotide sequences encoding a polypeptide of interest, where the different members of the plurality encode variants of the polypeptide of interest that differ from one another in amino acid sequence. In some cases, the library comprises variants of an antibody or an antigen-binding fragment such as a nanobody, a single-chain Fv, and the like. In some cases, the library comprises variants of an enzyme. In some cases, the library comprises variants of a receptor. In some cases, the library comprises variants of a protease inhibitor. In some cases, the library comprises variants of a nanobody. In some cases, the library comprises variants of an Affitin. In some cases, the library comprises variants of a bacterial periplasmic binding protein. In some cases, the library comprises variants of a fluorescent protein. In some cases, the library comprises variants of a T7 phage gene 2 protein.

In some cases, the library comprises variants of an ankyrin repeat protein. In some cases, the library comprises variants of an affibody. In some cases, the library comprises variants of a knottin. In some cases, the library comprises variants of a receptor ligand. In some cases, the library comprises variants of a viral capsid protein. In some cases, the library comprises variants of a nanobody. In some cases, the library comprises variants of a single -chain Fv polypeptide. In some cases, the library comprises variants of a camelid antibody.

[00281] For example, a library of the present disclosure can comprise a plurality of ribosome - ribonucleic acid complexes of the present disclosure, comprising nucleotide sequences encoding an antigen-binding portion of an antibody, where the different members of the plurality encode variants of the antigen-binding portions that differ from one another in amino acid sequence. For example, the antigen-binding portion of an antibody can be a nanobody. As another example, the antigen-binding portion of an antibody can be a single-chain Fv polypeptide.

[00282] For example, a library of ribonucleic acids of the present disclosure is translated to provide a library of stalled ribonucleic acid/ribosome/nascent polypeptide complexes. The library of library of stalled ribonucleic acid/ribosome/nascent polypeptide complexes can then be screened with a selective agent having affinity for the nascent polypeptide chain, so as to provide a pool of ribosome -bound nascent chains of interest. In some cases, generating a library of ribonucleic acids comprises generating a library of mRNAs encoding a protein. In some cases, the protein is a single-chain variable fragment. In some cases, the library of ribonucleic acids lacks a stop codon in the open reading frame. In such cases, the library of ribonucleic acids lacking a stop codon in the open reading frame is subjected to in vitro translation (IVT).

[00283] As another example, a library of the present disclosure can comprise a plurality of

ribosome -ribonucleic acid complexes of the present disclosure, comprising nucleotide sequences encoding an enzyme, where the different members of the plurality encode variants of the enzyme that differ from one another in amino acid sequence. As an example, the enzyme can be an enzyme in a metabolic pathway, such as a biosynthetic pathway. Suitable biosynthetic pathway enzymes include, but are not limited to, enzymes that form an isoprenoid biosynthetic pathway; enzymes that form an alkaloid biosynthetic pathway; enzymes that form a phenylpropanoid biosynthetic pathway; and enzymes that form a polyketide biosynthetic pathway. Alkaloid biosynthetic pathway enzymes are known in the art. See, e.g., ((2004) TRENDS Plant Sci. 9: 116; Pauli and Kutchan ((1998) Plant J. 13:793-801 ; Collu et al. ((2001) FEBS Lett. 508:215- 220; Schroder et al. ((1999) FEBS Lett. 458:97-102. Phenylpropanoid biosynthetic pathway enzymes are known in the art. See, e.g., Mizutani et al. ((1997) Plant Physiol. 113:755-763; and Gang et al. ((2002) Plant Physiol. 130: 1536-1544. Polyketide biosynthetic pathway enzymes are known in the art. See e.g., Ikeda et al. ((1999) Proc. Natl. Acad. Sci. USA 96:9509-9514; and

Ward et al. ((2004) Antimicrob. Agents Chemother. 48:4703-4712.

[00284] As another example, a library of the present disclosure can comprise a plurality of

ribosome -ribonucleic acid complexes of the present disclosure, comprising nucleotide sequences encoding a receptor, where the different members of the plurality encode variants of the receptor that differ from one another in amino acid sequence. Receptors include, e.g., neurotransmitter receptors, pain receptors, serotonin receptors, and the like.

[00285] In some cases, members of a library of the present disclosure are immobilized on an insoluble support. In some cases, members of a library of the present disclosure are immobilized on an insoluble support at discrete locations.

CELLS

[00286] The present disclosure provides a cell (e.g., a genetically modified host cell) comprising: i) a nucleic acid of the present disclosure (a ribonucleic acid of the present disclosure; or a nucleic acid (e.g., a DNA; a recombinant vector) comprising a nucleotide sequence encoding a ribonucleic acid of the present disclosure); or ii) a ribonucleic acid-ribosome complex of the present disclosure; or iii) a ribonucleic acid-ribosome-nascent chain polypeptide complex of the present disclosure; or iv) a nascent chain polypeptide of the present disclosure. In some cases, the cell is in vitro.

[00287] Suitable host cells include mammalian cells, insect cells, reptile cells, amphibian cells, arachnid cells, plant cells, bacterial cells, archaeal cells, yeast cells, algal cells, fungal cells, and the like.

[00288] In some cases, the host cell is a mammalian cell, e.g., a human cell, a non-human

primate cell, a rodent cell, a feline (e.g., a cat) cell, a canine (e.g., a dog) cell, an ungulate cell, an equine (e.g., a horse) cell, an ovine cell, a caprine cell, a bovine cell, etc. In some cases, the host cell is a rodent cell (e.g., a rat cell; a mouse cell). In some cases, the host cell is a human cell. In some cases, the host cell is a non-human primate cell.

[00289] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.

CRL1573), HLHepG2 cells, and the like.

[00290] Suitable eukaryotic host cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like. In some cases, subject genetically modified host cell is a yeast cell. In some instances, the yeast cell is Saccharomyces cerevisiae.

UTILITY

[00291] A nucleic acid of the present disclosure (e.g., a ribonucleic acid of the present

disclosure; or a nucleic acid (e.g., a DNA; a recombinant vector) comprising a nucleotide sequence encoding a ribonucleic acid of the present disclosure) finds use in a variety of applications, which applications are also provided. A complex (e.g., a ribonucleic acid-ribosome complex; a ribonucleic acid-ribosome -nascent chain polypeptide complex) of the present disclosure finds use in a variety of applications, which applications are also provided.

Research applications

[00292] A nucleic acid of the present disclosure (e.g., a ribonucleic acid of the present

disclosure; or a nucleic acid (e.g., a DNA; a recombinant vector) comprising a nucleotide sequence encoding a ribonucleic acid of the present disclosure) and/or a complex (e.g., a ribonucleic acid-ribosome complex; a ribonucleic acid-ribosome -nascent chain polypeptide complex) of the present disclosure can be used to track and observe early translational events, e.g., simultaneously with ribosome translation of a ribonucleic acid to produce a nascent polypeptide chain, in vitro in a cell-free system or in a living cell (which cell may be in vitro or in vivo). Thus, the present disclosure provides methods for tracking and observing translational events, protein genesis, polypeptide folding, polypeptide maturation, and related events. Methods of identifying a translation-stalling agent

[00293] The present disclosure provides a method of identifying a translation-stalling agent that stalls translation of a polypeptide of interest. The method generally comprises: a) contacting a ribonucleic acid/ribosome complex of the present disclosure with a test agent; and b) determining the effect of the test agent on translation of the polypeptide of interest. In some cases, the complex is in a cell-free composition in vitro. In some cases, the complex is in a living cell in vitro. In some cases, the complex is in a cell in vivo.

[00294] In some cases, the complex is in a cell-free composition in vitro. The in vitro cell-free composition can be a cell lysate. Suitable cell lysates include, but are not limited to, a wheat germ lysate, a reticulocyte lysate, a yeast cell lysate, a mammalian cell lysate, or an insect cell lysate.

[00295] In some cases, the complex is in a cell in vitro, e.g., a eukaryotic cell such as a yeast cell, a mammalian cell, etc.

[00296] The complex comprises a ribonucleic acid of the present disclosure. In some cases, the ribonucleic acid present in the complex comprises, in order from 5' to 3' : a) the ribosome stalling polypeptide-encoding nucleotide sequence; and b) the heterologous polypeptide - encoding nucleotide sequence. The heterologous polypeptide can be polypeptide whose translation would be desirable to inhibit. In some cases, a candidate stalling agent would be useful for inhibiting translation of the heterologous polypeptide, where such inhibition could provide clinical benefit.

[00297] The complex comprises a ribonucleic acid of the present disclosure. In some cases, the ribonucleic acid present in the complex comprises, in order from 5' to 3' : a) the ribosome stalling polypeptide-encoding nucleotide sequence; and b) the heterologous polypeptide- encoding nucleotide sequence. The heterologous polypeptide can be a "reporter" polypeptide, e.g., a polypeptide that provides a detectable signal. Suitable "reporter" polypeptides include, e.g., fluorescent proteins; chromogenic proteins; enzymes that act on a substrate to produce a fluorescent, luminescent, or colored product; and the like.

[00298] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein

(GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

[00299] Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, β-glucuronidase, invertase, Xanthine Oxidase, luciferase (e.g., firefly lucif erase), glucose oxidase (GO), and the like.

[00300] The effect of the test agent on translational stalling can be determined by detecting the amount of heterologous polypeptide produced, or detecting the amount of signal produced by the heterologous polypeptide.

[00301] A test agent that stalls (inhibits) translation by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more than 80%, compared to the extent of translation in the absence of the test agent is a candidate agent for stalling translation.

[00302] The terms "candidate agent," "test agent," "agent," "substance," and "compound" are used interchangeably herein. Candidate agents encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules. Candidate agents include those found in large libraries of synthetic or natural compounds. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co.

(Trevillet, Cornwall, UK), ComGenex (South San Francisco, CA), and MicroSource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, Wis.).

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Pan Labs (Bothell, WA) or are readily producible. [00303] Test agents may be small organic or inorganic compounds having a molecular weight of more than 50 and less than about 10,000 daltons, e.g., from about 50 daltons to about 100 daltons, from about 100 daltons to about 500 daltons, from about 500 daltons to about 1000 daltons, from about 1000 daltons to about 5000 daltons, or from about 5000 daltons to about 10,000 daltons. Test agents may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The test agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. A test agent can be a peptide, a polypeptide, a natural product, or a synthetic peptide (e.g., where the synthetic peptide comprises one or more non-coded amino acid residues and/or a non-peptidic backbone).

[00304] Screening methods of the present disclosure include controls, where suitable controls include a sample (e.g., a sample comprising a complex of the present disclosure) in the absence of the test agent. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

[00305] Agents that have an effect in a screening method of the present disclosure may be further tested for cytotoxicity, bioavailability, and the like, using well known assays. Agents that have an effect in an assay method of the invention may be subjected to directed or random and/or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Such structural analogs include those that increase bioavailability, and/or reduced cytotoxicity. Those skilled in the art can readily envision and generate a wide variety of structural analogs, and test them for desired properties such as increased

bioavailability and/or reduced cytotoxicity and/or ability to cross the blood-brain barrier.

[00306] A variety of other reagents may be included in the screening assay. These include

reagents like salts, neutral proteins, e.g. albumin, detergents, etc., that are used to facilitate optimal binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components is added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, e.g., between 4 and 40°C.

Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Generally between 0.1 and 1 hour will be sufficient.

[00307] A candidate agent is assessed for any cytotoxic activity it may exhibit toward a cell, using well-known assays, such as trypan blue dye exclusion, an MTT ([3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-2 H-tetrazolium bromide]) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.

[00308] A test agent is other than Compound 2, Compound 1, or Compound 3.

Methods of displaying and selecting polypeptides

[00309] The present disclosure provides a method of displaying and selecting polypeptides. The method comprises: a) translating RNA present in a ribosome/nucleic acid library of the present disclosure in an in vitro cell free system in the presence of a stalling agent (e.g., a compound of Formula I; e.g., Compound 1, Compound 2, or Compound 3), to produce a plurality of ribosome/nucleic acid/nascent polypeptide complexes; b) contacting the ribosome/nucleic acid/nascent polypeptide complexes with a ligand, an antigen, or an antibody; and c) selecting a ribosome/nucleic acid/nascent polypeptide complex of interest. In some cases, the method further comprises: d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected ribosome/nucleic acid/nascent polypeptide complex of interest.

[00428] In one aspect, the present disclosure provides a method comprising generating a

ribosomal display. For example, a library of ribonucleic acids of the present disclosure is translated to provide a library of stalled ribonucleic acid/ribosome/nascent polypeptide complexes. The library of library of stalled ribonucleic acid/ribosome/nascent polypeptide complexes can then be screened with a selective agent having affinity for the nascent polypeptide chain, so as to provide a pool of ribosome-bound nascent chains of interest. In some cases, generating a library of ribonucleic acids comprises generating a library of mRNAs encoding a protein. In some cases, the protein is a single -chain variable fragment. In some cases, the library of ribonucleic acids lacks a stop codon in the open reading frame. In such cases, the library of ribonucleic acids lacking a stop codon in the open reading frame is subjected to in vitro translation (IVT). Non-limiting examples of primers used for making libraries include : Lib_PTC (nucleotides corresponding to the amino acids near the peptidyl transferase center): 5'- NNKNNKNNKtttcttcggaggagagcggtgg-3' (SEQ ID NO: 128) and 5'-

MNNcagaatcagaattagcaaagcaagaattcctcc-3' (SEQ ID NO: 129); Lib_compound (nucleotides corresponding to the amino acids that interact with the compound): 5'- NNKNNKNNKattctgctgctcttgctgtttcttcg-3' (SEQ ID NO: 130) and 5'-

MNNagcaagaattcctccaagaatcccc-3' (SEQ ID NO: 131); Lib_Ribl (nucleotides corresponding to the amino acids that have direct interaction with the ribosome): 5'- NNKNNKNNKgctttgctaattctgattctgctgctc-3' (SEQ ID NO: 132) and 5'-

MNNaagaatccccagaatggcaggaatttg-3' (SEQ ID NO: 133); Lib_Rib2 (nucleotides corresponding to the amino acids that have direct interaction with the ribosome): 5'- NNKcttggaggaattcttgctttgctaattctg-3 ' (SEQ ID NO: 134) and 5 ' -

MNNMNNMNNggcaggaatttgcaatcctgc-3' (SEQ ID NO: 135). In these DNA libraries, M stands for A or C nucleotides; N is any nucleotide; and K is G or T nucleotides. Such DNA libraries can encode the open reading frame, with stretches of 4 random codons encoded by NNK sequences, and can be generated by PCR amplification, to introduce random mutations in different sites of a nascent chain within the ribosome exit tunnel.

[00310] In some cases, the selective agent having affinity for a nascent polypeptide chain is a ligand for the nascent polypeptide chain. In some cases, the selective agent having affinity for a nascent polypeptide chain is a receptor for the nascent polypeptide chain. In some cases, the selective agent having affinity for a nascent polypeptide chain is an antibody that specifically binds the nascent polypeptide chain. In some cases, the selective agent having affinity for a nascent polypeptide chain is an mRNA that specifically binds the nascent polypeptide chain. In some cases, the selective agent having affinity for a nascent polypeptide chain is an aptamer that specifically binds the nascent polypeptide chain.

[00311] In some cases, the selective agent having affinity for a nascent polypeptide chain is immobilized on an insoluble support. Suitable insoluble supports can comprise any of a variety of polymers. In some cases, the insoluble support comprises a polymer, where the polymer comprises polyethylene glycol, polymethacrylate, polymethylmethacrylate, polyvinyl alcohol, polyvinyl acetate, polystyrene, polyglutaraldehyde, polyacrylamide, agarose, chitosan, alginate, or a combination of two or more thereof. The insoluble support can be any of a variety of shapes/forms, including, e.g., spherical, planar, and the like. In some cases, the insoluble support is a bead.

[00312] The ribonucleic acid encoding the nascent polypeptide chain of interest and present in the stalled complex can be isolated and/or characterized. For example, a selected ribonucleic acid can be subjected to RT-PCR to obtain a DNA nucleic acid encoding the ribonucleic acid. As noted above, in some cases, the method further comprises conducting RT-PCR on the mRNA bound in the selected ribosome/nucleic acid/nascent polypeptide complex of interest. The DNA nucleic acid encoding the ribonucleic acid can be sequenced. The DNA nucleic acid encoding the ribonucleic acid can be inserted into an expression vector.

Methods of generating a stalled ribosome nascent chain complex

[00313] The present disclosure provides methods of generating a stalled ribosome nascent chain complex (RNC). The methods generally involve contacting a ribonucleic acid of the present disclosure with a ribosome and a stalling agent (e.g., a compound of Formula I; e.g., Compound 1, Compound 2, or Compound 3). The contacting can be in a cell-free system in vitro; in a living cell in vitro; or in a cell in vivo. The contacting step can occur under at room temperature, or a physiological temperatures (e.g., about 37°C). The contacting results in the generation of a ribosome/nascent polypeptide chain complex, which can be used for a variety of purposes. In some cases, the stalling agent is Compound 1. In some cases, the stalling agent is Compound 2. In some cases, the stalling agent is Compound 3.

Examples of Non-Limiting Aspects of the Disclosure

[00314] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-63 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[00315] Aspect 1. A ribonucleic acid comprising: a) a nucleotide sequence encoding a ribosome stalling polypeptide, wherein the ribosome stalling polypeptide comprises: i) a stalling signal protector polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in FIG. 3B and having a length of from about 95 amino acids to about 120 amino acids; and ii) a ribosome stalling signal; and b) a nucleotide sequence encoding a heterologous polypeptide of interest, or an insertion site for a nucleotide sequence encoding a heterologous polypeptide of interest.

[00316] Aspect 2. The ribonucleic acid of aspect 1, wherein the stalling signal protector

polypeptide comprises an amino acid substitution of at least one cysteine residue present in the amino acid sequence depicted in FIG. 3B.

[00317] Aspect 3. The ribonucleic acid of aspect 1, wherein the ribonucleic acid, when present in a eukaryotic cell comprising a translation-stalling agent, or when present in a cell-free in vitro translation system comprising a translation-stalling agent, is translated, and wherein the ribosome stalling polypeptide and the translation-stalling agent together provide for ribosome stalling.

[00318] Aspect 4. The ribonucleic acid of aspect 3, wherein the stalling agent is a compound of

Formula I, optionally wherein the stalling agent selected from the group consisting of Compound 2, Compound 1, and Compound 3.

[00319] Aspect 5. The ribonucleic acid of aspect 1, wherein the ribosome stalling signal

comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences:

[00320] i) EGAAGVCRKAQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO:2);

[00321] ii) MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDED (SEQ ID NO: 3);

[00322] iii) MQHRGFLLLTLLALLALTSAV (SEQ ID NO:4);

[00323] iv) MKFLLDILLLLPLLIVC (SEQ ID NO:5);

[00324] v) MGLWWVTVQPPARRMGWLPLLLLLTQCLGVPGQRSPLNDF (SEQ ID NO:6);

[00325] vi) MGKFMKPGKVVLVLAGRYSGRKAVIVK (SEQ ID NO:7) ;

[00326] vii) MDAGVTESGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO:8);

[00327] viii) MSLQWTAVATFLYAEVFVVLLLCIP (SEQ ID NO:9) ;

[00328] ix) MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO: 10);

[00329] x) MGLHLRPYRVGLLPDGLLFLLLLLMLLADPALPAGRHPP (SEQ ID NO: 11); [00330] xi) RMKPWFEVGDENSGWSAQKVTNLHLMLQLVRVLVSPTNPPGA (SEQ ID

NO: 12);

[00331] xii) MAAPALGLVCGRCPELGLVLLLLLLSLLCGAA (SEQ ID NO: 13);

[00332] xiii) MAAALWGFFPVLLLLLLS (SEQ ID NO: 14);

[00333] xiv) MARGAALALLLFGLLGVLVAAPDGGFDLSDA (SEQ ID NO: 15) ;

[00334] xv) MRAHAQRGRGCTRRSAAVLMARHGLPLLPLLSLLVGAWLKLG (SEQ ID

NO: 16);

[00335] xvi) MDTGVIEGGLNVTLTIRLLMHGKEVGSIIGKKG (SEQ ID NO: 17) ;

[00336] xvii) MGARASGGPLARAGLLLLLL (SEQ ID NO: 18); and

[00337] xviii) GPLKKSNAPLVNVTLYYEALCGGCRAFLIRELFPTWLLVMEI (SEQ ID

NO: 19),

[00338] wherein the ribosome stalling signal has a length of from about 15 amino acids to about

60 amino acids.

[00339] Aspect 6. The ribonucleic acid of aspect 1, wherein the ribosome stalling signal

comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences:

[00340] i) NSVGEACTDMKREYDQCFNRWFAEK (SEQ ID NO:38);

[00341] ii) SHIQIPPGLTELLQGYTVEVLRQQPP (SEQ ID NO:39);

[00342] iii) EWWASSPLRLWLLLRLLP (SEQ ID NO:40);

[00343] iv) NRVLCAPAAGAVRALRLIGWASRSLHP (SEQ ID NO:41);

[00344] v) FSSSKANPHRWSVGHTMGKGHQRPWWKVLPLSCFLVALIIWCY (SEQ ID

NO:42);

[00345] vi) SRPQLRRWRLVSSPP (SEQ ID NO:43);

[00346] vii) FEVFVFDVGQKTWKSYDWSQITTVATFGKYDSELMCYAHSKGARVV (SEQ

ID NO:44);

[00347] viii) SASVVSVISRFLEEYLSSTPQRLK (SEQ ID NO:45);

[00348] ix) HLLAILFCALWSAVL (SEQ ID NO:46);

[00349] x) TFLYGTPTMFVDILNQPDFSSYDISTMCGGVIAGSPAPPE (SEQ ID NO:47);

[00350] xi) NEEYDVIVLGTGLTECILSGIM (SEQ ID NO:48); [00351] xii) FPRVSTFLPLRPLSRHPLSSGSPETSAAAIMLLTVRH (SEQ ID NO:49);

[00352] xiii) ALFVRLLA (SEQ ID NO:50);

[00353] xiv) RIEKCYFCSGPIYP (SEQ ID NO:51);

[00354] xv) AAMASLGALALLLLSSLSRC (SEQ ID NO:52);

[00355] xvi) RSLGALLLLLSACLAVSAGPVPTPPD (SEQ ID NO:53);

[00356] xvii) MLLKTVLLLGHVAQVLMLDNGLLQTPPMGW (SEQ ID NO:54);

[00357] xviii) MGGRVFLVFLAFCVWLTLPGAE (SEQ ID NO:55);

[00358] xix) MGAVWSALLVGGGLAGALFVWLLRGGPG (SEQ ID NO:56);

[00359] xx) MGPAGSLLGSGQMQITLWGSLAAVAIFFVITFLIFLCS (SEQ ID NO:57);

[00360] xxi) MSRFLNVLRSWLVMVSIIAMGNTLQSFRDHTFLYEKLYTGKPNL (SEQ ID

NO:58);

[00361] xxii) MGLQACLLGLFALILSGKCSYSPEPD (SEQ ID NO:59);

[00362] xxiii) MGRLLRAARLPPLLSPLLLLLVGGAFLGACVA (SEQ ID NO: 10) ;

[00363] xxiv) MAADLNLEWISLPRSWTYGITRGGRVFFINEEAKS (SEQ ID NO:60);

[00364] xxv) MCTGGCARCLGGTLIPLAFFGFLANILLFFPGGKVIDDND (SEQ ID NO:61); and

[00365] xxvi) MGPVRLGILLFLFL (SEQ ID NO:62),

[00366] wherein the ribosome stalling signal has a length of from about 8 amino acids to about

60 amino acids.

