BROAD-SPECTRUM KINASE BINDING AGENTS

FIELD

Provided herein are compounds that are broad-spectrum protein kinase binding agents, detectable tracers comprising such compounds, and method of use thereof for the detection of protein kinases.

BACKGROUND

The human genome contains about 560 protein kinase genes, and they constitute about 2% of all human genes (Manning et al. (2002) Science 298 (5600): 1912-1934.; herein incorporated by reference in its entirety). Up to 30% of all human proteins may be modified by kinase activity, and kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction. The chemical activity of a kinase involves transferring a phosphate group from a nucleoside triphosphate (usually ATP) and covalently attaching it to specific amino acids with a free hydroxyl group. Most kinases act on both serine and threonine (serine/threonine kinases), others act on tyrosine (tyrosine kinases), and a number act on all three (dual-specificity kinases) (Dhanasekaran & Premkumar (September 1998). Oncogene. 17 (11 Reviews): 1447-55.; herein incorporated by reference in its entirety). Aberrant kinase signaling is associated with many diseases and conditions.

U.S. Pat. Pub No. 2014/0323346 and Zhang et al. ( J. Proteome Res. 2013, 12, 3104-3116), both of which are herein incorporated by reference in their entireties, describe protein kinase binding agents. Improved kinase ligands with broad specificity are needed to allow cellular kinase profiling, kinase purification, etc.

SUMMARY

Provided herein are compounds that are broad-spectrum protein kinase binding agents, detectable tracers comprising such compounds, and method of use thereof for the detection of protein kinases.

In some embodiments, provided herein are broad-spectrum kinase binding agents of formula:

(CC-1852). In some embodiments, provided herein are broad-spectrum kinase binding agents of formula:

(CC-1861).

In some embodiments, provided herein are broad-spectrum kinase binding agents of formula:

In some embodiments, provided herein are broad-spectrum kinase binding agents (e.g., CC-1852) attached to a functional element or solid surface, comprising a moiety of formula:

, attached to the functional element or solid surface

(e.g., CC-1816, CC-1817, etc.).

In some embodiments, provided herein are broad-spectrum kinase binding agents (e.g., CC-1861) attached to a functional element or solid surface, comprising a moiety of formula:

, attached to the functional element or solid surface

(e.g., CC-1803, CC-1804, etc.).

In some embodiments, provided herein are broad-spectrum kinase binding agents (e.g., CTx-0294885) attached to a functional element or solid surface, comprising a moiety of formula:

(), attached to the functional element or solid surface (e.g., CC-1290, CC-1294, etc.)·

In some embodiments, provided herein are the broad-spectrum kinase binding agents herein linked to a solid surface, wherein the solid surface is selected from a sedimental particle, a membrane, glass, a tube, a well, a self-assembled monolayer, a surface plasmon resonance chip, or a solid support with an electron conducting surface. In some

embodiments, the sedimental particle is a magnetic particle.

In some embodiments, provided herein are the broad-spectrum kinase binding agents herein linked to a functional element, wherein the functional element is selected from a detectable element, an affinity element, and a capture element. In some embodiments, the detectable element comprises a fluorophore, chromophore, radionuclide, electron opaque molecule, a MRI contrast agent, SPECT contrast agent, or mass tag. In some embodiments, a broad-spectrum kinase binding agent described herein (e.g., CC-1852, CC-1861, or CTx- 0294885) is attached to the functional element directly. In some embodiments, a broad- spectrum kinase binding agent described herein (e.g., CC-1852, CC-1861, or CTx-0294885)is attached to the functional element via a linker. In some embodiments, the linker comprises - [(CH ₂ ) ₂ 0]n-, wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, or ranges therebetween). In some embodiments, the linker is attached to the broad- spectrum kinase binding agent described herein (e.g., CC-1852, CC-1861, or CTx-0294885), and/or the functional element, by an amide bond.

In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

wherein X is a functional element or solid surface. In some embodiments, X is a fluorophore. In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

wherein X is a functional element or solid surface. In some embodiments, X is a fluorophore. In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

wherein X is a functional element or solid surface. In some embodiments, X is a fluorophore. In some embodiments, provided herein is a broad-spectrum kinase binding agent comprising a structure of:

In some embodiments, a broad-spectrum kinase binding agent herein comprises a non-natural abundance of one or more stable heavy isotopes.

In some embodiments, provided herein are methods of detecting or quantifying kinases in a sample, comprising contacting the sample with a broad-spectrum kinase binding agent herein and detecting or quantifying the functional element of a signal produced thereby. In some embodiments, the functional element of a signal produced thereby is detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer (e g., FRET, BRET, ALPHA).

In some embodiments, provided herein are methods of isolating kinases from a sample, comprising contacting the sample with a broad-spectrum kinase binding agent herein, and separating the functional element of the solid surface, as well as the bound kinases, from the unbound portion of the sample. In some embodiments, methods comprise isolating the kinases from a sample by a method described herein, and analyzing the isolated kinases by mass spectrometry.

In some embodiments, provided herein are methods of monitoring interactions between kinases and unmodified biomolecules comprising contacting the sample with a broad-spectrum kinase binding agent herein.

In some embodiments, methods herein are performed using a sample selected from a cell, cell lysate, body fluid, tissue, biological sample, in vitro sample, and environmental sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-B. Summary of the BRET results generated with kinase tracers against a large panel of kinase/NanoLuc® fusions expressed in live cells. Assay signals were assessed by taking the ratio of BRET in the presence of tracer/no tracer control. Assays are categorized as follows: >3-fold signal (green cells), 2-3 fold signal (yellow cells), and 1.5-2 fold signal (red cells).

Figure 1C-F. Individual BRET signals observed between the broad-spectrum kinase tracers and individual kinase/NanoLuc® fusions expressed in live cells. Assay signals were assessed by taking the ratio of BRET in the presence of tracer/no tracer control. Assays are categorized as follows: >3-fold signal (green cells), 2-3 fold signal (yellow cells), and 1.5-2 fold signal (red cells).

Figure 2A-C. BRET Target Engagement results in live cells with representative Kinase/NanoLuc® fusions. Cells transfected with plasmid DNA encoding each kinase/Nluc fusion was treated with serially diluted tracer 1804 (left panels), resulting in a dose-dependent increase of specific BRET. Target engagement can be observed for each kinase by titration of unlabeled test compound against a fixed concentration of tracer 1804 (right panels). For each example shown, the BRET signal was superior with tracer 1804 compared to 1294.

Figure 3. The chemical structures of broad-spectrum kinase binding agents CC-1852,

CC-1861 and CTx-02994885. Figure 4. Illustration of NanoBRET™ target engagement work flow for determining target engagement for CC-1861 vs CTx-0294885. NanoBRET™ tracers were introduced at approximately 50% target occupancy values. Occupancy of the test compounds are determined by competitive displacement of the NanoBRET™ Tracers in live cells

Figure 5. A condensed heat map of target engagement potency in live cells for 300 nM CC-1861 vs CTx-0294885. Target engagement was measured by competitive displacement against NanoBRET™ tracers in living HEK293 cells. Red indicates higher occupancy, and green indicates lower occupancy.

Figure 6 A detailed heat map of target engagement potency in live cells for 300 nM CC-1861 vs CTx-0294885. Target engagement was measured by competitive displacement against NanoBRET™ tracers in living HEK293 cells. Red indicates higher occupancy, and green indicates lower occupancy.

Figure 7. Comparison of intracellular affinity for CC-1861 vs CTx-0294884.

NanoBRET™ assays were run by competitive displacement of NanoBRET™ tracers in living HEK293 cells. Intracellular affinity of CC-1861 was stronger than CTx-0294885 for three of the four targets tested.

Figure 8. Detailed comparisons of intracellular affinity values for CC-1861 vs CTx- 0294885 for various kinases in living cells. Values were measured by competitive displacement of NanoBRET™ tracers in living cells.

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, 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. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms“a”,“an” and“the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to“a peptide” is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term“and/or” includes any and all combinations of listed items, including any of the listed items individually. For example,“A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement“A, B, and/or C.”

As used herein, the term“comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase“consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed“consisting of’ and/or“consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

As used herein, the term“CC-1852” refers to a compound of the structure:

(CC-1852);

or a moiety/substituent of a molecular entity, the moiety/substituent having the structure:

(e.g., CC-1816, CC-1817). As used herein, the term“CC-1861” refers to a compound of the structure:

(CC-1861); or

a moiety/substituent of a molecular entity, the moiety/substituent having the structure:

(e.g., CC-1803, CC-1804, etc.).

As used herein, the term“CTx-0294885” refers to a compound of the structure:

(CTx-0294885); or a moiety/substituent of a molecular entity, the moiety/substituent having the structure:

(e.g., CC-1294, CC-1290, etc.).

As used herein, the term“tracer” refers to a compound of interest or an agent that binds to an analyte of interest (e.g., protein of interest (e.g., kinase), etc.) and displays a quantifiable or detectable property (e.g., detected or quantified any suitable biochemical or biophysical technique (e.g., optically, magnetically, electrically, by resonance imaging, by mass, by radiation, etc.)). Tracers may comprise a compound of interest or an agent that binds to an analyte of interest linked (e.g., directly or via a suitable linker) to a fluorophore, radionuclide, mass tag, contrast agent for magnetic resonance imaging (MRI), planar scintigraphy (PS), positron emission tomography (PET), single photon emission computed tomography (SPECT), and computed tomography (CT) (e.g., a metal ion chelator with bound metal ion, isotope, or radionuclide), etc.

As used herein, the term“sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, and the like. Sample may also refer to cell lysates or purified forms of the enzymes, peptides, and/or polypeptides described herein. Cell lysates may include cells that have been lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Sample may also include cell-free expression systems. Environmental samples include environmental material such as surface matter, soil, water, crystals, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

As used herein, the term“linearly connected atoms” refers to the backbone atoms of a chain or polymer, excluding pendant, side chain, or H atoms that do not form the main chain or backbone.

As used herein, the term“functional element” refers to a detectable, reactive, affinity, or otherwise bioactive agent or moiety that is attached (e.g., directly or via a suitable linker) to a compound described herein (e.g., CC-1852, CC-1861, CTx-029485), derivatives or analogs thereof, etc.). Other additional functional elements that may find use in embodiments described herein comprise“localization elements”,“detection elements”, etc.

As used herein, the term“capture element” refers to a molecular entity that forms a covalent interaction with a corresponding“capture agent.”

As used herein, the term“affinity element” refers to a molecular entity that forms a stable noncovalent interaction with a corresponding“affinity agent.”

As used herein, the term“solid support” is used in reference to any solid or stationary material to which reagents such as substrates, mutant proteins, drug-like molecules, and other test components are or may be attached. Examples of solid supports include microscope slides, wells of microtiter plates, coverslips, beads, particles, resin, cell culture flasks, as well as many other suitable items. The beads, particles, or resin can be magnetic or paramagnetic.

As used herein, in chemical structures the indication:

represents a point of attachment of one moiety to another moiety.

DETAILED DESCRIPTION

Provided herein are compounds that are broad-spectrum protein kinase binding agents, detectable tracers comprising such compounds, and method of use thereof for the detection of protein kinases.

In some embodiments, provided herein are compounds that bind a broad spectrum of protein kinases (e.g., specific to protein kinases, but not specific among protein kinases). In some embodiments, compounds CC-1852, CC-1861, and/or CTx-0294885:

(CC-1852);

(CTx-0294885); are provided. In some embodiments, provided herein are compounds or molecular entities comprising a CC-1861, CC-1852, and/or CTx-0294885moiety:

(e.g., CC-1290, CC-1294); a functional element (e.g., attached to the nitrogen at the 4-position of the piperazine of CC- 1861, CC-1852, CTx-0294885). In some embodiments, provided herein are analogs or derivatives of CC-1852, CC-1861, CTx-0294885, and/or compounds or molecular entities comprising analogs or derivatives of CC-1852, CC-1861, CTx-0294885, and a functional element (e.g., analogs or derivatives of CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC-1294, etc.). In some embodiments, CC-1852, CC-1861, CTx-0294885, or an analog or derivative thereof is attached directly (via a single covalent bond) to a functional element. In some embodiments, CC-1852, CC-1861, CTx-0294885, or an analog or derivative thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.) is attached indirectly (via a linker) to a functional element.