[00367] Aspect 7. The ribonucleic acid of any one of aspects 1-6, wherein the ribosome stalling polypeptide comprises two or more ribosome stalling signals.

[00368] Aspect 8. The ribonucleic acid of aspect 7, wherein the two or more ribosome stalling signals comprise the same amino acid sequence.

[00369] Aspect 9. The ribonucleic acid of aspect 7, wherein the two or more ribosome stalling signals comprise different amino acid sequences.

[00370] Aspect 10. The ribonucleic acid of any one of aspects 1-9, comprising a nucleotide sequence encoding an affinity tag,

[00371] Aspect 11. The ribonucleic acid of aspect 10, wherein the affinity tag-encoding

nucleotide sequence is 5' of the ribosome stalling signal-encoding nucleotide sequence. [00372] Aspect 12. The ribonucleic acid of any one of aspects 1-11, comprising an internal ribosome entry site (IRES).

[00373] Aspect 13. The ribonucleic acid of any one of aspects 1-12, wherein the heterologous polypeptide of interest generates a detectable signal.

[00374] Aspect 14. The ribonucleic acid of aspect 13, wherein the heterologous polypeptide of interest is a luminescent polypeptide, a fluorescent protein, or a chromogenic polypeptide.

[00375] Aspect 15. The ribonucleic acid of any one of aspects 1-14, wherein the nucleic acid lacks a stop codon.

[00376] Aspect 16. The ribonucleic acid of any one of aspects 1-15, comprising, in order from 5' to 3' : a) the ribosome stalling polypeptide -encoding nucleotide sequence; and b) the

heterologous polypeptide -encoding nucleotide sequence.

[00377] Aspect 17. The ribonucleic acid of any one of aspects 1-16, comprising, in order from 5' to 3' : a) the heterologous polypeptide -encoding nucleotide sequence; and b) the ribosome stalling polypeptide-encoding nucleotide sequence.

[00378] Aspect 18. The nucleic acid of aspect 17, wherein the heterologous polypeptide

comprises one or more amino acid differences compared to the amino acid sequence of a reference polypeptide.

[00379] Aspect 19. The nucleic acid of aspect 18, wherein the reference polypeptide is a wild- type polypeptide.

[00380] Aspect 20. The nucleic acid of aspect 18 or 19, wherein the reference polypeptide is an antigen-binding portion of an antibody, a receptor, or an enzyme.

[00381] Aspect 21. The nucleic acid of any one of aspects 18-20, wherein the one or more amino acid differences are selected from one or more of an amino acid substitution, an amino acid insertion, and an amino acid deletion.

[00382] Aspect 22. A deoxyribonucleic acid comprising a nucleotide sequence encoding the ribonucleic acid of any one of aspects 1-21, comprising a promoter operably linked to the ribosome stalling polypeptide-encoding nucleotide sequence.

[00383] Aspect 23. The deoxyribonucleic acid of aspect 22, wherein the promoter is a

regulatable promoter. [00384] Aspect 24. The deoxyribonucleic acid of aspect 22 or aspect 23, wherein the nucleic acid is present in a recombinant expression vector.

[00385] Aspect 25. A polypeptide encoded by the ribonucleic acid of any one of aspects 1-21.

[00386] Aspect 26. A complex comprising:

[00387] a) the ribonucleic acid of any one of aspects 1-21 ; and

[00388] b) a ribosome; and, optionally

[00389] c) a nascent chain polypeptide encoded by the ribonucleic acid.

[00390] Aspect 27. A composition comprising:

[00391] a) the ribonucleic acid of any one of aspects 1-21 ; or

[00392] b) the complex of aspect 26.

[00393] Aspect 28. The composition of aspect 27, wherein the composition comprises a cell lysate.

[00394] Aspect 29. The composition of aspect 28, wherein the cell lysate is a wheat germ lysate, a reticulocyte lysate, a yeast cell lysate, a mammalian cell lysate, or an insect cell lysate.

[00395] Aspect 30. A eukaryotic cell comprising:

[00396] a) the ribonucleic acid of any one of aspects 1-21 ;

[00397] b) the nascent chain polypeptide of aspect 25; or

[00398] c) the complex of aspect 26.

[00399] Aspect 31. The eukaryotic cell of aspect 30, wherein the eukaryotic cell is a mammalian cell.

[00400] Aspect 32. A method of identifying a translation-stalling agent that stalls translation of a polypeptide of interest, the method comprising: a) contacting the complex of aspect 26 with a test agent; and b) determining the effect of the test agent on translation of the heterologous polypeptide of interest.

[00401] Aspect 33. The method of aspect 32, wherein the complex is in a cell-free composition in vitro.

[00402] Aspect 34. The method of aspect33, wherein the cell-free composition is a cell lysate.

[00403] Aspect 35. The method of aspect 34, wherein the cell lysate is selected from the group consisting of a wheat germ lysate, a reticulocyte lysate, a yeast cell lysate, a mammalian cell lysate, and an insect cell lysate. [00404] Aspect 36. The method of aspect 32, wherein the complex is in a eukaryotic cell in vitro.

[00405] Aspect 37. The method of any one of aspects 32-36, wherein the ribonucleic acid present in the complex comprises, in order from 5' to 3' : a) the ribosome stalling polypeptide -encoding nucleotide sequence; and b) the heterologous polypeptide-encoding nucleotide sequence.

[00406] Aspect 38. The method of any one of aspects 32-37, wherein the heterologous

polypeptide is a luminescent polypeptide or a chromogenic polypeptide.

[00407] Aspect 39. The method of any one of aspects 32-38, wherein the test agent is a peptide, a polypeptide, a natural product, or a synthetic peptide.

[00408] Aspect 40. The method of aspect 39, wherein the synthetic peptide comprises one or more non-coded amino acid residues and/or a non-peptidic backbone.

[00409] Aspect 41. The method of any one of aspects 32-38, wherein the test agent is a small molecule.

[00410] Aspect 42. A method of generating a stalled ribosome nascent chain complex (RNC), the method comprising contacting the complex of aspect 26 with a translation stalling agent.

[00411] Aspect 43. The method of aspect 42, wherein the translation stalling agent is a

compound of Formula I.

[00412] Aspect 44. The method of aspect 43, wherein the translation stalling agent is Compound

1 or Compound 2.

[00413] Aspect 45. The method of aspect 43, wherein the translation stalling agent is Compound

3.

[00414] Aspect 46. The method of any one of aspects 42-45, wherein the ribonucleic acid present in the complex comprises, in order from 5' to 3' : a) the heterologous polypeptide-encoding nucleotide sequence; and b) the ribosome stalling polypeptide-encoding nucleotide sequence.

[00415] Aspect 47. A library comprising a plurality of ribosome-ribonucleic acid complexes, wherein each of the plurality of ribosome -nucleic acid complexes comprises: a) a ribonucleic acid of any one of aspects 1-21 ; and b) a ribosome.

[00416] Aspect 48. The library of aspect 47, wherein each of the ribonucleic acids comprises a nucleotide sequence barcode.

[00417] Aspect 49. The library of aspect 47 or aspect 48, wherein the ribosome stalling

polypeptide encoded by the ribonucleic acid comprises a protein tag. [00418] Aspect 50. The library of aspect 49, wherein the protein tag is selected from an Avi tag

(GLNDIFEAQKIEWHE) (SEQ ID NO:98), a calmodulin tag

(KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO:99), a FLAG tag (DYKDDDDK) (SEQ ID NO:91), a His-FLAG tag (HHHHHHDYKDHDG) (SEQ ID NO: 142), a 3XFLAG tag (DYKDHDGDYKDHDIDYKDDDDK) (SEQ ID NO: 145), a hemagglutinin tag

(YPYDVPDYA) (SEQ ID NO:93), a poly(histidine) tag (HHHHHH) (SEQ ID NO: 89), a Myc tag (EQKLISEEDL) (SEQ ID NO:90), an S tag (KETAAAKFERQHMDS) (SEQ ID NO: 100), an SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ GQREP) (SEQ ID NO: 101), a Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 102), a Softag 3 (TQDPSRVG) (SEQ ID NO: 103), a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 104), an Xpress tag (DLYDDDDK) (SEQ ID NO: 105), an Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 106), a SpyTag (AHIVMVDAYKPTK) (SEQ ID NO: 107), a TwinStrep tag

(WSHPQFEKGAMTGWSHPQFEK) (SEQ ID NO: 144) and a streptactin tag (Strep-tag II:

WSHPQFEK) (SEQ ID NO:92).

[00419] Aspect 51. The library of any one of aspects 47-50, wherein the heterologous

polypeptide of interest is a variant of a selected reference polypeptide.

[00420] Aspect 52. The library of aspect 51 , wherein the selected reference polypeptide is an antigen-binding portion of an antibody or a non-antibody scaffold.

[00421] Aspect 53. The library of aspect 51, wherein the selected reference polypeptide is an enzyme.

[00422] Aspect 54. The library of aspect 53, wherein the enzyme is a metabolic pathway

enzyme.

[00423] Aspect 55. The library of aspect 51, wherein the selected reference polypeptide a

receptor.

[00424] Aspect 56. A method of selecting a polypeptide of interest, the method comprising: a) translating RNA present in a ribosome/nucleic acid library of any one of aspects 47-55 in an in vitro cell free system, in the presence of a stalling agent, to produce a plurality of

ribosome/nucleic acid/nascent polypeptide complexes; b) contacting the plurality of ribosome/nucleic acid/nascent polypeptide complexes with a binding agent that specifically binds the nascent polypeptide; and c) selecting a ribosome/nucleic acid/nascent polypeptide complex of interest.

[00425] Aspect 57. The method of aspect 56, wherein the stalling agent is a compound of

Formula I, optionally wherein the compound is selected from the group consisting of Compound

2, Compound 1, and Compound 3.