In some embodiments, compounds herein (e.g., comprising CC-1852, CC-1861, CTx- 0294885, analogs or derivatives thereof, etc.) incorporate reactive groups suitable for chemical conjugation to a functional element (e.g., detectable element, linker, etc.). These reactive groups may be present on the compound (e.g., at the 4-position of the piperazine of CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof, etc.) or connected by a suitable linker (e.g., connected to the 4-position of the piperazine of CC-1852, CC-1861, CTx-029488, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC-1294, etc.), etc.). In some embodiments, the reactive group on the compound is configured to react specifically (e.g., via bioorthogonal, or click chemistry) with a reactive partner that is present or has been introduced on the functional element. An exemplary click reaction is copper catalyzed click where the compound bears an alkyne or an azide, and the functional element bears the complementary group (e.g., an azide or an alkyne). Mixing these two species together in the presence of an appropriate copper catalyst causes the compound to be covalently conjugated to the functional element through a triazole. Many other bioorthogonal reactions have been reported (for example Patterson, D. M., et al. (2014). "Finding the Right (Bioorthogonal) Chemistry." ACS Chemical Biology 9(3): 592-605.; herein incorporated by reference in its entirety), and compounds (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) and functional elements incorporating

complementary reactive species are embodiments of the present invention.

In some embodiments, compounds herein (e.g., comprising CC-1852, CC-1861, CTx- 0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC- 1290, CC1294, etc.), etc.) are tethered to a functional element (e.g., detectable element, solid surface, etc.) by a suitable linker. In some embodiments, linkers connecting the compounds herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) to a functional element (e.g., detectable element, solid surface, etc.) are provided as part of the compositions herein. In some embodiments, a linker provides sufficient distance between compounds herein (e.g., CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-

1816, CC-1817, CC-1803, CC-1804, CC-1290, CC-1294, etc.), etc.) and functional elements (e.g., detectable element, solid surface, etc.) to allow each to function undisturbed (or minimally disturbed by the linkage to the other. For example, linkers provide sufficient distance to allow a kinase binding agent to bind a kinase and detectable moiety to be detectable (e.g., without or with minimal interference between the two). In some

embodiments, a linker separates a compound herein (e.g., CC-1852, CC-1861, CC- CTx- 0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC- 1290, CC1294, etc.), etc.) and a functional element (e.g., detectable element, solid surface, etc.) by 5 angstroms to 1000 angstroms, inclusive, in length. Suitable linkers separate a compound herein and a functional element by 5 A, 10 A, 20 A, 50 A, 100 A, 150 A, 200 A, 300 A, 400 A, 500 A, 600 A, 700 A, 800 A, 900 A, 1000 A, and any suitable ranges therein (e.g., 5-100 A, 50-500 A, 150-700 A, etc.). In some embodiments, the linker separates a compound herein and a functional element by 1-200 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable ranges therein (e.g., 2-20, 10-50, etc.)).

In some embodiments, a linker comprises 1 or more (e.g., 1-20 (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any ranges therebetween) -(CFk^O- (oxyethylene) groups (e.g., -(CH2)20-(CH2)20-(CH2)20-(CH2)20-, -(CH2)20-(CH2)20- (CH ₂ )20-(CH ₂ )20- CH ₂ ) ₂ 0-, -(CH ₂ )20-(CH2)20-(CH2)20-(CH ₂ )20- CH ₂ )20-(CH ₂ )20-, etc ). In some embodiments, the linker is -(CH2)20-(CH2)20-(CH2)20-(CH2)20-.

In some embodiments, a linker is attached to a compound herein (e.g., CC-1852, CC- 1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC- 1804, CC-1290, CC1294, etc.), etc.) at the 4-position of the piperazine of a compound herein (e.g., CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-

1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.). In some embodiments, the N at the 4-position of the piperazine of a compound herein (e.g., CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) forms an amide bond with the terminus of a linker. In some

embodiments, a linker comprises one or more (e.g., 2, 3, 4, 5, 6, or more or ranges therebetween) amides.

In some embodiments, a linker comprises two or more“linker moieties” (L ¹ , L ² , etc.). In some embodiments, a linker comprises a cleavable (e.g., enzymatically cleavable, chemically cleavable, etc.) moiety (Y) and 0, 1, 2, of more“linker moieties” (L ¹ , L ² , etc.). In some embodiments, linker moieties are straight or branched chains comprising any combination of alkyl, alkenyl, or alkynyl chains, and main-chain heteroatoms (e.g., O, S, N,

P, etc.). In some embodiments, linker moieties comprises one or more backbone groups selected from of: -0-, -S-, -CH=CH-, =C=, a carbon-carbon triple bond, C=0, NH, SH, OH, CN, etc. In some embodiments, a linker moiety comprises one or more substituents, pendants, side chains, etc., comprising any suitable organic functional groups (e.g., OH,

NH2, CN, =0, SH, halogen (e.g. Cl, Br, F, I), COOH, CH3, etc ).

In particular embodiments, a linker moiety comprises an alkyl carbamate group (e.g., (CH2)nOCONH, (CH2)nNHC00, etc.). In some embodiments, the alkyl carbamate is oriented such the COO end is oriented toward the compound herein (e.g., CC-1852, CC- 1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC- 1804, CC-1290, CC1294, etc.), etc.), and the NH end is oriented toward the functional element. In some embodiments, the alkyl carbamate is oriented such the NH end is oriented toward the compound and the COO end is oriented toward the functional element. In some embodiments, a linker or linker moiety comprises a single alkyl carbamate group. In some embodiments, a linker or linker moiety comprises two or more alkyl carbamate groups (e.g., 2, 3, 4, 5, 6, 7, 8, etc.).

In some embodiments, a linker moiety comprises more than 1 linearly connected C, S, N, and/or O atoms. In some embodiments, a linker moiety comprises one or more alkyl carbamate groups. In some embodiments, a linker moiety comprises one or more alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.). In some embodiments, a linker moiety comprises 1-200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable ranges therein (e.g., 2-20, 10-50, 6-18)). In some embodiments, a linker moiety is 1-200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable ranges therein (e.g., 2-20, 10-50, 6-18)) in length.

In some embodiments, a compound herein (e.g., CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) is attached (e.g., directly or via a linker) to a functional element (e.g., detectable element, capture element, solid surface, etc.).

In some embodiments, the compositions described herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) are biocompatible (e.g., cell compatible) and/or cell permeable. Therefore, in some embodiments, suitable functional elements (e.g., detectable, capture elements) are ones that are cell compatible and/or cell permeable within the context of such compositions. In some embodiments, a composition comprising an addition element, when added extracellularly, is capable of crossing the cell membrane to enter a cell (e.g., via diffusion, endocytosis, active transport, passive transport, etc.). In some embodiments, suitable functional elements and linkers are selected based on cell compatibility and/or cell permeability, in addition to their particular function.

In certain embodiments, functional elements have a detectable property that allows for detection of the compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290,

CC1294, etc.), etc.) or an analyte (e.g., kinase) bound thereto. Detectable functional elements include those with a characteristic electromagnetic spectral property such as emission or absorbance, magnetism, electron spin resonance, electrical capacitance, dielectric constant, or electrical conductivity as well as functional groups which are ferromagnetic, paramagnetic, diamagnetic, luminescent, electrochemiluminescent, fluorescent, phosphorescent, chromatic, antigenic, or have a distinctive mass. A functional element includes, but is not limited to, a nucleic acid molecule (e.g., DNA or RNA (e.g., an oligonucleotide or nucleotide), a protein (e.g., a luminescent protein, a peptide, a contrast agent (e.g., MRI contract agent), a radionuclide, an affinity tag (e.g., biotin or streptavidin), a hapten, an amino acid, a lipid, a lipid bilayer, a solid support, a fluorophore, a chromophore, a reporter molecule, a radionuclide, an electron opaque molecule, a MRI contrast agent (e.g., manganese, gadolinium(III), or iron-oxide particles), or a coordinator thereof, and the like. Methods to detect a particular functional element, or to isolate a composition comprising a particular functional element and anything bound thereto, are understood.

In some embodiments, a functional group is or comprises a solid support. Suitable solid supports include a sedimental particle such as a magnetic particle, a sepharose, or cellulose bead; a membrane; glass, e.g., glass slides; cellulose, alginate, plastic, or other synthetically prepared polymer (e.g., an Eppendorf tube or a well of a multi-well plate); self- assembled monolayers; a surface plasmon resonance chip; or a solid support with an electron conducting surface; etc.

Exemplary detectable functional elements include haptens (e.g., molecules useful to enhance immunogenicity such as keyhole limpet hemacyanin), cleavable labels (e.g., photocleavable biotin) and fluorescent labels (e.g., N-hydroxysuccinimide (NHS) modified coumarin and succinimide or sulfonosuccinimide modified BODIPY (which can be detected by UV and/or visible excited fluorescence detection), rhodamine (R110, rhodols, CRG6, Texas Methyl Red (TAMRA), Rox5, FAM, or fluorescein), coumarin derivatives (e.g., 7 aminocoumarin, and 7-hydroxy coumarin, 2-amino-4-methoxynapthalene, 1 -hydroxy pyrene, resorufm, phenalenones or benzphenalenones (U.S. Pat. No. 4,812,409)), acridinones (U.S. Pat. No. 4,810,636), anthracenes, and derivatives of alpha and beta-naphthol, fluorinated xanthene derivatives including fluorinated fluoresceins and rhodols (e.g., U.S. Pat. No.

6,162,931), and bioluminescent molecules (e.g., luciferase (e.g., Oplophorus-derive luciferase (See e.g., U.S. App. Ser. No. 12/773,002; U.S. App. Ser. No. 13/287,986; herein incorporated by reference in their entireties) or GFP or GFP derivatives). A fluorescent (or

bioluminescent) functional element may be used to sense changes in a system, like phosphorylation, in real-time. A fluorescent molecule, such as a chemosensor of metal ions, may be employed to label proteins which bind the composition. A bioluminescent or fluorescent functional group such as BODIPY, rhodamine green, GFP, or infrared dyes, finds use as a functional element and may, for instance, be employed in interaction studies (e.g., using BRET, FRET, LRET or electrophoresis).

Another class of functional elements includes molecules detectable using

electromagnetic radiation and includes, but is not limited to, xanthene fluorophores, dansyl fluorophores, coumarins and coumarin derivatives, fluorescent acridinium moieties, benzopyrene-based fluorophores as well as 7-nitrobenz-2-oxa-l, 3-diazole, and 3-N-(7- nitrobenz-2-oxa-l,3-diazol-4-yl)-2, 3-diamino-propionic acid. Preferably, the fluorescent molecule has a high quantum yield of fluorescence at a wavelength different from native amino acids and more preferably has high quantum yield of fluorescence that can be excited in the visible, or in both the UV and visible, portion of the spectrum. Upon excitation at a preselected wavelength, the molecule is detectable at low concentrations either visually or using conventional fluorescence detection methods. Electrochemiluminescent molecules such as ruthenium chelates and its derivatives or nitroxide amino acids and their derivatives are detectable at femtomolar ranges and below.

In some embodiments, a functional element is a fluorophore. Suitable fluorophores for linking to the compounds herein (e.g., to form a fluorescent tracer) include, but are not limited to: xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, Texas red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole,

benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphin, phtalocyanine, bilirubin, etc.), CF dye (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes)(Columbia

Biosciences), APC, APCXL, RPE, BPE (Phy co-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc. In some embodiments, a fluorophore is a rhodamine analog (e.g., carboxy rhodamine analog) such as those described in U.S. Pat. App. Ser. No. 13/682,589, herein incorporated by reference in its entirety.

In addition to fluorescent molecules, a variety of molecules with physical properties based on the interaction and response of the molecule to electromagnetic fields and radiation find use in the compositions and methods described herein. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, presence of chromophores that are Raman active and can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity, and nuclear magnetic resonances and molecular mass, e.g., via a mass spectrometer.

In some embodiments, a functional element is a capture element. In some

embodiments, a capture element is a substrate for a protein (e.g., enzyme), and the capture agent is that protein. In some embodiments, a capture element is a“covalent substrate” or one that forms a covalent bond with a protein or enzyme that it reacts with. The substrate may comprise a reactive group (e.g., a modified substrate) that forms a covalent bond with the enzyme upon interaction with the enzyme, or the enzyme may be a mutant version that is unable to reconcile a covalently bound intermediate with the substrate. In some

embodiments, the substrate is recognized by a mutant protein (e.g., mutant dehalogenase), which forms a covalent bond thereto. In such embodiments, while the interaction of the substrate and a wild-type version of the protein (e.g., dehalogenase) results in a product and the regeneration of the wild-type protein, interaction of the substrate (e.g., haloalkane) with the mutant version of the protein (e.g., dehalogenase) results in stable bond formation (e.g., covalent bond formation) between the protein and substrate. The substrate may be any suitable substrate for any mutant protein that has been altered to form an ultra-stable or covalent bond with its substrate that would ordinarily only transiently bound by the protein. In some embodiments, the protein is a mutant hydrolase or dehalogenase. In some embodiments, the protein is a mutant dehalogenase and the substrate is a haloalkane. In some embodiments, the haloalkane comprises an alkane (e.g., C2-C20) capped by a terminal halogen (e.g., Cl, Br, F, I, etc.)· In some embodiments, the haloalkane is of the formula A-X, wherein X is a halogen (e.g., Cl, Br, F, I, etc.), and wherein A is an alkane comprising 2-20 carbons.

In certain embodiments, A comprises a straight-chain segment of 2-12 carbons. In certain embodiments, A is a straight-chain segment of 2-12 carbons. In some embodiments, the haloalkane may comprise any additional pendants or substitutions that do not interfere with interaction with the mutant dehalogenase.

In some embodiments, a capture agent is a SNAP-Tag and a capture element is benzyl guanine (See, e.g., Crivat G, Taraska JW (January 2012). Trends in Biotechnology 30 (1): 8- 16.; herein incorporated by reference in its entirety). In some embodiments, a capture agent is a CLIP-Tag and a capture element is benzyl cytosine (See, e.g., Gautier, et al. Chem Biol. 2008 Feb;l5(2): 128-36.; herein incorporated by reference in its entirety).

Systems comprising mutant proteins (e.g., mutant hydrolases (e.g., mutant dehalogenases) that covalently bind their substrates (e.g., haloalkane substrates) are described, for example, in U.S. Pat. No. 7,238,842; U.S. Pat. No. 7,425,436; U.S. Pat. No. 7,429,472; U.S. Pat. No. 7,867,726; each of which is herein incorporated by reference in their entireties.

In some embodiments, a functional element is an affinity element (e.g., that binds to an affinity agent). Examples of such pairs would include: an antibody as the affinity agent and an antigen as the affinity element; a His-tag as the affinity element and a nickel column as the affinity agent; a protein and small molecule with high affinity as the affinity agent and affinity element, respectively (e.g., streptavidin and biotin), etc. Examples of affinity molecules include molecules such as immunogenic molecules (e.g., epitopes of proteins, peptides, carbohydrates, or lipids (e.g., any molecule which is useful to prepare antibodies specific for that molecule)); biotin, avidin, streptavidin, and derivatives thereof; metal binding molecules; and fragments and combinations of these molecules. Exemplary affinity molecules include His5 (HHHHH)(SEQ ID NO: 1), HisX6 (HHHHHH)(SEQ ID NO: 2), C- myc (EQKLISEEDL) (SEQ ID NO: 3), Flag (DYKDDDDK) (SEQ ID NO: 4), SteptTag (WSHPQFEK)(SEQ ID NO: 5), HA Tag (YPYDVPDYA) (SEQ ID NO: 7), thioredoxin, cellulose binding domain, chitin binding domain, S-peptide, T7 peptide, calmodulin binding peptide, C-end RNA tag, metal binding domains, metal binding reactive groups, amino acid reactive groups, inteins, biotin, streptavidin, and maltose binding protein. Another example of an affinity molecule is dansyllysine. Antibodies that interact with the dansyl ring are commercially available (Sigma Chemical; St. Louis, Mo.) or can be prepared using known protocols such as described in Antibodies: A Laboratory Manual (Harlow and Lane, 1988).

In some embodiments, provided herein are methods of using the compounds herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC- 1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) alone or attached to a functional element (e.g., directly of via a suitable linker) to detect, isolate, analyze, characterize, etc., kinases within a system (e.g., a cell, a cell lysate, a sample, a biochemical solution or mixture, a tissue, an organism, etc.).

In some embodiments, provided herein are methods of detecting one or more kinases in a sample, the method comprising contacting the sample with a compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.). In some embodiments, provided herein are methods to isolate one or more kinases from a sample.

In some embodiments, methods are provided for characterizing a sample by analyzing the presence, quantity, and or population of kinases in the sample (e.g., what kinases are present and/or at what quantities) by contacting the sample with a compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.).

In some embodiments, provided herein are methods of diagnosing a disease of condition comprising detecting the presence or quantity of one or more kinases in a sample from the subject by contacting the sample with a compound herein (e.g., comprising CC- 1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC- 1803, CC-1804, CC-1290, CC1294, etc.), etc.), wherein the presence or quantity of the one or more of the kinases in the sample is indicative of the disease, condition, or a predisposition thereto.

In some embodiments, provided herein are methods of monitoring a subject's response to a therapeutic treatment comprising: (a) detecting the presence or quantity of one or more kinases in a sample from the subject by contacting the sample with compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC- 1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) prior to administration of the therapeutic treatment, and (b) detecting the presence or quantity of one or more kinases in a sample from the subject by contacting the sample with compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) following administration of the therapeutic treatment, wherein a change in the presence of quantity of the one or more kinases is indicative of the subject's response to the therapeutic treatment.

In some embodiments, kinases bound by the compounds herein are detected, quantified, and/or isolated by taking advantage of unique properties of the compound and/or the functional element bound thereto by any means including electrophoresis, gel filtration, high-pressure or fast-pressure liquid chromatography, mass spectroscopy, affinity

chromatography, ion exchange chromatography, chemical extraction, magnetic bead separation, precipitation, hydrophobic interaction chromatography (HIC), or any combination thereof. The isolated kinase(s) may be employed for structural and functional studies, for diagnostic applications, for the preparation biological or pharmaceutical reagents, as a tool for the development of drugs, and for studying protein interactions, for the isolation and characterization of protein complexes, etc.

In some embodiments, methods are provided for detecting and/or quantifying a compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) and/or analyte (e.g., kinases) bound thereto in a sample. In some embodiments, techniques for detection and/or quantification of a compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) and/or analyte (e.g., kinases) bound thereto depend upon the identity of the functional element attached to the compound (e.g., capture element, affinity element, detectable element (e.g., fluorophore, luciferase, chelated radionuclide, chelated contrast agent, etc.) and/or specific modifications to the compound (e.g., mass tags (e.g., heavy isotopes (e.g., ¹³ C, ¹⁵ N, ² H, etc.). For example, when a compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) is linked to a fluorophore or other light emitting functional element, the compound and/or analyte (e.g., kinases) bound thereto may be detected/quantified in a sample using systems, devices, and/or apparatuses that are provided to detect, quantitate, or monitor, the amount of light (e.g., fluorescence) emitted, or changes thereto. In some embodiments, detection, quantification, and/or monitoring are provided by a device, system or apparatus comprising one or more of a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode,

nephlometer, photon counter, electrodes, ammeter, voltmeter, capacitative sensors, flow cytometer, CCD, etc. In addition to fluorescent functional elements, a variety of functional elements with physical properties based on the interaction and response of the functional elements to electromagnetic fields and radiation can be used to detect the compound herein (e.g., comprising CC-1852, CC-1861, CTx-0294885, analogs or derivatives thereof (e.g., CC-1816, CC-1817, CC-1803, CC-1804, CC-1290, CC1294, etc.), etc.) and/or a bound kinase. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, presence of chromophores that are Raman active and can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity and nuclear magnetic resonances and molecular mass, e.g., via a mass spectrometer.

In some embodiments, systems are provided comprising: (a) a fusion of a protein kinase (e.g., of Table 1A-0 or a variant thereof) and a bioluminescent protein; and (b) a broad-spectrum kinase binding moiety herein linked to a fluorophore; wherein the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorophore, such that BRET is detectable between the bioluminescent protein and the fluorophore when the broad-spectrum kinase binding moiety binds to the protein kinase. Similar BRET systems (e.g., utilizing a NANOLUC® luciferase) are described in, for example, Inti. Pat. App.

PCT/US13/74765 (herein incorporated by reference in its entirety); embodiments of which will find use in the systems and methods herein.

In some embodiments, the compounds herein bind a broad spectrum of kinases, including protein kinases are of the following common families or subgroups: AGC (e.g., containing the PKA, PKG and PKC subfamilies), CAMK (e.g., calcium/calmodulin- dependent protein kinases), CK1 (e.g., casein kinase 1), CMGC (e.g., containing the CDK, MAPK, GSK3 and CLK subfamilies), NEK, RGC (e.g., receptor guanylate cyclases), STE, TKL (e.g., tyrosine protein kinase-like), and Tyr (e.g., tyrosine protein kinase). In some embodiments, the compounds herein bind to one or more kinases of atypical kinase families, such as, ADCK, alpha-type, FAST, PDK/BCKDK, PI3/PI4-kinase, RIO-type, etc. In some embodiments, the compounds herein bind to kinases of any suitable organism. In some embodiments, compounds herein bind to human and/or mouse kinases, such as those listed in Tables 1A-0, and/or homologs and analogs from other organisms. Table 1A. AGC Ser/Thr protein kinase family

AKT1 AKT 1 HUMAN (P31749 ) AKT1 MOUSE (P31750)

AKT2 AKT2 HUMAN (P31751 ) AKT2 MOUSE (Q60823)

AKT3 AKT3 HUMAN (Q9Y243 ) AKT3 MOUSE (Q9WUA6)

CDC42BPA MRCKA HUMAN (Q5VT25 ) MRCKA MOUSE (Q3UU96)

CDC42BPB MRCKB HUMAN (Q9Y5S2 ) MRCKB MOUSE (Q7TT50) CDC42BPG MRCKG HUMAN (Q6DT37 ) MRCKG MOUSE (Q80UW5) CIT CTRO HUMAN (014578 ) CTRO MOUSE (P49025)

DMPK DMPK HUMAN (Q09013 ) DMPK MOUSE (P54265)

GRK1 RK HUMAN (Q 15835 ) RK MOUSE (Q9WVL4)

GRK2 ARBK1 HUMAN (P25098 ) ARBK1 MOUSE (Q99MK8)

GRK3 ARBK2 HUMAN (P35626 ) ARBK2 MOUSE (Q3UYH7)

GRK4 GRK4 HUMAN (P32298 ) GRK4 MOUSE (070291)

GRK5 GRK5 HUMAN (P34947 ) GRK5 MOUSE (Q8VEB1)

GRK6 GRK6 HUMAN (P43250 ) GRK6 MOUSE (070293)

GRK7 GRK7 HUMAN (Q8WTQ7 )

LATS1 LATS1 HUMAN (095835 ) L ATS 1 MOUSE (Q8BYR2)

LATS2 LATS2 HUMAN (Q9NRM7 ) LATS2 MOUSE (Q7TSJ6) MAST1 MAST1 HUMAN (Q9Y2H9 ) MAST1 MOUSE (Q9R1L5)

MAST2 MAST2 HUMAN (Q6P0Q8 ) MAST2 MOUSE (Q60592)

MAST3 MAST3 HUMAN (060307 ) MAST3 MOUSE (Q3U214)