[00426] Aspect 58. The method of aspect 56 or 57, wherein the binding agent is a ligand, an antibody, an antigen, an mRNA, or a receptor.

[00427] Aspect 59. The method of any one of aspects 56-58, wherein the method further

comprises: d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the

RNA bound in the selected ribosome/ribonucleic acid/nascent polypeptide complex of interest, to generate a DNA molecule encoding the nascent polypeptide.

[00428] Aspect 60. The method of aspect 59, wherein the method further comprises inserting the nascent polypeptide -encoding DNA molecule into a cloning and/or an expression vector.

[00429] Aspect 61. The method of any one of aspects 56-60, wherein the in vitro system

comprises a cell lysate.

[00430] Aspect 62. The method of aspect 61, wherein the cell lysate is a wheat germ lysate, a reticulocyte lysate, a yeast cell lysate, a mammalian cell lysate, and an insect cell lysate.

[00431] Aspect 63. The method of any one of aspects 56-62, wherein the binding agent is

immobilized on an insoluble support.

EXAMPLES

[00432] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.

Example 1

MATERIALS AND METHODS DNA constructs and in vitro transcription

[00429] The DNA fragments needed to assemble the construct encoding the affinity tagged

CDHl stalling nascent chain (EMCV IRES, CDHl sequence encoding amino acids 586-750 and Nano luciferase; see FIG. 1A) were amplified from previous plasmids encoding full length PCSK9 and CDHl (Lintner et al., 2017) and fused together by overlapping PCR. In CDHl sequences in which four cysteine residues were substituted for other amino acids (residues 603, 686, 688 and 695 as shown in Table 1), mutations were generated by two-step PCR. For chimeras of domain 5 of CDHl and PCSK9, CDHl amino acids 716-750 were replaced with PCSK9 amino acids 1-35, by PCR with the amplified sequence for PCSK9(l-35) and primers overlapping the 3' end of CDHl domain 5 and the 5' end of the luciferase gene. The luciferase DNA was obtained by fusing the IRES and luciferase fragments. Primers used for these constructs are listed in Table 1.

[00430] In vitro transcription reactions were performed using PCR products generated with

primers flanking the T7 promoter and a poly-A tail. Reactions were set up with 20 mM Tris-HCl pH 7.5, 35 mM MgCl ₂ , 2 mM spermidine, 10 mM DTT, 1 U/mL pyrophosphatase (Sigma), 7.5 mM each NTP, 0.2 U/L SUPERaseln RNase Inhibitor (Thermofisher), 0.1 mg/mL T7 RNA polymerase and 40 ng/μΕ DNA. Reactions were incubated for 3 hours at 37 °C. To remove the template DNA, 0.1 U/μΕ DNasel (Promega) was added into the reactions, which were then incubated at 37 °C for 30 min. RNA was precipitated for 2 hours at -20 °C by adding ½ volume of 7.5 M LiCl/50 mM EDTA, followed by washing with cold 70% ethanol and suspended with RNase free water. The mRNA was further purified by Zymo RNA Clean and Concentrator (Zymo research) before using for in vitro translation reactions. In vitro translation reactions

[00431] The HeLa cell extract was made as described previously (Lintner et al., 2017). Briefly, a frozen HeLa cell pellet was thawed and suspended with equal volume of lysis buffer (20 mM HEPES pH 7.5, 10 mM potassium acetate, 1.8 mM magnesium acetate, and 1 mM DTT). After incubation on ice for 10 min, the cells were lysed with a Dounce homogenizer 150 times, followed by centrifugation twice at 1 ,200 x g for 5 min. The supernatant after centrifugation was aliquoted to avoid freeze-thaw cycles, and flash-frozen in liquid nitrogen. For a 10 Ε reaction, 5 μΕ Hela cell extract was used in a buffer containing final concentrations of 20 mM Hepes pH 7.4, 120 mM KOAc, 2.5 mM Mg(OAc) ₂ , 1 mM ATP/GTP, 2 mM creatine phosphate (Roche), 10 ng/μΕ creatine kinase (Roche), 0.21 mM spermidine, 0.6 mM putrescine, 2 mM TCEP, 10 μΜ amino acids mixture (Promega), 1 U/μΕ murine RNase inhibitor (NEB), 300 ng mRNA, and 1 μΕ 0.5 mM or 1 mM Compound 1 stock solution in 10% DMSO, or 10% DMSO control. Translation reactions were incubated for 20 min at 30 °C, after which luciferase activity was assayed.

Pull down and western blot

[00432] 700 μΕ translation reactions were incubated with 30 μΕ (packed volume) of anti-FLAG

M2 beads (Sigma) for 1 hour at room temperature with gentle mixing. The anti-FLAG beads were washed at room temperature three times with 500 RNC buffer (20 mM Hepes pH 7.4, 300 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT, 0.2 mM Compound 1), then three times with 500 μΐ, RNC buffer plus 0.1 % TritonX-100, then three times with 300 μΐ, RNC buffer plus 0.5% TritonX-100, and finally three times with 300 μL· RNC buffer. Two elutions were carried out at room temperature for 30 min each, with 30 μL· 0.2 mg/mL 3xFLAG peptide (Sigma) in RNC buffer. The eluted fractions were combined and centrifuged at 100,000 rpm at 4 °C in a sucrose cushion buffer of 1 M sucrose, 1 mM DTT, 0.2 mM Compound 1 , 120 mM KOAc and 2.5 mM Mg(OAc) ₂ for 50 min in an MLA rotor (Beckman Coulter). After centrifugation, the ribosome pellet was suspended in 40 μL· RNC buffer and used for western blot analysis. DNA libraries with random sequences to test stalling mechanism of the CDHl stalling sequence

[00433| DNA libraries encoding the open reading frame in Fig. 1A, with stretches of 4 random codons encoded by NNK sequences (N=any nucleotide, K=G or T) were generated by PCR amplification, to introduce random mutations in different sites of the CDHl nascent chain within the ribosome exit tunnel. For the NNK libraries, any nucleotide can be presented in the first two codon positions (represented by N in the primers), and either T or G can be possible for the third position (represented by K in the primers). PCR products were purified via spin columns (Qiagen, catalog number: 28506) before Dpnl nuclease treatment to remove template DNA, which was done at 37 °C for 1 hour. The treated DNA was then ligated on the 5' end to a double- stranded DNA fragment encoding a T7 RNA polymerase promoter, and to a double-stranded DNA encoding a poly-A tail on the 3' end, using T4 DNA ligase and T4 Polynucleotide Kinase at 16 °C overnight. The ligation reaction was followed by PCR amplification using primers flanking the T7 promoter and a poly-A tail.

[00434] The following primers were used for making the NNK DNA libraries. Lib_PTC

(nucleotides corresponding to the amino acids near the peptidyl transferase center): 5'- NNKNNKNNKtttcttcggaggagagcggtgg-3' (SEQ ID NO: 128) and 5'-

MNNcagaatcagaattagcaaagcaagaattcctcc-3' (SEQ ID NO: 129); Lib_compound (nucleotides corresponding to the amino acids that interact with the compound): 5'- NNKNNKNNKattctgctgctcttgctgtttcttcg-3' (SEQ ID NO: 130) and 5'-

MNNagcaagaattcctccaagaatcccc-3' (SEQ ID NO: 131); Lib_Ribl (nucleotides corresponding to the amino acids that have direct interaction with the ribosome): 5'- NNKNNKNNKgctttgctaattctgattctgctgctc-3' (SEQ ID NO: 132) and 5'-

MNNaagaatccccagaatggcaggaatttg-3' (SEQ ID NO: 133); Lib_Rib2 (nucleotides corresponding to the amino acids that have direct interaction with the ribosome): 5'- NNKcttggaggaattcttgctttgctaattctg-3 ' (SEQ ID NO: 134) and 5 ' -

MNNMNNMNNggcaggaatttgcaatcctgc-3' (SEQ ID NO: 135). In these libraries, M stands for A or C nucleotides.

Ribosome nascent chain complexes with random libraries stalled by PF-06446846

[00435] The mRNAs encoding the NNK libraries were prepared by transcription using T7 RNA polymerase, using the PCR products of the NNK DNA libraries described above. After making the mRNA using the protocol described before, a 1.0 mL in vitro translation reaction with 50 μΜ PF-06446846846 was incubated at 30 °C for 28 min. Stalled RNCs were purified using anti- FLAG peptide beads as described for Fig. 2, with the following modifications. Stalled ribosome nascent-chain complexes (RNCs) were bound to the beads and treated with RNase H during the binding reaction. During the binding step, 1 μΜ of the DNA oligonucleotide 5'- TCTCCTCCGAAGAAA-3 ' (targeting DNA) (SEQ ID NO: 136) and 2 μΐ. RNase H (NEB) were added to the reaction. After the sucrose cushion pelleting step, the pellet was resuspended with 40 μΕ RNase H buffer (20 mM Tris 7.5, 150 mM KC1, 2.5 mM MgC12, 2mM DTT) and treated with 1 μΕ RNase H and 1 μΜ of the targeting DNA oligonucleotide described above, at 37 °C for 40 min. Total RNA was then extracted from the purified and RNase H treated RNCs and input mRNAs were isolated using TRIzol LS (Thermo Fisher Scientific, cat. number: 10296010). First strand cDNAs were synthesized using a primer specific to the nucleotide sequence in the open reading frame of Fig. 1A, using Superscript II (Invitrogen, cat. number: 18064014), according to the manufacture's protocol. The cDNA was then used in a 70 μL· PCR reaction with Q5 DNA polymerase with optimized cycles of 10 s at 98 °C, 30 s at 66 °C and 15 s at 72 °C, followed by a two step purification with SPRIselect beads (Beckman Coulter, cat. number: B23317), first by adding 42 μΕ beads (0.6X amount of the PCR reaction) and then applying the flowthrough to another 56 μΕ beads (0.8X amount of the PCR reaction). The resulting bound DNA was eluted in 15 μΕ molecular biology grade water. The DNA libraries were sequenced using an Illumina HiSeq 4000 at the University of California, Berkeley. The following oligonucleotides were used: RT primer (used for synthesizing the first strand cDNA):

5 'AGACGTGTGCTCTTCCGATCTNNNNCTCTTTGACC ACCGCTCTCC-3 ' (SEQ ID NO: 137); Universal P5 adaptor (Forward primer for PCR):

5'AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTC CGATCTNNNNNCGAAGCAGGATTGCAAATTCCT-3' (SEQ ID NO: 138); Indexed P7 adapter (Reverse primer for PCR):

5'CAAGCAGAAGACGGCATACGAGATCCTTGGAAGTGACTGGAGTTCAGACGTGTGC TCTTCCGATCT-3 ' (SEQ ID NO: 139) (the underlined nucleotides indicate the index sequence for one sample. Different index sequences were used for every sample). Table 1: Primers used in this study

Primer Sequence

1 Amplify IRES fragment with FLAG overlap

IRES with T7 Agcttaatacgactcactatagggcgaattaattccggttattttccaccatattg (SEQ ID NO: 108) promoter

-Forward

IRES with 3xFLAG Gtcgtggtccttgtagtccatattatcatcgtgtttttcaaaggaaaacc (SEQ ID NO: 109) overlap -Reverse