MAST4 MAST4 HUMAN (015021 ) MAST4 MOUSE (Q811L6)

MASTL GWL HUMAN (Q96GX5 ) GWL MOUSE (Q8C0P0)

PDPK1 PDPK1 HUMAN (015530 ) PDPK1 MOUSE (Q9Z2A0) PDPK2P PDPK2 HUMAN (Q6A1A2 )

PKN1 PK 1 HUMAN (Q16512 ) PKN1 MOUSE (P70268)

PKN2 PKN2 HUMAN (Q16513 ) PKN2 MOUSE (Q8BWW9)

PKN3 PKN3 HUMAN (Q6P5Z2 ) PKN3 MOUSE (Q8K045)

PRKACA KAPCA HUMAN (P 17612 ) KAPCA MOUSE (P05132) PRKACB KAPCB HUMAN (P22694 ) KAPCB MOUSE (P68181) PRKACG KAPCG HUMAN (P22612 ) PRKCA KP C A HUM AN (P 17252 ) KPCA MOUSE (P20444)

PRKCB KPCB HUMAN (P05771 ) KPCB MOUSE (P68404)

PRKCD KPCD HUMAN (Q05655 ) KPCD MOUSE (P28867)

PRKCE KPCE HUMAN (Q02156 ) KPCE MOUSE (P 16054)

PRKCG KPCG HUMAN (P05129 ) KPCG MOUSE (P63318)

PRKCH KPCL HUMAN (P24723 ) KPCL MOUSE (P23298)

PRKCI KPCI HUMAN (P41743 ) KPCI MOUSE (Q62074)

PRKCQ KPCT HUMAN (Q 04759 ) KPCT MOUSE (Q02111)

PRKCZ KPCZ HUMAN (Q05513 ) KPCZ MOUSE (Q02956)

PRKG1 KGP1 HUMAN (Q 13976 ) KGP1 MOUSE (P0C605)

PRKG2 KGP2 HUMAN (Q 13237 ) KGP2 MOUSE (Q61410)

PRKX PRKX HUMAN (P51817 ) PRKX MOUSE (Q922R0)

PRKY PRKY HUMAN (043930 )

ROCK1 ROCK1 HUMAN (Q 13464 ) ROCK1 MOUSE (P70335)

ROCK2 ROCK2 HUMAN (075116 ) ROCK2 MOUSE (P70336)

RPS6KA1 KS 6 A 1 HUMAN (Q15418 ) KS6A1 MOUSE (P18653)

RPS6KA2 KS 6 A2 HUMAN (Q15349 ) KS6A2 MOUSE (Q9WUT3)

RPS6KA3 KS 6 A3 HUMAN (P51812 ) KS6A3_MOUSE (P 18654)

RPS6KA4 KS 6 A4 HUMAN (075676 ) KS6A4 MOUSE (Q9Z2B9)

RPS6KA5 KS 6 A5 HUMAN (075582 ) KS6A5 MOUSE (Q8C050)

RPS6KA6 KS 6 A6 HUM AN (Q9UK32 ) KS6A6 MOUSE (Q7TPS0)

RPS6KB1 KS 6B 1 HUMAN (P23443 ) KS6B1 MOUSE (Q8BSK8)

RPS6KB2 KS 6B2 HUM AN (Q9UBS0 ) KS6B2 MOUSE (Q9Z1M4)

SGK1 SGK1 HUMAN (000141 ) SGK1 MOUSE (Q9WVC6)

SGK2 S GK2 HUM AN (Q9HBY8 ; l SGK2 MOUSE (Q9QZS5)

SGK3 SGK3 HUMAN (Q96BR1 ) SGK3 MOUSE (Q9ERE3)

STK38 STK38 HUMAN (Q 15208 ) STK38 MOUSE (Q91VJ4)

STK38L ST38L HUMAN (Q9Y2H1 ) ST38L MOUSE (Q7TSE6) Table IB. CAMK Ser/Thr protein kinase family

SMKX MOUSE (Q8C0X8)

BRSK1 BRSK1 HUMAN (Q8TDC3 ) BRSK1 MOUSE (Q5RJI5) BRSK2 BRSK2 HUMAN (Q8IWQ3 ) BRSK2 MOUSE (Q69Z98) CAMK1 KCC1A HUMAN (Q 14012 ) KCC1A MOUSE (Q91YS8) CAMK1D KCC1D HUMAN (Q8IU85 ) KCC1D MOUSE (Q8BW96)

CAMK1G KCC1G HUMAN (Q96NX5 ) KCC1G MOUSE (Q91VB2)

CAMK2A KCC2A HUMAN (Q9UQM7 ) KCC2A MOUSE (P11798) CAMK2B KCC2B HUMAN (Q13554 ) KCC2B MOUSE (P28652) CAMK2D KCC2D HUMAN (Q13557 ) KCC2D MOUSE (Q6PHZ2) CAMK2G KCC2G HUMAN (Q13555 ) KCC2G MOUSE (Q923T9)

CAMK4 KCC4 HUMAN (Q16566 ) KCC4 MOUSE (P08414) CAMKV CAMKV HUMAN (Q8NCB2 ) CAMKV MOUSE (Q3UHL1) CASK C SKP HUMAN (014936 ) CSKP MOUSE (070589)

CHEK1 CHK1 HUMAN (014757 ) CHK1 MOUSE (035280)

CHEK2 CHK2 HUMAN (096017 ) CHK2 MOUSE (Q9Z265)

DAPK1 D APK 1 HUMAN (P53355 ) DAPK1 MOUSE (Q80YE7)

DAPK2 DAPK2 HUMAN (Q9UIK4 ) DAPK2 MOUSE (Q8VDF3)

DAPK3 DAPK3 HUMAN (043293 ) DAPK3 MOUSE (054784)

DCLK1 DCLK1 HUMAN (015075 ) DCLK1 MOUSE (Q9JLM8)

DCLK2 D CLK2 HUM AN (Q8N568 ) DCLK2 MOUSE (Q6PGN3)

DCLK3 D CLK3 HUM AN (Q9C098 ) DCLK3 MOUSE (Q8BWQ5)

Gm4922 SMKZ_MOUSE (Q8C0N0)

Gm7l68 SMKY_MOUSE (A0AUV4)

HUNK HUNK HUMAN (P57058 ) HUNK MOUSE (088866)

KALRN KALRN HUMAN (060229 ) KALRN MOUSE (A2CG49) MAPKAPK2 MAPK2 HUMAN (P49137 ) MAPK2 MOUSE (P49138)

MAPKAPK3 MAPK3 HUMAN (Q 16644 ) MAPK3 MOUSE (Q3UMW7) MAPKAPK5 MAPK5 HUMAN (Q8IW41 ) MAPK5 MOUSE (054992) MARK1 MARK1 HUMAN (Q9P0L2 ) MARK 1 _MOU S E (Q8VHJ5) MARK2 MARK2 HUMAN (Q7KZI7 ) MARK2 MOUSE (Q05512) MARK3 MARK3 HUMAN (P27448 ) MARK3 MOUSE (Q03141) MARK4 MARK4 HUMAN (Q96L34 ) MARK4 MOUSE (Q8CIP4) MELK MELK HUMAN (Q 14680 ) MELK MOUSE (Q61846) MKNK1 MKNK1 HUMAN (Q9BUB5 ) MKNK1 MOUSE (008605) MKNK2 MKNK2 HUMAN (Q9HBH9 ) MKNK2 MOUSE (Q8CDB0) MYLK MYLK HUMAN (Q 15746 ) MYLK MOUSE (Q6PDN3) MYLK2 MYLK2 HUMAN (Q9H1R3 ) MYLK2 MOUSE (Q8VCR8)

MYLK3 MYLK3 HUMAN (Q32MK0 ) MYLK3 MOUSE (Q3UIZ8) MYLK4 MYLK4 HUMAN (Q86YV6 ) MYLK4 MOUSE (Q5SUV5) NIM1K NIM1 HUMAN (Q8IY84 ) NIM1 MOUSE (Q8BHI9) NUAK1 NUAK1 HUMAN (060285 ) NUAK1 MOUSE (Q641K5)

NUAK2 NUAK2 HUMAN (Q9H093 ) NUAK2 MOUSE (Q8BZN4)

OBSCN OB S CN HUM AN (Q5VST9 ) OBSCN MOUSE (A2AAJ9)

PASK PASK HUMAN (Q96RG2 ) PASK MOUSE (Q8CEE6) PHKG1 PHKG1 HUMAN (Q 16816 ) PHKG1 MOUSE (P07934) PHKG2 PHKG2 HUMAN (P 15735 ) PHKG2 MOUSE (Q9DB30)

PIM1 PIM1 HUMAN (P11309 ) PIM1 MOUSE (P06803)

PIM2 PIM2 HUMAN (Q9P1W9 ) PIM2 MOUSE (Q62070)

PIM3 PIM3 HUMAN (Q86V86 ) PIM3 MOUSE (P58750)

PNCK KCC1B HUMAN (Q6P2M8 ) KCC1B MOUSE (Q9QYK9)

PRKAA1 AAPK1 HUMAN (Q13131 ) AAPK1 MOUSE (Q5EG47)

PRKAA2 AAPK2 HUMAN (P54646 ) AAPK2 MOUSE (Q8BRK8)

PRKD1 KPCD1 HUMAN (Q15139 ) KPCD1 MOUSE (Q62101) PRKD2 KP CD2 HUM AN (Q9BZL6 ) KPCD2 MOUSE (Q8BZ03) PRKD3 KPCD3 HUMAN (094806 ) KPCD3 MOUSE (Q8K1Y2) PSKHl KPSH1 HUMAN (P 11801 ) KPSH1 MOUSE (Q91YA2)

PSKH2 KP SH2 HUM AN (Q96QS6 )

SIK1 SIK1 HUMAN (P57059 ) SIK1 MOUSE (Q60670)

SIK2 SIK2 HUMAN (Q9H0K1 ) SIK2 MOUSE (Q8CFH6)

SIK3 SIK3 HUMAN (Q9Y2K2 ) SIK3 MOUSE (Q6P4S6)

SNRK SNRK HUMAN (Q9NRH2 ) SNRK MOUSE (Q8VDU5) SPEG SPEG HUMAN (Q 15772 ) SPEG MOUSE (Q62407)

STK11 STK11 HUMAN (Q15831 ) STK11 MOUSE (Q9WTK7)

STK17A ST17A HUMAN (Q9UEE5 )

STK17B ST17B HUMAN (094768 ) ST17B MOUSE (Q8BG48) STK33 STK33 HUMAN (Q9BYT3 ) STK33 MOUSE (Q924X7)

STK40 STK40 HUMAN (Q8N2I9 ) STK40 MOUSE (Q7TNL3)

Smok2a SMK2A MOUSE (Q9QYZ6)

Smok2b SMK2B MOUSE (Q9QYZ3)

Smok3a SMK3A MOUSE (C0HKC8)

Smok3b SMK3B MOUSE (C0HKC9)

Stk-ps2 SMKW MOUSE (Q8C0V7)

TRIB1 TRIB1 HUMAN (Q96RU8 ) TRIB1 MOUSE (Q8K4K4)

TRIB2 TRIB2 HUMAN (Q92519 ) TRIB2 MOUSE (Q8K4K3)

TRIB3 TRIB 3 HUMAN (Q96RU7 ) TRIB3 MOUSE (Q8K4K2)

TRIO TRIO HUMAN (075962 ) TRIO MOUSE (Q0KL02)

TSSK1B TSSK1 HUMAN (Q9BXA7 ) TSSK1 MOUSE (Q61241)

TSSK2 TS SK2 HUMAN (Q96PF2 ) TSSK2 MOUSE (054863)

TSSK3 TSSK3 HUMAN (Q96PN8 ) TSSK3 MOUSE (Q9D2E1)

TSSK4 TSSK4 HUMAN (Q6SA08 ) TSSK4 MOUSE (Q9D411)

TSSK6 TS SK6 HUMAN (Q9BXA6 ) TSSK6 MOUSE (Q925K9)

TTN TITIN HUMAN (Q8WZ42 ) TITIN MOUSE (A2ASS6)

Tssk5 TSSK5 MOUSE (Q8C1R0)

Table 1C. CK1 Ser/Thr protein kinase family

CSNK1A1 KC 1 A HUM AN (P48729 ) KC1A MOUSE (Q8BK63) CSNK1A1L KC 1 AL HUMAN (Q8N752 )

CSNK1D KC 1 D HUMAN (P48730 ) KC1D MOUSE (Q9DC28) CSNK1E KC1E HUMAN (P49674 ) KC1E MOUSE (Q9JMK2) CSNK1G1 KC1G1 HUMAN (Q9HCP0 ) KC1G1 MOUSE (Q8BTH8) CSNK1G2 KC1G2 HUMAN (P78368 ) KC1G2 MOUSE (Q8BVP5) CSNK1G3 KC1G3 HUMAN (Q9Y6M4 ) KC1G3 MOUSE (Q8C4X2) TTBK1 TTBK1 HUMAN (Q5TCY1 ) TTBK1 MOUSE (Q6PCN3) TTBK2 TTBK2 HUMAN (Q6IQ55 ) TTBK2 MOUSE (Q3UVR3)

VRK1 VRK1 HUMAN (Q99986 ) VRK1 MOUSE (Q80X41)

VRK2 VRK2 HUMAN (Q86Y07 ) VRK2 MOUSE (Q8BN21)

VRK3 VRK3 HUMAN (Q8IV63 ) VRK3 MOUSE (Q8K3G5) Table ID. CMGC Ser/Thr protein kinase family

CDK1 CDK1 HUMAN (P06493 ) CDK1 MOUSE (P 11440) CDK10 CDK10 HUMAN (Q15131 ) CDKlO MOUSE (Q3UMM4)

CDK11A CD11A HUMAN (Q9UQ88 )

CDK11B CD11B HUMAN (P21127 ) CD11B MOUSE (P24788)

CDK12 CDK12 HUMAN (Q9NYV4 ) CDK12 MOUSE (Q14AX6) CDK13 CDKl3_HUMAN (Q 14004 ) CDK13 MOUSE (Q69ZA1) CDK14 CDK14 HUMAN (094921 ) CDK14 MOUSE (035495)

CDK15 CDK15 HUMAN (Q96Q40 ) CDK15 MOUSE (Q3V3A1) CDK16 CDK16 HUMAN (Q00536 ) CDK16 MOUSE (Q04735) CDK17 CDK17 HUMAN (Q00537 ) CDK17 MOUSE (Q8K0D0) CDK18 CDKl8_HUMAN (Q07002 ) CDKl8_MOUSE (Q04899) CDK19 CDK19 HUMAN (Q9BWU1 ) CDK19 MOUSE (Q8BWD8) CDK2 CDK2 HUMAN (P24941 ) CDK2 MOUSE (P97377)

CDK20 CDK20 HUMAN (Q8IZL9 ) CDK20 MOUSE (Q9JHU3) CDK3 CDK3 HUMAN (Q00526 ) CDK3 MOUSE (Q80YP0)

CDK4 CDK4 HUMAN (P11802 ) CDK4 MOUSE (P30285)

CDK5 CDK5 HUMAN (Q00535 ) CDK5 MOUSE (P49615)

CDK6 CDK6 HUMAN (Q00534 ) CDK6 MOUSE (Q64261)

CDK7 CDK7 HUMAN (P50613 ) CDK7 MOUSE (Q03147)

CDK8 CDK8 HUMAN (P49336 ) CDK8 MOUSE (Q8R3L8) CDK9 CDK9 HUMAN (P50750 ) CDK9 MOUSE (Q99J95) CDKL1 CDKL1 HUMAN (Q00532 ) CDKL1 MOUSE (Q8CEQ0) CDKL2 CDKL2 HUMAN (Q92772 ) CDKL2 MOUSE (Q9QUK0) CDKL3 CDKL3 HUMAN (Q8IVW4 ) CDKL3 MOUSE (Q8BLF2) CDKL4 CDKL4 HUMAN (Q5MAI5 ) CDKL4 MOUSE (Q3TZA2) CDKL5 CDKL5 HUMAN (076039 ) CDKL5 M0USE (Q3UTQ8) CLK1 CLK1 HUMAN (P49759 ) CLK1 M0USE (P22518)

CLK2 CLK2 HUMAN (P49760 ) CLK2 M0USE (035491)

CLK3 CLK3 HUMAN (P49761 ) CLK3 M0USE (035492)

CLK4 CLK4 HUMAN (Q9HAZ1 ) CLK4 M0USE (035493) DYRK1A DYR1A HUMAN (Q13627 ) DYR1A M0USE (Q61214) DYRK1B DYR1B HUMAN (Q9Y463 ) DYR1B MOUSE (Q9Z188)

DYRK2 DYRK2 HUMAN (Q92630 ) DYRK2 MOUSE (Q5U4C9) DYRK3 DYRK3 HUMAN (043781 ) DYRK3 MOUSE (Q922Y0) DYRK4 DYRK4 HUMAN (Q9NR20 ) DYRK4 MOUSE (Q8BI55) GSK3A GSK3A HUMAN (P49840 ) GSK3A MOUSE (Q2NL51)

GSK3B GSK3B HUMAN (P49841 ) GSK3B MOUSE (Q9WV60) HIPK1 HIPK1 HUMAN (Q86Z02 ) HIPK1 MOUSE (088904) HIPK2 HIPK2 HUMAN (Q9H2X6 ) HIPK2 MOUSE (Q9QZR5) HIPK3 HIPK3 HUMAN (Q9H422 ) HIPK3 MOUSE (Q9ERH7) HIPK4 HIPK4 HUMAN (Q8NE63 ) HIPK4 MOUSE (Q3V016) ICK ICK HUMAN (Q9UPZ9 ) ICK MOUSE (Q9JKV2) MAK MAK HUMAN (P20794 ) MAK MOUSE (Q04859) MAPK1 MKO 1 HUMAN (P28482 ) MKOl MOUSE (P63085) MAPK10 MK10 HUMAN (P53779 ) MKlO MOUSE (Q61831) MAPK11 MK11 HUMAN (Q15759 ) MK11 MOUSE (Q9WUI1) MAPK12 MK12 HUMAN (P53778 ) MK12 MOUSE (008911) MAPK13 MK13 HUMAN (015264 ) MK13 MOUSE (Q9Z1B7) MAPK14 MK14 HUMAN (Q16539 ) MK14 MOUSE (P47811) MAPK15 MK15 HUMAN (Q8TD08 ) MK15 MOUSE (Q80Y86) MAPK3 MK03 HUMAN (P27361 ) MK03 MOUSE (Q63844) MAPK4 MK04 HUMAN (P31152 ) MK04 MOUSE (Q6P5G0) MAPK6 MK06 HUMAN (Q 16659 ) MK06 MOUSE (Q61532) MAPK7 MK07 HUMAN (Q13164 ) MK07 MOUSE (Q9WVS8) MAPK8 MK08 HUMAN (P45983 ) MKO 8_MOU S E (Q91Y86) MAPK9 MK09 HUMAN (P45984 ) MK09 MOUSE (Q9WTU6) MOK MOK HUMAN (Q9UQ07 ) MOK MOUSE (Q9WVS4) NLK NLK HUMAN (Q9UBE8 ) NLK MOUSE (054949) PRPF4B PRP4B HUMAN (Q 13523 ) PRP4B MOUSE (Q61136) SRPK1 SRPK1 HUMAN (Q96SB4 ) SRPK1 MOUSE (070551) SRPK2 SRPK2 HUMAN (P78362 ) SRPK2 MOUSE (054781) SRPK3 SRPK3 HUMAN (Q9UPE1 ) SRPK3 MOUSE (Q9Z0G2) Table IE. NEK Ser/Thr protein kinase family

NEK1 NEK1 HUMAN (Q96PY6 ) NEK1 MOUSE (P51954) NEK10 NEK10 HUMAN (Q6ZWH5 ) NEK10 MOUSE (Q3UGM2) NEK11 NEK11 HUMAN (Q8NG66 ) NEK11 MOUSE (Q8C0Q4)

NEK2 NEK2 HUMAN (P51955 ) NEK2 MOUSE (035942)

NEK3 NEK3 HUMAN (P51956 ) NEK3 M0USE (Q9R0A5)

NEK4 NEK4 HUMAN (P51957 ) NEK4 M0USE (Q9Z1J2)

NEK5 NEK5 HUMAN (Q6P3R8 ) NEK5 M0USE (Q7TSC3)

NEK6 NEK6 HUMAN (Q9HC98 ) NEK6 M0USE (Q9ES70)

NEK7 NEK7 HUMAN (Q8TDX7 ) NEK7 M0USE (Q9ES74)

NEK8 NEK8 HUMAN (Q86SG6 ) NEK8 M0USE (Q91ZR4)

NEK9 NEK9 HUMAN (Q8TD19 ) NEK9 M0USE (Q8K1R7)

Table IF. STE Ser/Thr protein kinase family

MAP2K1 MP2K1 HUMAN (Q02750 ) MP2K1 M0USE (P31938) MAP2K2 MP2K2 HUMAN (P36507 ) MP2K2 MOUSE (Q63932)

MAP2K3 MP2K3 HUMAN (P46734 ) MP2K3 MOUSE (009110) MAP2K4 MP2K4 HUMAN (P45985 ) MP2K4 MOUSE (P47809)

MAP2K5 MP2K5 HUMAN (Q13163 ) MP2K5 MOUSE (Q9WVS7)

MAP2K6 MP2K6 HUMAN (P52564 ) MP2K6 MOUSE (P70236)

MAP2K7 MP2K7 HUMAN (014733 ) MP2K7 MOUSE (Q8CE90)

MAP3K1 M3K1 HUMAN (Q13233 ) M3K1 M0USE (P53349)

MAP3K10 M3KlO_HUMAN (Q02779 ) M3K10 MOUSE (Q66L42) MAP3K11 M3Kl l_HUMAN (Q16584 ) M3K11 M0USE (Q80XI6) MAP3K12 M3K12 HUMAN (Q12852 ) M3K12 MOUSE (Q60700) MAP3K13 M3K13 HUMAN (043283 ) M3K13 MOUSE (Q1HKZ5)

MAP3K14 M3K14 HUMAN (Q99558 ) M3K14 MOUSE (Q9WUL6)

MAP3K15 M3K15 HUMAN (Q6ZN16 ) M3K15 MOUSE (A2AQW0) MAP3K19 M3K19 HUMAN (Q56UN5 ) M3K19 MOUSE (E9Q3S4)

MAP3K2 M3K2 HUMAN (Q9Y2U5 ) M3K2 MOUSE (Q61083)

MAP3K20 M3K20 HUMAN (Q9NYL2 ) M3K20 MOUSE (Q9ESL4) MAP3K21 M3K21 HUMAN (Q5TCX8 ) M3K21 MOUSE (Q8VDG6) MAP3K3 M3K3 HUMAN (Q99759 ) M3K3 MOUSE (Q61084)

MAP3K4 M3K4 HUMAN (Q9Y6R4 ) M3K4 MOUSE (008648)

MAP3K5 M3K5 HUMAN (Q99683 ) M3K5 MOUSE (035099)

MAP3K6 M3K6 HUMAN (095382 ) M3K6 MOUSE (Q9WTR2)

MAP3K7 M3K7 HUMAN (043318 ) M3K7 MOUSE (Q62073)

MAP3K8 M3K8 HUMAN (P41279 ) M3K8 MOUSE (Q07174)

MAP3K9 M3K9 HUMAN (P80192 ) M3K9 MOUSE (Q3U1V8)

MAP4K1 M4K1 HUMAN (Q92918 ) M4Kl_MOUSE (P70218)

MAP4K2 M4K2 HUMAN (Q12851 ) M4K2 MOUSE (Q61161)

MAP4K3 M4K3 HUMAN (Q8IVH8 ) M4K3 MOUSE (Q99JP0)