IRES with lxFLAG Gcgttgtcattcacatcagacttatcgtcgtcatccttgtaatccatattatcatcgtgt ttttcaaagg (SEQ ID overlap -Reverse NO: 110)

2 FLAG tag with IRES and CDH1 ⁵⁸⁶ overlap

FLAG with IRES Aaaaacacgatgataatatggactacaaggaccacgacggcgactac (SEQ ID NO: 111) overlap -Forward

3xFLAG oligo Gactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgac gacaag -Forward (SEQ ID NO: 112)

CDHl ⁵⁸⁶ with FLAG Gttgtcattcacatcagacttgtcgtcgtcgtccttgtagtcgatg (SEQ ID NO: 113)

overlap -Reverse

3 CDHl ^{586 750} fragment with 3xFLAG and Nano Luciferase (Luc) overlap

CDHl ⁵⁸⁶ with Aaggacgacgacgacaagtctgatgtgaatgacaacgccccc (SEQ ID NO: 114)

3xFLAG overlap - Forward

CDHl ⁷⁵⁰ with Luc Ttcgagtgtgaagacgtcccgggtgtcatc (SEQ ID NO: 115)

overlap -Reverse

4 CDHl ^{586 750} fragment with lxFLAG and Luc overlap

lxFLAG with Aaaaacacgatgataatatggattacaaggatgacgacgataagtctgatgtgaatgaca acgcccccatacca CDHl ⁵⁸⁶ overlap - (SEQ ID NO: 116)

Forward

CDHl ⁷⁵⁰ with Luc Ttcgagtgtgaagacgtcccgggtgtcatc (SEQ ID NO: 115)

overlap -Reverse

5 Luc for fusing with CDHl domain 5

Luc-Forward Gtcttcacactcgaa (SEQ ID NO: 117)

Luc-PA-Reverse Ttttttttttttttttttttttttttttttttacgccagaatgcgttcgca (SEQ ID NO: 118)

6 Luc for fusing with IRES

IRES-Luc-Forward Aaaaacacgatgataatatggtcttcacactcgaagatttc (SEQ ID NO: 119)

Luc-PA-Reverse Ttttttttttttttttttttttttttttttttacgccagaatgcgttcgca (SEQ ID NO: 118)

7 CDHl Cysteine mutations

CDH1 ^C603S -Forward Cgaactatattcttctctgaga (SEQ ID NO: 120)

CDH1 ^C603S -Reverse Tggattcctctcagagaag (SEQ ID NO: 121)

CDHl ^C686S > ^C688V > ^C695F Tcagcgtgagtgacgttgaaggggccgctggcgtctttaggaaggcacagcctgt (SEQ ID NO: 122) -Forward Primer Sequence

CDH1 ^C686S > ^C688V > ^C695F Gccttcctaaagacgccagcggccccttcaacgtcactcacgctgacctct (SEQ ID NO: 123) -Reverse

8 Cdhl-PCSK9 chimera

Replace CDH1 ⁷¹⁶ Ggagctgacggtgcccattccaagaatccccagaatggcaggaatttg (SEQ ID NO: 124) ⁷⁵⁰ with PCSK9 ^{1 3S} - Forward

Replace Cdhl ⁷¹⁶ Ggagctgacggtgcccataattcctccaagaatccccagaatggcaggaa (SEQ ID NO: 125) ⁷⁵⁰ with PCSK9 ^{1 3S} - Reverse

PCSK9 ^{1 35} -Forward Atgggcaccgtcagctccagg (SEQ ID NO: 126)

PCSK9 ^{1 35} -Reverse GTCCTCgtcctcctgcgcacgg (SEQ ID NO: 127)

DNA constructs and in vitro transcription

[00437] The DNA fragments needed to assemble the constructs encoding the affinity tagged nascent chains (EMCV IRES, CDH1 sequence encoding amino acids 586-750 and PCSK9 sequence encoding amino acids 1-35; FIG. 5 A) were amplified from previous plasmids encoding full length PCSK9 and CDH1 in Lintner et al. For the CDH1 nascent chain used for structural studies, the EMCV IRES, affinity tags, CDH1 sequence encoding amino acids 586-750 and a NanoLuc luciferase reporter gene were fused together by overlap PCR. For the PCSK9 nascent chain, CDH1 amino acids 716-750 were replaced with PCSK9 amino acids 1-35, leaving the CDHl-V domain intact. The NanoLuc control without stalling sequence was obtained by fusing the EMCV IRES and the NanoLuc open reading frame using overlap PCR. All PCR products were purified via spin columns (Qiagen) before their use in in vitro transcription reactions.

[00438] In vitro transcription reactions were performed using PCR products generated with primers encoding a flanking T7 promoter and a poly-A tail. Reactions were set up with 20 mM Tris-HCl pH 7.5, 35 mM MgCl ₂ , 2 mM spermidine, 10 mM DTT, 1 U/mL pyrophosphatase (Sigma), 7.5 mM each NTP, 0.2 U/L SUPERaseIn RNase Inhibitor (ThermoFisher), 0.1 mg/mL T7 RNA polymerase and 40 ng/μΕ DNA. After a 3 hours incubation at 37 °C, 0.1 υ/μί DNase I (Promega) was added to the reactions and incubated at 37 °C for 30 min to remove the template DNA. RNA was precipitated for 2-3 hours at -20 °C after adding 1/2 volume of 7.5 M LiCl/50 mM EDTA, and the resulting pellet was washed with cold 70% ethanol and dissolved with RNase free water. The mRNA was further purified by Zymo RNA Clean and Concentrator (Zymo research) before use in in vitro translation reactions. In vitro translation reactions

[00439] The HeLa cell extract was made as described previously in Lintner et al. Briefly, a

frozen HeLa cell pellet was thawed and suspended with an equal volume of lysis buffer (20 mM HEPES pH 7.5, 10 mM potassium acetate, 1.8 mM magnesium acetate, and 1 mM DTT). After incubation on ice for 20 min, the cells were lysed with a Dounce homogenizer 150 times, followed by centrifugation twice at 1 ,200 x g for 5 min. The supernatant after centrifugation was aliquoted to avoid freeze-thaw cycles, and flashfrozen in liquid nitrogen. For a 10 μL· reaction, 5 μΕ HeLa cell extract was used in a buffer containing final concentrations of 20 mM Hepes pH 7.4, 120 mM KOAc, 2.5 mM Mg(OAc) ₂ , 1 mM ATP/GTP, 2 mM creatine phosphate (Roche), 10 ng/μΕ creatine kinase (Roche), 0.21 mM spermidine, 0.6 mM putrescine, 2 mM TCEP, 10 μΜ amino acids mixture (Promega), 1 Ό/μL· murine RNase inhibitor (NEB), 200 ng mRNA, and 0.1 mM PF846 in 1 % DMSO, or 1 % DMSO as control. Translation reactions were incubated for 23 min at 30 °C, after which luciferase activity was monitored with a Microplate Luminometer (Veritas).

Purification of stalled RNCs

[00440] 1.5 mL in vitro translation reactions with 0.2 mM PF846 were incubated at 30 °C for 23 min and then centrifuged at 11 ,400 r.p.m. for 5 min. The supernatant was incubated with 50 μL· (packed volume) of anti-FLAG M2 agarose beads (Sigma) for 30 min at room temperature with gentle mixing. To avoid the binding of nonspecific ribosomes and other proteins, the beads were washed at room temperature three times with 500 μL· RNC buffer (20 mM Hepes pH 7.4, 300 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT, 0.2 mM PF846), then three times with 500 μΐ, RNC buffer plus 0.1% TritonX-100, followed by three times with 300 μΐ, RNC buffer plus 0.5% TritonX-100, and finally washed twice with 300 μΐ, RNC buffer. The PF846 stalled RNCs, bound to the FLAG® beads by the N-terminal 3xFLAG® tag, were eluted twice at room temperature for 20 min each time, with 30 μL· 0.2 mg/mL 3xFLAG® peptide (Sigma) in RNC buffer. The eluted fractions were combined and loaded onto a 50% sucrose cushion prepared with cushion buffer (25 mM HEPES -KOH pH 7.5, 120 mM KOAc and 2.5 mM Mg(OAc) ₂ , 1 M sucrose, 1 mM DTT, 0.2 mM PF846), and centrifuged for 1 hour in an MLA rotor (Beckman Coulter) with 100,000 r.p.m. (-603,000 g) at 4 °C. The pellet was suspended in ice-cold RNC buffer and was immediately used for cryo-EM grid preparation. The concentration of purified RNC was determined using a NanoDrop Microvolume Spectrophotometer and calculated using an A260 unit around 1 corresponding to 20 pmol of ribosome.

[00441] RNaseA digestion and western blot RNCs released from the anti-FLAG beads and

pelleted through the sucrose cushion were resuspended in 20 μL· with an A260 around 1 and then incubated with or without 100 μg/mL DNase free Rnase A (ThermoFisher) and 50 mM EDTA at 37 °C for 30 min followed by western blot of the FLAG-tagged peptides. For the western blots, Monoclonal anti-FLAG® M2-Peroxidase (HRP) antibody (SIGMA) with 1 : 10,000 was used.

Cryo-sample preparation and Data collection

[00442] Approximately 3 μΕ of freshly made RNC at a concentration of 40 to 60 nM were

incubated for 1 min on plasma-cleaned 300-mesh holey carbon grids (C-flat R2/2), on which a home-made continuous carbon film was pre-deposited. Grids were blotted for 4 seconds with 100% humanity at 4 °C and plunge -frozen in liquid ethane using an FEI Vitrobot. Automated data collection was performed on a Titan Krios electron microscope (FEI) equipped with a K2 Summit direct detector and GIF Quantum filter (Gatan) at 300 kV (FIG. 9). The total exposure time was 9 seconds, with a total dose of 50 electrons per A2 (frame dose 1.3 electrons per A2).

Data Processing

[00443] Frames were aligned using MotionCor2 with FtBin 2. Gautomatch (www(dot)mrc- lmb(dot)cam(dot)ac(dot)uk/kzhang/) was used for automatic particle picking. Powerspectra, defocus values and astigmatism were determined with Gctf software with per-particle CTF estimation. All the subsequent data processing was performed in RELION 2.0 unless specifically noted. To speed up computing, the particles were binned by 8 times during particle extraction. Reference free 2D classification was performed to remove particles with ice or other contaminants, followed by 3D classification to select RNC particles. The initial 3D classification roughly separated the ribosome particles from the non-ribosome particles without yielding structurally distinct classes.

[00444] For the CDH1-RNC data set, the whole data set was divided into three subsets and

carried out particles sorting as shown in FIG. 7A. After initial 3D classification and refinement, an additional round of 3D classification was performed with a local angular search that separated CDHl-RNCs in rotated states from a class with apo-80S ribosomes or ribosomes with weak density for tRNA (FIG. 7A). A total number of 84,437 particles representing the CDH1-RNC in the rotated state were combined and subjected to 3D refinement, which generated a map of 4.0 A without the use of a mask. Signal subtraction was conducted to improve the density of the nascent peptide chain. Starting with the aligned 84,437 particles, a region with CDH1 nascent chain, PF846 and A/P NC-tRNA was extracted and classified by applying a soft mask generated in RELION. Focused 3D classification separated the rotated state into two major classes, one with A/P NC-tRNA and P/E tRNA (-84% of particles), and the other with A/A NC-tRNA and P/E tRNA (-15% of the particles). The more-abundant class with A/P NC-tRNA had good electron density for the CDH1 nascent chain, which was used for model building (FIG. 7B-7C). The overall resolutions of the CDHl-RNC's were 3.7 A and 4.3 A using the gold-standard Fourier shell correlation (FSC=0.143) criterion (FIG. 7B-7C, and FIG 9).

[00445] To better sort the particles, the remaining classes were combined after sorting the RNC with hybrid tRNAs (FIG. 7A) for an additional 3D refinement and classification with a local angular search, from which three major classes were observed: 80S ribosome with GTPase bound in the A site (24%), CDH1-RNC with P/P tRNA (26%), and the large (60S) subunit (15%). Another round of signal subtraction for the CDH1-RNC with P/P tRNA was conducted based on the P/P tRNA density, resulting in -61% particles which were chosen for the final reconstruction.

[00446] Two data sets for the PCSK9-RNC were collected on the same Titan Krios microscope within a week of each other. An additional round of 3D classification was performed with a local angular research after overall refinement, which separated RNCs in the rotated state (FIG. 8A). A total of 53,247 particles were combined with hybrid state tRNAs from both data sets for another round of 3D auto refinement. The postprocessing step implemented in RELION 2.0 was used to generate the final maps using the FSC 0.143 criterion indicating an average resolution of 3.7 A (B factor -112A ₂ ) (FIG. 8A and FIG. 9). Signal subtraction for PCSK9_RNC with either hybrid tRNAs or P/P tRNA was conducted in the same way as described for the CDH1-RNC complexes (FIG. 8A). For the data set of stalled PCSK9-RNC which was prepared using a short time interval, the selected particles after RELION 2D classification were processed using CryoSPARC for further 3D classification and homogeneous refinement, which generated two maps representing different states of ribosome at an average resolution of 4.6 A and 4.8 A respectively according to the "gold standard" FSC = 0.143 (FIG. 8B and FIG. 9).

Model Building and Refinement

[00447] Initial rigid body fitting of the human 40S ribosomal subunit structure (PDB: 6ek0) and

60S subunit structure (PDB: 5aj0) was done for all the rotated RNCs (CHDl/PCSK9_RNCs with both A/A NC-tRNA and A/P NC-tRNA), using UCSF Chimera. The ribosomal proteins and RNAs of the 40S and 60S subunits were docked separately into the EM map, with parts of the model manually adjusted to the EM map using Coot. All the tRNAs (A/A, A/P, P/E tRNA) for the RNCs in the rotated state were docked into the EM maps using reported tRNA structures (PDB: 3j7r), followed by manual rebuilding in Coot . The anticodon nucleotides from 34 to 36 in the tRNA were modeled as one sequence (CCG). RNCs with P/P site tRNA were initially modeled using rigid-body docking of the human ribosome structure (PDB: 5aj0) and then corrected based on the EM density in Coot . The nascent chains were manually built into the density in one sequence register (CDH1_NC sequence:

CRKAQPVEAGLQIPAILGILGGILALLILILLLLLF (SEQ ID NO: 140) and PCSK9_NC sequence: WWPLPLLLLLLLLLGPAGARAQED (SEQ ID NO: 141) in Coot. All RNC structures were refined in PHENIX using phenix.real_space_refine with secondary structure restrains imposed. Model refinement and validation statistics were provided in FIG. 9.

[00448J To model the interaction of E. coli 23S rRNA and PF846, the high-resolution model

(PDB: 4YBB) was docked into the map by rigid-body docking. For the analysis of the pairing between the A/P-tRNA 3'-CCA end and 28S rRNA, the high resolution tRNA (PDB: lvy4) was docked as a rigid body, using nucleotides in the ribosomal P loop as reference. Subsequent modeling of C75 used manual rebuilding in Coot followed by real-space refinement, enforcing minimal changes to the positions of C74 and A76.

Difference map calculation for codon-anticodon base pairs

[00449] Correlations between modeled nucleotides and observed cryo-EM density at 3.7-4.0 A resolution was dominated by the phosphate-ribose backbone and the common position of the nucleobase in pyrimidines and five-membered ring in purines. Thus, it was difficult to distinguish purines from pyrimidines at this resolution using only real-space correlations. Helix h24 in human 18S rRNA (nts 1037-1078) was used to assess the ability of real-space difference density maps to distinguish purines and pyrimidines in base pairs, and guanosines from adenosines in base pairs, in -3.7-4.0 A resolution cryo-EM maps. Map fragments were generated from the CDH1-RNC cryo-EM map with A/P NC-tRNA and made mutations in nucleotides using Coot, followed by real-space refinement of models in Phenix

(phenix.real_space_refine), with secondary structure restraints generated by

phenix.secondary_structure_restraints and verified manually. Real-space difference maps were generated in Chimera. The minimum threshold of the observed cryo-EM map was set to zero (vop threshold minimum 0), and the model coordinates were used to generate a calculated map at 3.7-4.0 A resolution (molmap). Then, difference maps were generated by minimizing the root- mean-square of the differences (vop subtract minRMS true). From these maps, negative difference density was found to be diagnostic for positions that were incorrectly modeled as purine, due to negative density on the N2, C2, Nl, C6, and 06/N6 positions of the modeled purine ring. Negative difference density was also diagnostic for positions incorrectly modeled as guanosine rather than adenosine, with negative difference density appearing over the exocyclic N2 position of the purine. A clear signal could not always be detected in positive difference density maps. Without intending to be bound by any particular theory, it is proposed that this may indicate the position of a purine incorrectly modeled as a pyrimidine, possibly due to the distribution in density in the map calculated from model coordinates with ADP values grouped at the nucleotide level, currently the only setting available in phenix.real_space_refine.

Quantum mechanical calculations of PF846 conformations

[00450] All torsional assessments and geometry optimizations of PF846 were performed by quantum mechanical calculations using TeraChem using a DFT-(b31yp) level of theory and a 6- 31gs basis set. Dihedral angle energy profiles from model systems derived from truncation of the small molecule X-ray structure of PF846 were used to create low energy poses for fitting to the density. All poses were geometry optimized before fitting. FIG. 15 provided the calculated energies for the poses in FIG. 14A-14F.

Figure preparation

[00451J Figures were generated with UCSF Chimera, PyMOL, and ChemDraw (PerkinElmer). Example 2:

Stalling of human ribosome on CDHl polypeptides with different affinity tags

[00452] Different mRNAs were designed to encode an N-terminal affinity tag followed by the

Cadherin-5 domain of CDHl and the C-terminus of CDHl, comprised of amino acids 586-750. This sequence was followed by the sequence for luciferase. The transcribed mRNAs were used in human in vitro translation extracts, in the absence and presence of Compound 1. The presence of the compound inhibited synthesis of luciferase, as expected for the mechanism of action of this compound (Lintner et al., 2017). CDHl with various affinity tags also showed Compound 1 dependent stalling, as determined by reduction of luciferase expression. The Sequence for the affinity tags include: His-FLAG tag:HHHHHHDYKDHDG (SEQ ID NO: 142); SBP tag:

MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 143); TwinStrep tag WSHPQFEKGAMTGWSHPQFEK (SEQ ID NO: 144); and 3XFLAG tag

DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 145).

Example 3

Stalling of human ribosome on chimeric CDH1-PCSK9

[00453] Additionally, mRNAs were designed to encode N-terminal IX and 3X FLAG tags, followed by the Cadherin-5 domain and then fused to the N-terminal sequence of PCSK9, followed by the sequence of nano-luciferase. Translation of these mRNAs in human in vitro translation extracts also resulted in stalling of RNCs by Compound 1, as evidenced by the reduced luciferase production in the presence of the compound. Anti-FLAG antibodies conjugated to anti-FLAG M2 beads were used to affinity purify stalled RNCs, demonstrating that stalled RNCs could be isolated through a round of selection as in ribosome display methods.

[00454] FIG. 1A-1B. Stalling of ribosome nascent chains on N-terminally tagged CDH1- derived polypeptides. Diagram of mRNA encoding nascent chain substrates used in this study (FIG. 1A). The internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV) is shown in yellow, followed by the affinity tag (yellow). The CDHl domain5 (cyan) is modified to contain an N-terminal tag for affinity purification. The stalling site of CDHl is indicated in the diagram, with CDHl amino acid residues indicated above the diagram. In vitro translation of nano-luciferase, affinity tagged CDHl and CDHl with 4 Cysteines residues removed (CDH1- mut-), in HeLa-based cell-free translation reactions in the absence (black bars) or presence (white bars) of 100 μΜ Compound 1 (FIG. IB).

[00455] FIG. 2A-2C. Stalling of ribosome nascent chains on N-terminally tagged CDHl chimeric polypeptides. Diagram of mRNA encoding chimeric CDHl -PCS K9 nascent chain (CP) (FIG. 2A). The C terminal stalling sequence of CDHl is substituted by amino acids 1-35 of PCSK9 (purple), which causes stalling of PCSK9 when used in isolation. CDHl Domain 5 is comprised of amino acids 586-696 of the CDHl sequence (cyan). In vitro translation of IxFLAG and 3xFLAG tagged chimeric polypeptides in HeLa-based cell-free translation reactions in the absence (black bars) or presence (white bars) of 100 μΜ Compound 1 (FIG. 2B). Western blot of fractions from 3xFLAG-tag affinity purifications. Lane 1, flowthrough: lane 2, anti-FLAG M2 beads after washing with wash buffers; lane 3, beads after releasing with 3xFLAG peptide; lane 4, polypeptide fraction released from the beads; lane 4, sample from lane 5, sample from lane 4, pelleted by using a sucrose cushion (FIG. 2C).

Example 4:

Additional chimeric polypeptides stalled by Compound 1

[00456] Based on results in (Lintner et al., 2017), Compound 1 is highly selective and stalls translation of just 18 polypeptides, of those that could be assayed using ribosome profiling as a readout (Figure 6 of Lintner et al., 2017). Of these 18, only 3 have polypeptide sequences that would extend beyond the ribosome exit tunnel, which can enclose -35-40 amino acids at most due to its -100 A length (Wilson & Beckmann, 2011). In Example 2 and Example 3, CDHl, in its cellular context, was shown to stall at amino acid 729, and can be dissected to isolate a single domain that enables robust in vitro stalling of RNCs, with engineered N-terminal extensions and alternate stall sequences appended C-terminal to the Cadherein-5 domain. N-terminal sequence of USO 1 up to amino acid 298 could be used to stall RNCs, with additional sequences appended N-terminal to this segment of USO 1. Alternatively, the N-terminal 92 amino acids of IFI30 could be used to form stalled RNCs.