MAP4K4 M4K4 HUMAN (095819 ) M4K4_MOUSE (P97820) MAP4K5 M4K5 HUMAN (Q9Y4K4 ) M4K5 MOUSE (Q8BPM2) MINK1 MINK1 HUMAN (Q8N4C8 ) MINK1 MOUSE (Q9JM52) MY03A MY 03 A HUMAN (Q8NEV4 ) MY03A M0USE (Q8K3H5) MY03B MY03B HUMAN (Q8WXR4 ) MY03B M0USE (Q1EG27) NRK NRK HUMAN (Q7Z2Y5 ) NRK MOUSE (Q9R0G8)

OXSR1 OXSR1 HUMAN (095747 ) OXSR1 MOUSE (Q6P9R2) PAK1 PAK1 HUMAN (Q13153 ) PAK1 MOUSE (088643)

PAK2 PAK2 HUMAN (Q13177 ) PAK2 MOUSE (Q8CIN4)

PAK3 PAK3 HUMAN (075914 ) PAK3 MOUSE (Q61036)

PAK4 PAK4 HUMAN (096013 ) PAK4 MOUSE (Q8BTW9)

PAK5 PAK5 HUMAN (Q9P286 ) PAK5 MOUSE (Q8C015)

PAK6 PAK6 HUMAN (Q9NQU5 ) PAK6 MOUSE (Q3ULB5)

PBK T OPK HUM AN (Q96KB5 ) TOPK MOUSE (Q9JJ78)

SLK SLK HUMAN (Q9H2G2 ) SLK MOUSE (054988)

STK10 STKlO_HUMAN (094804 ) STKlO_MOUSE (055098)

STK24 STK24 HUMAN (Q9Y6E0 ) STK24 MOUSE (Q99KH8)

STK25 STK25 HUMAN (000506 ) STK25 MOUSE (Q9Z2W1)

STK26 STK26 HUMAN (Q9P289 ) STK26 MOUSE (Q99JT2)

STK3 STK3 HUMAN (Q13188 ) STK3 MOUSE (Q9JI10)

STK39 STK39 HUMAN (Q9UEW8 ) STK39 MOUSE (Q9Z1W9) STK4 STK4 HUMAN (Q13043 ) STK4 MOUSE (Q9JI11)

STRADA STRAA HUMAN (Q7RTN6 ) STRAA MOUSE (Q3UUJ4) STRADB STRAB HUMAN (Q9C0K7 ) STRAB MOUSE (Q8K4T3)

TAOK1 TAOK1 HUMAN (Q7L7X3 ) TAOK1 MOUSE (Q5F2E8)

TAOK2 T AOK2 HUM AN (Q9UL54 ) TAOK2 MOUSE (Q6ZQ29) TAOK3 T AOK3 HUM AN (Q9H2K8 ) TAOK3 MOUSE (Q8BYC6) TNIK TNIK HUMAN (Q9UKE5 ) TNIK MOUSE (P83510)

Table 1G. TKL Ser/Thr protein kinase family

ACVR1 ACVR1 HUMAN (Q04771 ) ACVR1 MOUSE (P37172)

ACVR1B ACV1B HUMAN (P36896 ) ACV1B MOUSE (Q61271) ACVR1C ACV1C HUMAN (Q8NER5 ) ACV1C MOUSE (Q8K348) ACVR2A AVR2A HUMAN (P27037 ) AVR2A MOUSE (P27038)

ACVR2B AVR2B HUMAN (Q 13705 ) AVR2B MOUSE (P27040)

ACVRL1 ACVL1 HUMAN (P37023 ) ACVL1 MOUSE (Q61288)

AMHR2 AMHR2 HUMAN (Q 16671 ) AMHR2 MOUSE (Q8K592)

ANKK1 ANKK1 HUMAN (Q8NFD2 ) ANKK1 MOUSE (Q8BZ25) ARAF ARAF HUMAN (P 10398 ) ARAF MOUSE (P04627) BMPR1A BMR1A HUMAN (P36894 ) BMR1A MOUSE (P36895) BMPR1B BMR1B HUMAN (000238 ) BMR1B MOUSE (P36898) BMPR2 BMPR2 HUMAN (Q13873 ) BMPR2 MOUSE (035607)

BRAF BRAF HUMAN (P 15056 ) BRAF MOUSE (P28028)

ILK ILK HUMAN (Q13418 ) ILK MOUSE (055222)

IRAK1 IRAK1 HUMAN (P51617 ) IRAK1 MOUSE (Q62406) IRAK2 IRAK2 HUMAN (043187 ) IRAK2 MOUSE (Q8CFA1)

IRAK3 IRAK3 HUMAN (Q9Y616 ) IRAK3 MOUSE (Q8K4B2)

IRAK4 IRAK4 HUMAN (Q9NWZ3 ) IRAK4 MOUSE (Q8R4K2) KSR1 KSR1 HUMAN (Q8IVT5 ) KSR1 MOUSE (Q61097)

KSR2 KSR2 HUMAN (Q6VAB6 ) KSR2 MOUSE (Q3UVC0) LIMK1 LIMK 1 HUMAN (P53667 ) LIMK1 MOUSE (P53668)

LIMK2 LIMK2 HUMAN (P53671 ) LIMK2 MOUSE (054785)

LRRK1 LRRK1 HUMAN (Q38SD2 ) LRRK1 MOUSE (Q3UHC2) LRRK2 LRRK2 HUMAN (Q5S007 ) LRRK2 MOUSE (Q5S006) RAF1 RAF 1 HUMAN (P04049 ) RAF1 MOUSE (Q99N57)

RIPK1 RIPK1 HUMAN (Q 13546 ) RIPK1 MOUSE (Q60855) RIPK2 RIPK2 HUMAN (043353 ) RIPK2 MOUSE (P58801) RIPK3 RIPK3 HUMAN (Q9Y572 ) RIPK3 MOUSE (Q9QZL0) RIPK4 RIPK4 HUMAN (P57078 ) RIPK4 MOUSE (Q9ERK0) TESK1 TESK1 HUMAN (Q15569 ) TESK1 MOUSE (070146) TESK2 TESK2 HUMAN (Q96S53 ) TESK2 MOUSE (Q8VCT9)

TGFBR1 TGFR1 HUMAN (P36897 ) TGFR1 MOUSE (Q64729)

TGFBR2 TGFR2 HUMAN (P37173 ) TGFR2 MOUSE (Q62312)

TNNI3K TNI3K HUMAN (Q59H18 ) TNI3K MOUSE (Q5GIG6)

Table 1H. Tyr protein kinase family

AATK LMTK1 HUMAN (Q6ZMQ8 ) LMTK1 MOUSE (Q80YE4) ABL1 ABL1 HUMAN (P00519 ) ABL1 MOUSE (P00520)

ABL2 ABL2 HUMAN (P42684 ) ABL2 MOUSE (Q4JIM5)

ALK ALK HUMAN (Q9UM73 ) ALK MOUSE (P97793)

AXL UFO HUMAN (P30530 ) UFO MOUSE (Q00993)

BLK BLK HUMAN (P51451 ) BLK MOUSE (P16277)

BMX BMX HUMAN (P51813 ) BMX MOUSE (P97504)

BTK BTK HUMAN (Q06187 ) BTK MOUSE (P35991)

CSF1R CSF1R HUMAN (P07333 ) CSF1R MOUSE (P09581)

CSK CSK HUMAN (P41240 ) CSK MOUSE (P41241)

DDR1 DDR1 HUMAN (Q08345 ) DDR1 MOUSE (Q03146)

DDR2 DDR2 HUMAN (Q16832 ) DDR2 MOUSE (Q62371)

EGFR EGFR HUMAN (P00533 ) EGFR MOUSE (Q01279)

EPHA1 EPHA1 HUMAN (P21709 ) EPHA1 MOUSE (Q60750) EPHA10 EPHAA HUMAN (Q5JZY3 ) EPHAA MOUSE (Q8BYG9) EPHA2 EPHA2 HUMAN (P29317 ) EPHA2 MOUSE (Q03145)

EPHA3 EPHA3 HUMAN (P29320 ) EPHA3 MOUSE (P29319)

EPHA4 EPHA4 HUMAN (P54764 ) EPHA4 MOUSE (Q03137)

EPHA5 EPHA5 HUMAN (P54756 ) EPHA5 MOUSE (Q60629)

EPHA6 EPHA6 HUMAN (Q9UF33 ) EPHA6 MOUSE (Q62413)

EPHA7 EPHA7 HUMAN (Q 15375 ) EPHA7 MOUSE (Q61772)

EPHA8 EPHA8 HUMAN (P29322 ) EPHA8 MOUSE (009127)

EPHB1 EPHB1 HUMAN (P54762 ) EPHB1 MOUSE (Q8CBF3) EPHB2 EPHB2 HUMAN (P29323 ) EPHB2 MOUSE (P54763)

EPHB3 EPHB3 HUMAN (P54753 ) EPHB3 MOUSE (P54754)

EPHB4 EPHB4 HUMAN (P54760 ) EPHB4 MOUSE (P54761)

EPHB6 EPHB6 HUMAN (015197 ) EPHB6 MOUSE (008644)

ERBB2 ERBB2 HUMAN (P04626 ) ERBB2 MOUSE (P70424)

ERBB3 ERBB3 HUMAN (P21860 ) ERBB3 MOUSE (Q61526)

ERBB4 ERBB4 HUMAN (Q15303 ) ERBB4 MOUSE (Q61527)

FER FER HUMAN (P16591 ) FER MOUSE (P70451)

FES FES HUMAN (P07332 ) FES MOUSE (P16879)

FGFR1 FGFR1 HUMAN (P11362 ) FGFR1 MOUSE (P 16092)

FGFR2 FGFR2 HUMAN (P21802 ) FGFR2 MOUSE (P21803)

FGFR3 FGFR3 HUMAN (P22607 ) FGFR3 MOUSE (Q61851)

FGFR4 FGFR4 HUMAN (P22455 ) FGFR4 MOUSE (Q03142)

FGR FGR HUMAN (P09769 ) FGR MOUSE (P14234)

FLT1 VGFR1 HUMAN (P 17948 ) VGFR1 MOUSE (P35969)

FLT3 FLT3 HUMAN (P36888 ) FLT3 MOUSE (Q00342)

FLT4 VGFR3 HUMAN (P35916 ) VGFR3 MOUSE (P35917)

FRK FRK HUMAN (P42685 ) FRK MOUSE (Q922K9)

FYN FYN HUMAN (P06241 ) FYN MOUSE (P39688)

HCK HCK HUMAN (P08631 ) HCK MOUSE (P08103)

IGF1R IGF1R HUMAN (P08069 ) IGF1R MOUSE (Q60751)

INSR INSR HUMAN (P06213 ) INSR MOUSE (P15208)

INSRR INSRR HUMAN (P 14616 ) INSRR MOUSE (Q9WTL4)

ITK ITK HUMAN (Q08881 ) ITK MOUSE (Q03526)

JAK1 JAK1 HUMAN (P23458 ) JAK1 MOUSE (P52332)

JAK2 JAK2 HUMAN (060674 ) JAK2 MOUSE (Q62120)

JAK3 JAK3 HUMAN (P52333 ) JAK3 MOUSE (Q62137)

KDR VGFR2 HUMAN (P35968 ) VGFR2 MOUSE (P35918)

KIT KIT HUMAN (P10721 ) KIT MOUSE (P05532)

LCK LCK HUMAN (P06239 ) LCK MOUSE (P06240)

LMTK2 LMTK2 HUMAN (Q8IWU2 ) LMTK2 MOUSE (Q3TYD6) LMTK3 LMTK3 HUMAN (Q96Q04 ) LMTK3 MOUSE (Q5XJV6) LTK LTK HUMAN (P29376 ) LTK MOUSE (P08923)

LYN LYN HUMAN (P07948 ) LYN MOUSE (P25911) MATK MATK HUMAN (P42679 ) MATK MOUSE (P41242) MERTK MERTK HUMAN (Q12866 ) MERTK MOUSE (Q60805) MET MET HUMAN (P08581 ) MET MOUSE (P16056)