[00457] Using structure prediction algorithms, for example Phyre2 (Kelley et al., 2015), it would be to minimize the sequences of USO 1 or IFI30 that are needed to form RNCs. Furthermore, the prediction of USO 1 or IFI30 domain structure, coupled with information on other stalled sequences that reside solely in the exit tunnel (i.e. PCSK9, RPL27, MDK in Fig. 6 of Lintner et al., 2017), allowed for the design of other protein sequences that would be stalled by PF- 06446846. The linker length could be altered between the Cadherin-5 folding domain in CDHl, the N-terminal domain of USOl, or of IFI30 and any of the stalling sequences in Figure 6 of (Lintner et al., 2017) linked to the C-terminus. Finally, for a new chemical compound that induces stalling of ribosome nascent chains, i.e. derivatives of PF-06446846, it could be possible to use ribosome profiling to identify the full spectrum of protein sequences stalled by the compound, as described in (Lintner et al, 2017). These newly-identified stalling sequences could be appended to the Cadherin-5 domain of CDHl, or the folding domains of USOl or IFI30, analogously to the results in Example 3. Ribosome profiling with these new compounds identifies additional proteins that extend beyond the ribosome exit tunnel, i.e. whose stall site resides ~40 or more amino acids from the N-terminus, preferably 80 or more amino acids from the N-terminus. These could be to make chimeric protein sequences that form RNCs in the presence of the new compound.

Example 5:

Chimeric polypeptides stalled PF846

PF846 blocked production of proprotein convertase subtilisin/kexin type 9 (PCSK9)-an important target for regulating plasma low-density lipoprotein cholesterol levels-by interfering with the elongation phase of translation. PF846 selectively stalled the ribosome on very few translated protein nascent chains, generally early in their formation and with no definitive sequence pattern. To gain insight into the mode of action of PF846 in targeting the human ribosome and specific nascent chains, and to identify principles for future drug development, structures of PF846 stalled ribosome nascent chain (RNC) complexes were determined using single -particle cryo-electron microscopy (EM). Since the stalling sequences in most proteins affected by PF846 occurred near the N- terminus and therefore resided in the ribosome exit tunnel, conditions for affinity purification of stable PF846-stalled RNCs were first identified from in vitro translation reactions in human cell extracts. The calcium-dependent cell-cell adhesion glycoprotein Cadherin-1 (CDHl) was stalled near its C-terminus by PF846, enabling the extension of the peptide N-terminus beyond the confines of the ribosomal exit tunnel.

Appending an N-terminal affinity tag followed by the CDHl cadherin-V domain and the nascent chain sequences targeted by PF846 did not disrupt the ability of PF846 to stall translation and allowed formation and purification of stable PF846-stalled RNCs (FIG. 5A-5B and FIG. 6A-6B).

[00459] To investigate how PF846 stalled specific nascent chain sequences in the human

ribosome, cryo-EM was used to determine structures of PF846-stalled CDH1 or PCSK9 ribosome nascent chain complexes (CDH1-RNC or PCSK9-RNC, respectively) (FIG. 7A-7C, FIG. 8A-8E, and FIG. 9). In both CDH1-RNC and PCSK9-RNC samples, particle sorting of the purified RNCs revealed two populations of ribosomes in the rotated state: one with A/A site peptidyl-tRNA (i.e. nascent chain-tRNA, or NC-tRNA), and the other with hybrid A/P site NC- tRNA. Both also included tRNA in the hybrid P/E site and PF846 bound in the ribosome exit tunnel (FIG. 5C, FIG. 10A-10B).

[00460] The following analyses described the CDHl-RNCs due to the higher-quality CDH1-

RNC cryo-EM maps, although similar behavior was observed for the PCSK9-RNCs. The CDH1- RNC complexes with hybrid A/P site NC-tRNA or with A/A site NC-tRNA yielded cryo-EM maps with an average resolution of 3.7 A and 4.3 A, respectively (FIG. 5C-5D and FIG. 7A-7C). The hybrid A/P NC-tRNA represented a mixture of tRNAs, as inferred from the EM map density for nucleotide bases in the codon-anticodon base pairs (FIG. 11 A-l IF and FIG 12A-12I), consistent with the clustering of stall sites spanning multiple codons observed by ribosome profiling. A small population of stalled RNCs was also observed in the non-rotated state bearing a P/P-site NC-tRNA and PF846 in our samples (FIG. 7A-7C, FIG. 8A-8E and FIG. IOC). This population appeared in higher abundance when the RNC sample was purified using a short isolation time prior to making cryo-EM grids (FIG. 8B). These results suggested that the non- rotated RNC was a transient state during PF846 mediated translation stalling. Collectively, PF846 preferentially stalled RNCs in the rotated state, while the non-rotated state with P/P-site NC-tRNA could be resolved during translation in the presence of PF846.

[00461] FIG. 5A-5D. Structural analysis of PF846 stalled ribosome nascent chains.

Schematic representation of the DNA construct used to prepare PF846 stalled RNCs (FIG. 5A). Stalling of the chimeric PCSK9 nascent chain in in vitro translation reactions in the absence (black bar) or presence (white bar) of 100 μΜ PF846 (FIG. 5B). Cryo-EM structure of the stalled CDH1-RNC in the rotated state showing the A/P tRNA (green), P/E tRNA (magenta), CDH1 nascent chain (purple) and PF846 (dark green) with small and large subunit colored cyan and grey, respectively (FIG. 5C). Structure of the stalled CDH1-RNC in the rotated state with A/A tPvNA, with A/A and P/E tRNA colored in orange and magenta, respectively (FIG. 5D).

[00462] FIG. 6A-6B. PF846-mediated stalling of RNCs. In vitro translation assays of PCSK9 and CDHl nascent chains with different affinity tags in the absence (black bar) or presence (grey bar) of 100 μΜ PF846 (FIG. 6A). Significance value: * p<0.1, **p<0.001. Western blot with anti-FLAG® antibody showing R ase A treatment of affinity purified stalled PCSK9-RNCs after elution and pelleting (FIG. 6B).

[00463] FIG. 7A-7C. Cryo-EM data processing and model validation of PF846 stalled

CDHl- RNCs. The EM micrographs were first divided into 3 subsets for classification and refinement (FIG. 7A). The selected classes after refinement (labeled with red asterisk) were combined for an overall refinement. Signal subtraction using the A/P NC-tRNA, nascent chain and PF846 allowed classification and refinement of separate classes with A/A NC-tRNA or A/P NC-tRNA. The rest of the particles remaining after selecting the rotated state RNCs (labeled with blue asterisk) were merged and used for another refinement and 3D classification, which generated 4 different classes, including a non-rotated state with P/P NC-tRNA. Final FSC curves of CDHl-RNCs, with the "gold standard" value of 0.143 were used to define the resolution indicated (FIG. 7B). where FIG. 7C depicted model to map correlation at FSC value of 0.5.

[00464] FIG. 8A-8E. Cryo-EM data processing of PF846 stalled PCSK9-RNC. Data

processing steps for stalled PCSK9-RNC, as described in FIG. 7A-7C were shown in FIG. 8A. Final FSC curves of CDHl_RNCs, with the "gold standard" value of 0.143 were used to define the resolution indicated (FIG. 8B), where FIG. 8C depicted model to map correlation at FSC value of 0.5. Data processing steps for sample prepared with a short incubation time generated both non-rotated and rotated states of the ribosome (FIG. 8D). Final FSC curves of RNCs, with the "gold standard" value of 0.143 were used to define the resolution as indicated (FIG. 8E). A table of data collection, structure model refinement, and validation statistics of the CDHl-RNCs including CDH1-RNC AP tRNA, CDH1-RNC AA tRNA, and CDH11-RNC PP tRNA was shown in FIG. 9.

[00465] FIG. lOA-lOC. Cryo-EM reconstructions of PF846-stalled PCSK9 RNCs. PCSK9-

RNC in the rotated state with A/P and P/E tRNA, with a 40S subunit (cyan), a 60S subunit (grey), an A/P site tRNA (purple), a P/E site tRNA (magenta), a stalled PCSK9 nascent chain (blue) and PF846 (dark green) (FIG. 10A); PCSK9-RNC in the rotated state with A/A tRNA (red) and P/E tRNA (magenta) (FIG. 10B); and a non-rotated state of the PCSK9-RNC stalled by PF846, with P/P tRNA (orange) (FIG. IOC).

[00466] FIG. 11A-11F. Analysis of the interactions of A/P tRNAs with mRNA in PF846 stalled CDH1-RNC. Observed density for representative A-U (A1049 and U1069), G-U (G680 and U1025), and G-C (G1051 and C1067) base pairs in 18S rRNA in the 40S head and platform domains, near the P site (FIG. 1 lA-11C). Observed density for tRNA anticodon-mRNA codon base pairs at the 3rd position of the codon in the A and P sites (for A/P and P/E tRNAs modeled as tRNA ^phe GAA and tRNA ^Leu i _AG , respectively) (FIG. 1 lD-1 IF). Two U-G pairs were shown with FIG. 11D refined in PHENIX with G-U hydrogen -bonding enforced, and FIG. HE with rigid- body docking of a G-U (G680 and U1025) base pair from 18S rRNA.

[00467] FIG. 12A-12I. Difference density maps defining purine and pyrimidine bases.

Representative negative difference densities were shown for an incorrectly placed G-C base pair (18S rRNA nucleotides A1077 and U1038 modeled as G-C base pair). The negative difference map shown was the observed CDH1-RNC map minus the calculated map for the model (Materials and Methods), contoured at -8.8 standard deviations from the mean. The negative difference density for the wild-type sequence was shown in blue and negative difference density for the mutated sequence was shown in red. Representative negative difference density for an incorrect U-A pair (18S rRNA nucleotides G1054 and C1064 modeled as U-A base pair) (FIG. 12B); for an incorrectly placed G instead of U in a base pair (18S rRNA nucleotides 1042 and 1073 modeled as C-G base pair rather than A-U) (FIG. 12C); and for an incorrect G-C pair (18S rRNA nucleotides C1074 and G1041 modeled as G-C base pair) (FIG. 12D) were shown.

Negative difference density maps of tRNA anticodon-mRNA codon base pairs at the 3rd position of the codon in the A and P sites, with the A/P (upper pair in every panel) and P/E tRNA base pairs (lower pair in every panel) were modeled (FIG. 12E-12I). mRNA was colored in orange and tRNA in green.

Example 6:

CDH1 nascent chain library-based selection

[00468] Four different CDH1 nascent chain libraries were designed based on the high-resolution cryo-EM structures to understand the selectivity of the small molecule (PF-06446846). All designs were driven by using the same strategies for making the stalled eukaryotic ribosome nascent chain complexes.

[00469] RNA library design for CDH1 nascent chains. Molecular model of CDH1 nascent chain (magenta) following the 3' -CCA end of A/P tRNA (green) in the ribosome exit tunnel with 28S rRNA (grey), uL22 (orange), uL4 (light green) and PF846 (dark green) highlighted (FIG. 17A). Superposition of hCMV (blue) and SRP (cyan) nascent chain within the exit tunnel, showing the predicted steric clashes with PF846 (dark green), using with van der Waals surfaces (FIG. 17B-17C). Black boxes indicate the regions of the nascent chain libraries (FIG. 17A): 1) Peptidyl transferase center (Lib PTC), 2) Compound binding site (Lib compound), 3) Exit tunnel interacting site (Lib Ribl), 4) Exit tunnel interacting site (Lib_Rib2). FIG. 17D shows stalling of the libraries induced by PF-06446846, reported in relative luciferase units.

Example 7:

PF846's specificity for eukaryotic ribosomes

[00470] In the rotated-state RNCs with A P NC-tRNAs, PF846 occupied a binding pocket in a groove of the ribosomal exit tunnel formed by universally conserved 28S rRNA nucleotides and a stretch of the stalled nascent chain (FIG. 13A-13H). PF846 was fit to the density by evaluating its X-ray structures and low energy conformations based on quantum mechanical assessment of accessible torsion angles (FIG. 14A-14F and FIG. 15). Consistent with PF846 binding to non- translating 80S ribosomes, contacts with 28S rRNA make up the bulk of the small molecule interactions with the ribosome (FIG. 13A-13H and FIG. 14A-14F). The piperidine ring extended into the rRNA binding pocket, contacting nucleotides C1576, G1577, G1579, A1583 and U1584 (FIG. 13E). The chloro-pyridine and amide linkage also formed hydrogen bonds with highly conserved nucleotides G1577 and G1579 (FIG. 13F-13G). Additionally, the interaction was stabilized by U4517, which formed a stacking interaction with the chloro-pyridine ring (FIG. 13H). To explore the structural basis for PF846 action on eukaryotic but not bacterial ribosomes, the small molecule-binding pocket was aligned with the corresponding region in the E. coli ribosome (FIG. 13A-13B and FIG. 16A-16E). Phylogenetic analysis revealed key changes to the nucleotide pattern in 23S rRNA in the large ribosomal subunit of bacteria (FIG. 13A-13B). Whereas nucleotides CI 576, G1577 and G1579 in the human ribosome were highly complementary to the conformation of PF846 (FIG. 13F-13G), nucleotides Nl-methyl-G745 and ψ746 in bacterial 23S rRNA clashed with the compound (FIG. 16C-16D). Furthermore, whereas U4517 in the human ribosome stacked on the chloropyridine ring in PF846 (FIG. 13H), the corresponding nucleotide in bacterial 23S rRNA (U2609) sterically clashed with PF846 (FIG. 16E). Taken together, these distinctions in nucleotide identity and positions helped explain PF846's specificity for eukaryotic ribosomes.

[00471] FIG. 13A-13H. Interactions of PF846 with human ribosome. Phylogenetic analysis of the 28S rRNA binding pocket was performed in eukaryotes (blue) (FIG. 13A) and bacteria (red) (FIG. 13B). Nucleotides that interacted with PF846 were labeled with an asterisk (*). Nucleotides with capital and small letters (AUCG, aucg), were over 98% conserved or 90%-98% conserved, respectively. PF846 chemical and three-dimensional structures were shown in FIG. 13C-13D, respectively. 28S rRNA residues with direct interactions with the piperidine ring of PF846 were shown in FIG. 13E. Surfaces represented the van der Waals radii of the C, N and O atoms. Interactions of PF846 and nucleotides from 28S rRNA were shown in FIG. 13F-13G. Interaction of the chloropyridine ring with U4517 was shown in FIG. 13H, with van der Waals surfaces as in FIG. 13E.

[00472] FIG. 14A-14F. Docking of different conformations of PF846 into density extracted from the CDH1-RNC cryo-EM map. X-ray structures of PF846, with FIG. 14A as molecule 1 and FIG. 14B as molecule 2. Low energy conformations of PF846 were based on quantum mechanical calculations starting from the X-ray structures of molecule 2 (FIG. 14C-14F). From FIG. 14C to 14F corresponded to poses 1 to 4 in FIG. 15, which provided a table of the calculated energies of the various conformations of PF846.

[00473] FIG 16A-16E. Modeling of PF846 in the predicted binding pocket of E. coli

ribosome rRNA. Cartoon representation of the conserved small molecule binding loop from E. coli and human ribosomes, was colored in dark cyan and gray, respectively (FIG. 16A). Residues that contributed to the binding of PF846 were shown in stick format (PDB: 4YBB) (FIG. 16B). 16S rRNA residues from E. coli, which have direct interactions with PF846 in the corresponding positions in the human ribosome, led to steric clashes (FIG. 16C-16E). Dashed lines in FIG. 16C-16E indicated the closest distances between the nucleotides and PF846. In E. coli, G745 was modified to Nl-methyl-G, and U746 to ψ746, although the steric clashes occurred with unmodified nucleotides.

Example 8:

PF846 stalled nascent chains

] The triazolopyridine ring system of PF846 faced the ribosome tunnel and was the only part of the molecule with direct interaction with the stalled nascent chain (FIG. 17A-17C, FIG. 18A-18B, and FIG. 19A-19B). The triazolopyridine moiety had incomplete density that may be caused by the flexibility of this region during nascent chain elongation (FIG. 14A-14F). The density for the nascent chains in the RNCs with A/P NC-tRNA was mostly well defined in the ribosome exit tunnel with a local resolution of ~4 A, although they were likely comprised of different sequences superimposed on each other corresponding to the cluster of stalled states spanning multiple mRNA codons (FIG. 11A-11F and FIG. 12A-12I). Both the CDH1 and PCSK9 nascent chains had similar geometry and adopted a predominantly extended

conformation in the exit tunnel (FIG. 18A-18B and FIG. 19A-19B). The nascent chain spanned ~9 residues between the C-terminus of the nascent chains bonded to the 3' -CCA end of A/P site tRNA in the peptidyl transferase center (PTC) and the small molecule binding site (FIG. 17A and FIG. 19A). These residues engaged in multiple interactions with nucleotides in 28S rRNA of the 60S subunit, including U4493, U4414, and A3879 (FIG. 17A and FIG. 19A). Past the compound-binding pocket (i.e. in the N-terminal direction), the nascent chain was stabilized by ribosomal proteins uL4 and uL22, along with A1582 and C2773 in 28S rRNA (FIG. 17A and FIG. 19A). Most reported RNC structures had "kinks" in the ribosome exit tunnel proximal to the PTC. The CDH1 and PCSK9 nascent chains also had a kink in their structures between the PTC active site and PF846-binding pocket (FIG. 17A, FIG. 18A-18B, and FIG. 19A-19B), but these adopted a different geometry when compared to stalled human cytomegalovirus (hCMV) and signal recognition particle (SRP) nascent chains (FIG. 17B-17C). In the CDH1-RNC and PCSK9-RNC structures, the "kinks" assumed more acute angles, to make enough space for the bound small molecule (FIG. 17A, FIG. 18A-18B, and FIG. 19A-19B), whereas the hCMV and SRP NC positions occluded PF846 binding (FIG. 17B-17C). [00475] FIG. 18A-18B. Cryo-EM densities of PF846 stalled nascent chains. Density extracted from maps of the CDHl-RNC (FIG. 18 A) and PCSK9-RNC (FIG. 18B) in the rotated state with A/P NC- tRNA, shown in the same orientation. The maps were low-pass filtered to 4.0 A with the asterisks indicate the location of peptidyl transferase center. The coloring scheme was the same as previously described.

[00476] FIG. 19A-19B. Interactions between the nascent chains and the ribosome exit tunnel. Density maps in the region showed where the CDH1 (purple) (FIG. 19 A) and PCSK9 (blue) (FIG. 19B) nascent chains interacted with the ribosome exit tunnel. Map densities were shown in mesh, ribosomal proteins and rRNA nucleotides that had an interaction with NC were shown in stick form (uL4 in green, uL22 in orange, uL16 in pink and 28S rRNA in cyan). The atomic models of the nascent chains were also shown in stick representation.

Example 9:

Mechanism of PF846-induced stalling

[00477] PF846 stalled translation of the ribosome predominantly in the rotated state, yet

somehow blocked the action of eEF2, the GTPase which promoted mRNA and tRNA translocation to the next codon position. In bacterial translation, translocation proceeded through a series of ribosome rotated states, in which the peptidyl-tRNA body and 3' -CCA end moved independently from one another, transiting from an A/A site with 3' -CCA end base pairing with the large subunit rRNA A- loop, to an A/A site with 3' -CCA end base pairing with the large subunit rRNA P-loop, and finally to an A/P site with 3' -CCA base pairing with the P-loop. At this point, EF-G (the bacterial homologue of eEF2) catalyzed the remaining conformational changes required for complete translocation of the mRNA and tRNAs. In the PF846-stalled complexes, populations with A/A and A/P NC -tRNAs were observed. However, the A/P-tRNA exhibited flexible base pairing of the 3' -CCA end with the ribosomal P-loop (FIG. 20A-20C and FIG 21A-21B). Without intending to be bound by any particular theory, it is proposed that PF846 inhibited rapid translocation of specific nascent chains by preventing the peptidyl-tRNA from binding stably in the hybrid A/P site, which required the tRNA 3' -CCA end to properly base pair with 28S rRNA nucleotides in the P-loop.

I l l [00478] The present structures of PF846-stalled CDH1 and PCSK9-RNCs provided the first molecular detail for the selective stalling of nascent chains in human ribosomes by a small molecule. PF846 bound a newly-identified small molecule -binding pocket in the exit tunnel of the human ribosome and traps select RNCs predominantly in the rotated state (FIG. 20C).

However, redirection of the nascent chain by PF846 appeared to prevent stable base pairing of the NC-tRNA 3' -CCA end with the ribosomal P-loop. Without intending to be bound by any particular theory, it is proposed that these RNCs poor substrates for the GTPase eEF2 were responsible for mRNA and tRNA translocation. Stalling of RNCs in the rotated state was a determinant of PF846 selectivity by also evading cellular quality control pathways that recognize aberrantly stalled ribosomes and initiate recycling. For example, Pelota (PELO, Dom34 in yeast) and its cofactors were involved in ribosome-associated quality control pathways that recognized stalled ribosome with an empty A site, to which Pelota bound and promoted ribosome subunit dissociation. Consistent with the proposal that quality control pathways resolved PF846-stalled RNCs, ribosome profiling experiments did not reveal a buildup of stalled RNCs in cells treated with PF846. The experimental methods presented here provided a means for probing the sequence specificity of PF846-like compounds and should enable the discovery of other compounds that target the translating human ribosome for therapeutic effect.

[00479] FIG. 20A-20C. Model of the mechanism of PF846-induced stalling. Poor positioning of A/P NC-tRNA 3 ' -CCA end in the ribosomal P-loop was shown in FIG. 20A. High -resolution structure of the 3'-CCA end of peptidyl-tRNA (PDB: lvy4) was docked as a rigid body, using nucleotides from 28S rRNA in the PTC as reference. C75 was flipped to nearby density without pairing with the P-loop (starting model from PDB: lvy34) (FIG. 20B). PF846 binding to the exit tunnel stalled the elongation of the nascent chains in the rotated state, with either A/A NC-tRNA or A/P NC-tRNA (FIG. 20C). Poor positioning of the 3'-CCA end prevented eEF2-GTP from catalyzing the translocation of hybrid A/P and P/E tRNAs into P/P tRNA and E/E tRNA sites. PF846 could also stabilize the resulting non-rotated ribosome with an empty A site that may signal quality control (QC) pathways.

[00480] FIG. 21A-21B. Poor base pairing of the 3'-CCA end of A/P NC-tRNA in PCSK9- stalled RNCs. Interactions between the 3' -CCA end of A/P tRNA and 28S rRNA nucleotides in the P-loop was shown in FIG. 21 A, with a high-resolution structure of the 3' -CCA end of peptidyl-tRNA (PDB: lvy4) rigid docked. C75 was flipped to nearby continuous density without pairing with the P-loop (starting model from PDB: lvy4) (FIG. 21B).

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While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.