MST1R RON HUMAN (Q04912 ) RON MOUSE (Q62190) MUSK MUSK HUMAN (015146 ) MUSK MOUSE (Q61006) NTRK1 NTRK1 HUMAN (P04629 ) NTRK1 MOUSE (Q3UFB7) NTRK2 NTRK2 HUMAN (Q 16620 ) NTRK2 MOUSE (P 15209) NTRK3 NTRK3 HUMAN (Q 16288 ) NTRK3 MOUSE (Q6VNS1) PDGFRA PGFRA HUMAN (P 16234 ) PGFRA MOUSE (P26618) PDGFRB PGFRB HUMAN (P09619 ) PGFRB MOUSE (P05622) PTK2 FAK1 HUMAN (Q05397 ) FAK1 MOUSE (P34152)

PTK2B F AK2 HUM AN (Q 14289 ) FAK2 MOUSE (Q9QVP9)

PTK6 PTK6 HUMAN (Q 13882 ) PTK6 MOUSE (Q64434)

PTK7 PTK7 HUMAN (Q13308 ) PTK7 MOUSE (Q8BKG3)

RET RET HUMAN (P07949 ) RET MOUSE (P35546)

ROR1 ROR1 HUMAN (Q01973 ) ROR1 MOUSE (Q9Z139)

ROR2 ROR2 HUMAN (Q01974 ) ROR2 MOUSE (Q9Z138)

ROS1 ROS1 HUMAN (P08922 ) ROS1 MOUSE (Q78DX7)

RYK RYK HUMAN (P34925 ) RYK MOUSE (Q01887)

SRC SRC HUMAN (P 12931 ) SRC MOUSE (P05480)

SRMS SRMS HUMAN (Q9H3Y6 ) SRMS MOUSE (Q62270) STYK1 STYK1 HUMAN (Q6J9G0 ) STYK1 MOUSE (Q6J9G1) SYK KSYK HUMAN (P43405 ) KSYK MOUSE (P48025)

Smokl SMOK1 MOUSE (Q9QYZ4)

Smokier SMKTR MOUSE (A2KF29)

TEC TEC HUMAN (P42680 ) TEC MOUSE (P24604)

TEK TIE2 HUMAN (Q02763 ) TIE2 MOUSE (Q02858)

TIE1 TIE 1 HUMAN (P35590 ) TIE 1 MOUSE (Q06806)

TNK1 TNK1 HUMAN (Q13470 ) TNK1 MOUSE (Q99ML2)

TNK2 ACK1 HUMAN (Q07912 ) ACK1 MOUSE (054967)

TXK TXK HUMAN (P42681 ) TXK MOUSE (P42682)

TYK2 TYK2 HUMAN (P29597 ) TYK2 MOUSE (Q9R117) TYR03 TYR03 HUMAN (Q06418 ) TYR03 MOUSE (P55144) YES1 YES HUMAN (P07947 ) YES_MOUSE (Q04736) ZAP 70 Z AP7 O HUMAN (P43403 ) ZAP70_MOUSE (P43404)

Table II. Other kinases.

AAK1 AAK1 HUMAN (Q2M2I8 ) AAK1 MOUSE (Q3UHJ0)

AURKA AURKA HUMAN (014965 ) AURKA MOUSE (P97477)

AURKB AURKB HUMAN (Q96GD4 ) AURKB MOUSE (070126) AURKC AURKC HUMAN (Q9UQB9 ) AURKC MOUSE (088445) BMP2K BMP2K HUMAN (Q9NSY1 ) BMP2K MOUSE (Q91Z96) BUB1 BUB 1 HUMAN (043683 ) BUB1 MOUSE (008901)

BUB1B BUB1B HUMAN (060566 ) BUB1B MOUSE (Q9Z1S0) CAMKK1 KKCC1 HUMAN (Q8N5S9 ) KKCC1 MOUSE (Q8VBY2) CAMKK2 KKCC2 HUMAN (Q96RR4 ) KKCC2 MOUSE (Q8C078) CDC7 CDC7 HUMAN (000311 ) CDC7 MOUSE (Q9Z0H0)

CHUK IKKA HUMAN (015111 ) IKKA MOUSE (Q60680)

CSNK2A1 CSK2l_HUMAN (P68400 ) CSK2l_MOUSE (Q60737) CSNK2A2 CSK22_HUMAN (P 19784 ) CSK22 MOUSE (054833) CSNK2A3 CSK23 HUMAN (Q8NEV1 )

DSTYK DUSTY HUMAN (Q6XUX3 ) DUSTY MOUSE (Q6XUX1) EIF2AK1 E2AK1 HUMAN (Q9BQI3 ) E2AK1 MOUSE (Q9Z2R9) EIF2AK2 E2AK2 HUMAN (P 19525 ) E2AK2 MOUSE (Q03963)

EIF2AK3 E2AK3 HUMAN (Q9NZJ5 ) E2AK3 MOUSE (Q9Z2B5) EIF2AK4 E2AK4 HUMAN (Q9P2K8 ) E2AK4 MOUSE (Q9QZ05) ERN1 ERN 1 HUMAN (075460 ) ERN1 MOUSE (Q9EQY0)

ERN2 ERN2 HUMAN (Q76MJ5 ) ERN2 MOUSE (Q9Z2E3)

GAK GAK HUMAN (014976 ) GAK MOUSE (Q99KY4) HASPIN HASP HUMAN (Q8TF76 ) HASP MOUSE (Q9Z0R0) IKBKB IKKB HUMAN (014920 ) IKKB MOUSE (088351) IKBKE IKKE HUMAN (Q14164 ) IKKE MOUSE (Q9R0T8)

MLKL MLKL HUMAN (Q8NB16 ) MLKL MOUSE (Q9D2Y4) MOS MOS HUMAN (P00540 ) MOS MOUSE (P00536)

NRBP1 NRBP HUMAN (Q9UHY1 ) NRBP MOUSE (Q99J45) NRBP2 NRBP2 HUMAN (Q9NSY0 ) NRBP2 MOUSE (Q91V36) PAN3 PAN 3 HUMAN (Q58A45 ) PAN3 MOUSE (Q640Q5) PDIK1L PDK1L HUMAN (Q8N165 ) PDK1L MOUSE (Q8QZR7) PEAK1 PEAK1 HUMAN (Q9H792 ) PEAK1 MOUSE (Q69Z38) PIK3R4 PI3R4 HUMAN (Q99570 ) PI3R4 MOUSE (Q8VD65) PINK1 PINK1 HUMAN (Q9BXM7 ) PINK1 MOUSE (Q99MQ3) PKDCC PKDCC HUMAN (Q504Y2 ) PKDCC MOUSE (Q5RJI4) PKMYT1 PMYT 1 HUMAN (Q99640 ) PMYT1 MOUSE (Q9ESG9) PLK1 PLK1 HUMAN (P53350 ) PLK1 MOUSE (Q07832)

PLK2 PLK2 HUMAN (Q9NYY3 ) PLK2 MOUSE (P53351)

PLK3 PLK3 HUMAN (Q9H4B4 ) PLK3 MOUSE (Q60806)

PLK4 PLK4 HUMAN (000444 ) PLK4 MOUSE (Q64702)

PLK5 PLK5 HUMAN (Q496M5 ) PLK5 MOUSE (Q4FZD7)

POMK SG196 HUMAN (Q9H5K3 ) SG196 MOUSE (Q3TUA9) PRAG1 PRAG1 HUMAN (Q86YV5 ) PRAG1 MOUSE (Q571I4) PXK PXK HUMAN (Q7Z7A4 ) PXK MOUSE (Q8BX57) RNASEL RN5A HUMAN (Q05823 ) RN5A MOUSE (Q05921) RPS6KC1 KS6Cl_HUMAN (Q96S38 ) KS6C1 MOUSE (Q8BLK9) RPS6KL1 RPKL 1 HUMAN (Q9Y6S9 ) RPKL1 MOUSE (Q8R2S1) SBK1 SBK1 HUMAN (Q52WX2 ; I SBK1 MOUSE (Q8QZX0) SBK2 SBK2 HUMAN (P0C263 ) SBK2 MOUSE (P0C5K1)

SBK3 SBK3 HUMAN (P0C264 ) SBK3 MOUSE (P0C5K0)

SCYL1 S C YL 1 HUMAN (Q96KG9 ) SCYL1 MOUSE (Q9EQC5) SCYL2 S C YL2 HUMAN (Q6P3W7 ) SCYL2 MOUSE (Q8CFE4) SCYL3 PACE1 HUMAN (Q8IZE3 ) PACE1 MOUSE (Q9DBQ7) SGK494 SG494 HUMAN (Q96LW2 ) SG494 MOUSE (Q5SYL1) STK16 STK16 HUMAN (075716 ) STK16 MOUSE (088697) STK31 STK31 HUMAN (Q9BXU 1 ) STK31 MOUSE (Q99MW1) STK32A ST32A HUMAN (Q8WU08 ) ST32A MOUSE (Q8BGW6) STK32B ST32B HUMAN (Q9NY57 ) ST32B MOUSE (Q9JJX8) STK32C ST32C HUMAN (Q86UX6 ) ST32C MOUSE (Q8QZV4) STK35 STK35 HUMAN (Q8TDR2 ) STK35 MOUSE (Q80ZW0) STK36 STK36 HUMAN (Q9NRP7 ) STK36 MOUSE (Q69ZM6) STKLD1 S TKL 1 HUMAN (Q8NE28 ) STKL1 MOUSE (Q80YS9) TBCK TBCK HUMAN (Q8TEA7 ) TBCK MOUSE (Q8BM85) TBK1 TBK1 HUMAN (Q9UHD2 ) TBK1 MOUSE (Q9WUN2) TEX 14 TEX14 HUMAN (Q8IWB6 ) TEX14 MOUSE (Q7M6U3) TLK1 TLK1 HUMAN (Q9UKI8 ) TLK1 MOUSE (Q8C0V0)

TLK2 TLK2 HUMAN (Q86UE8 ) TLK2 MOUSE (055047)

TP53RK PRPK HUMAN (Q96S44 ) PRPK MOUSE (Q99PW4) TTK TTK HUMAN (P33981 ) TTK MOUSE (P35761)

UHMK1 UHMK1 HUMAN (Q8TAS1 ) UHMK1 MOUSE (P97343) ULK1 ULK1 HUMAN (075385 ) ULK1 MOUSE (070405)

ULK2 ULK2 HUMAN (Q8IYT8 ) ULK2 MOUSE (Q9QY01)

ULK3 ULK3 HUMAN (Q6PHR2 ) ULK3 MOUSE (Q3U3Q1)

ULK4 ULK4 HUMAN (Q96C45 ) ULK4 MOUSE (Q3V129)

WEE1 WEE 1 HUMAN (P30291 ) WEE1 MOUSE (P47810)

WEE2 WEE2 HUMAN (P0C1S8 ) WEE2 MOUSE (Q66JT0)

WNK1 WNK1 HUMAN (Q9H4A3 ) WNK1 MOUSE (P83741)

WNK2 WNK2 HUMAN (Q9Y3S1 ) WNK2 MOUSE (Q3UH66)

WNK3 WNK3 HUMAN (Q9BYP7 ) WNK3 MOUSE (Q80XP9)

WNK4 WNK4 HUMAN (Q96J92 ) WNK4 MOUSE (Q80UE6)

Table 1J. ADCK protein kinase family

ADCK1 ADCK1 HUMAN (Q86TW2 ) ADCK1 MOUSE (Q9D0L4) ADCK2 AD CK2 HUMAN (Q7Z695 ) ADCK2 MOUSE (Q6NSR3)

ADCK5 AD CK5 HUMAN (Q3MIX3 ) ADCK5 MOUSE (Q80V03) COQ8A COQ8A HUMAN (Q8NI60 ) COQ8A MOUSE (Q60936) COQ8B COQ8B HUMAN (Q96D53 ) COQ8B MOUSE (Q566J8)

Table IK. Alpha-type protein kinase family

ALPK1 ALPK1 HUMAN (Q96QP1 ) ALPK1 MOUSE (Q9CXB8)

ALPK2 ALPK2 HUMAN (Q86TB3 ) ALPK2 MOUSE (Q91ZB0)

ALPK3 ALPK3 HUMAN (Q96L96 ) ALPK3 MOUSE (Q924C5) EEF2K EF2K HUMAN (000418 ) EF2K MOUSE (008796)

TRPM6 TRPM6 HUMAN (Q9BX84 ) TRPM6 MOUSE (Q8CIR4) TRPM7 TRPM7 HUMAN (Q96QT4 ) TRPM7 MOUSE (Q923J1) Table 1L. FAST protein kinase family

FASTK FASTK HUMAN (Q 14296 ) FASTK MOUSE (Q9JIX9)

Table 1M. PDK/BCKDK protein kinase family

BCKDK B CKD HUM AN (014874 ) BCKD MOUSE (055028)

PDK1 PDK1 HUMAN (Q15118 ) PDK1 MOUSE (Q8BFP9)

PDK2 PDK2 HUMAN (Q15119 ) PDK2 MOUSE (Q9JK42)

PDK3 PDK3 HUMAN (Q15120 ) PDK3 MOUSE (Q922H2)

PDK4 PDK4 HUMAN (Q16654 ) PDK4 MOUSE (070571)

Table IN. PI3/PI4-kinase family

ATM ATM HUMAN (Q13315 ) ATM MOUSE (Q62388)

ATR ATR HUMAN (Q13535 ) ATR MOUSE (Q9JKK8)

MTOR MTOR HUMAN (P42345 ) MTOR MOUSE (Q9JLN9)

PIK3CA PK3 C A HUM AN (P42336 ) PK3CA MOUSE (P42337)

PIK3CG PK3CG HUMAN (P48736 ) PK3CG MOUSE (Q9JHG7)

PRKDC PRKDC HUMAN (P78527 ) PRKDC MOUSE (P97313)

SMG1 SMG1 HUMAN (Q96Q15 ) SMG1 MOUSE (Q8BKX6)

Table lO. RIO-type Ser/Thr kinase family

RIOK1 RIOK1 HUMAN (Q9BRS2 ) RIOK1 MOUSE (Q922Q2)

RIOK2 RIOK2 HUMAN (Q9BVS4 ) RIOK2 MOUSE (Q9CQS5)

RIOK3 RIOK3 HUMAN (014730 ) RIOK3 MOUSE (Q9DBU3)

EXPERIMENTAL

All starting materials and reagents were purchased from commercial sources and used as received unless indicated otherwise. Abbreviations used in the examples that follow include the following: Boc is tert-butyloxy carbonyl; DCM is dichloromethane; DMF is N.N- dimethylformamide; ESI is electrospray ionization; HATU is 1- |bis(dimethylamino)methylene|- 1//- 1.2.3-tria/olo|4.5-/ |pyridinium 3-oxid hexafluorophosphate; HPLC is high-performance liquid chromatography; MS is mass spectrometry; RT is room temperature; and TFA is trifluoroacetic acid.

Example 1

Tert-buty\ (15-(4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyr imidin-

2 yl)amino)phenyl)piperazin-l-yl)- 15-oxo-3,6,9,12- tetraoxapentadecyl)carbamate

Compound CC-1290

2-((5-chloro-2-((4-(piperazin-l-yl)phenyl)amino)pyrimidin -4-yl)amino)-N-methylbenzamide (200.0 mg, 0.46 mmol) and 2,2-dimethyl-4-oxo-3,8,l l,l4,l7-pentaoxa-5-azaicosan-20-oic acid (250.0 mg, 0.69 mmol) and HATU (208.4 mg, 0.55 mmol) were taken up in DMF (5 mL), and /V./V-diisopropylethylamine (238.7 pL, 1.37 mmol) was added. The mixture was stirred for 3 hrs, and volatiles removed under reduced pressure. The crude was taken up in a minimum volume of dichloromethane, absorbed on Celite, and dried to a free flowing solid. The mixture was subjected to silica gel flash chromatography giving the desired product (192.0 mg, 53.3%) as a white solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C38H54CIN8O8: 785.38, found 785.60.

Example 2

Compound CC-1294

7cT/ -butyl (l5-(4-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyr imidin-2- yl)amino)phenyl)piperazin-l -yl)- 15-oxo-3,6,9, 12-tetraoxapentadecyl)carbamate (125.0 mg, 0.16 mmol) in DCM (2.0 mL) was treated with 100 uL of triisopropylsilane followed by trifluoroacetic acid (2 mL). The mixture was capped, allowed to stir for 3 hrs, and volatiles removed under reduced pressure to give a yellow oil. The mixture was dried under high vacuum, and the residue treated with diethyl ether to give a yellow solid. The diethylether was decanted, and the residue dried under high vacuum for 30 min. The crude was taken up in anhydrous DMF (5 mL) with stirring and treated with N. V- di i s o p ro py 1 e th y 1 am i n e (277 pL, 1.59 mmol). The reaction was allowed to stir for 10 min, NanoBRET™ 590 SE tracer (Promega Corporation; 74.5 mg, 0.17 mmol) added, and the reaction stirred in the dark for 2 hrs. Volatiles were removed under reduced pressure giving a blue film that was purified by amine-modified silica gel flash chromatography to give the product (118.5 mg 74.8%) as a blue solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C49H58BCIF2N11O7: 996.46, found 996.4.

Example 3

Tert-butyl (15-(4-(3-aminophenyl)piperazin-l-yl)-15-oxo-3,6,9,12

tetraoxapentadecyl)carbamate

Compound CC-1815

2,5-dioxopyrrolidin- 1 -y 1 2,2-dimethyl-4-oxo-3 ,8,11,14,17 -pentaoxa-5 -azaicosan-20-oate (525.0 mg, 1.14 mmol) and 3-(piperazin-l-yl)aniline (200.0 mg, 1.13 mmol) were dissolved in dichloromethane (10.0 mL) giving a yellow suspension. V.V-di isopropyl ethyl amine (590 pL, 3.39 mmol) was added, and the mixture stirred for 2 hrs. The crude mixture was absorbed on Celite, volatiles removed under reduced pressure, and the crude subjected to silica gel flash chromatography giving the product (590.0 mg, 100%) as a yellow solid.

Example 4

2-((2-((3-(4-(l-amino-3,6,9,12-tetraoxapentadecan-15-oyl)pip erazin-l- yl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)benzamide

Compound CC-1816

2-((2,5-dichloropyrimidin-4-yl)amino)benzamide (294.3 mg, 1.04 mmol) and /e/V-butyl (15- (4-(3-aminophenyl)piperazin-l-yl)-l5-oxo-3,6,9,l2-tetraoxape ntadecyl)carbamate (600.0 mg, 1.14 mmol) in 2-methoxy ethanol (8 mL) were heated to l00°C overnight. The mixture was cooled to RT, and volatiles removed under reduced pressure giving a red solid that was dried under high vacuum. The Boc-deprotected crude residue was dissolve in a minimum volume of dichloromethane, absorbed on Celite, dried to a free flowing solid, and purified by silica gel flash chromatography to give the desired product (270.0 mg, 33.7%) as a red-yellow solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C32H44CIN8O6 671.31 found 671.30.

Example 5

Compound CC-1817

2-((2-((3-(4-( 1 -amino-3 ,6,9, 12-tetraoxapentadecan- 15 -oy l)piperazin- 1 -y l)pheny l)amino)-5 - chloropyrimidin-4-yl)amino)benzamide (250.0 mg, 0.037 mmol) was taken up in anhydrous DMF (5 mL) with stirring. To the mixture, /V./V-diisopropylethylamine (22.2 pL, 0.03 mmol) was added, and the reaction allowed to stir for 10 min. NanoBRET™ 590 SE tracer

(Promega Corp.; 17.5 mg, 0.038 mmol) was added, and the reaction stirred in the dark for 2 hrs. Volatiles were removed under reduced pressure giving a blue film that was taken up in 8 mL of 1 : 1 :0.01 acetonitrile, water, trifluoroacetic acid and subjected to reverse phase preparative HPLC purification to give the product (12.0 mg 32.8%) as a blue solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C48H56BCIF2N11O7: 982.41, found 982.67.

Example 6

Tert-butyl 4-(4-((4-((2-carbamoylphenyl)amino)-5-chloropyrimidin-2- yl)amino)phenyl)piperazine-l-carboxylate

Compound CC-1790

2-((2,5-dichloropyrimidin-4-yl)amino)benzamide (500 mg, 1.77 mmol) and /e/V-butyl 4-(4- aminophenyl)piperazine-l-carboxylate (583.8, 1.94 mmol) in 2-methoxy ethanol (6 mL) were heated to l00°C overnight. The mixture was cooled to RT, and volatiles removed under reduced pressure to give a brown solid that was dried under high vacuum. The crude residue was dissolved in a minimum volume of dichloromethane, absorbed on Celite, and dried to a free flowing solid. The mixture was purified by silica gel flash chromatography to give the desired product (200.0 mg, 21.6%) as a yellow solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C26H31CIN7O3: 524.22, found 524.19.

Example 7

2-((5-chloro-2-((4-(piperazin-l-yl)phenyl)amino)pyrimidin-4- yl)amino)benzamide-TFA

Compound CC-1795

7cT/-butyl 4-(4-((4-((2-carbamoylphenyl)amino)-5-chloropyrimi din-2- yl)amino)phenyl)piperazine-l-carboxylate (200.0 mg, 0.38 mmol) in DCM (3.0 mL) was treated with 300 pL of triisopropylsilane followed by trifluoroacetic acid (3 mL). The mixture was capped, allowed to stir for 4 hrs, and volatiles removed under reduced pressure to give a purple oil. The mixture was dried under high vacuum, and the residue treated with diethyl ether, giving a dark solid. Ether was decanted, and an additional portion of diethyl ether added and decanted before drying the residue under hi vacuum overnight. The resulting solid was dissolved in 1: 1 :0.01 acetonitrile, water, trifluoroacetic acid and subjected to reverse phase-preparative HPLC purification to give the desired product (136.0 mg, 84.1%) as a yellow solid. Example 8

tert-butyl (15-(4-(4-((4-((2-carbamoylphenyl)amino)-5-chloropyrimidin-2 - yl)amino)phenyl)piperazin-l-yl)- 15-oxo-3,6,9, 12-tetraoxapentadecyl)carbamate

Compound CC-1796

2-((5-chloro-2-((4-(piperazin-l-yl)phenyl)amino)pyrimidin -4-yl)amino)benzamide (100.0 mg, 0.24 mmol), 2,5-dioxopyrrolidin-l-yl 2,2-dimethyl-4-oxo-3,8,l l,l4,l7-pentaoxa-5- azaicosan-20-oate (163.7 mg, 353.9 mmol) and /V^V-diisopropylethylamine (127.3 pL, 0.71 mmol) in dichloromethane (10 mL) were stirred for 2 hrs. The crude was absorbed on Celite, dried to a free flowing solid, and the mixture subjected to silica gel flash chromatography to give the desired product (156.0 mg, 85.7%) as a yellow solid. MS (ESI+) m/z calc’d for [M+H] ⁺ CsvffeClNsOs: 771.38, found 771.35.

Example 9

2-((2-((4-(4-(l-amino-3,6,9,12-tetraoxapentadecan-15-oyl)pip erazin-l-yl)phenyl)amino)-

5-chloropyrimidin-4-yl)amino)benzamide-TFA

Compound CC-1803

Tert-butyl (l5-(4-(4-((4-((2-carbamoylphenyl)amino)-5-chloropyrimi din-2- yl)amino)phenyl)piperazin-l-yl)-l5-oxo-3,6,9,l2-tetraoxapent adecyl)carbamate (100.0 mg, 0.13 mmol) in DCM (3.0 mL) was treated with 300 pL of triisopropylsilane followed by trifluoroacetic acid (3 mL). The mixture was capped and allowed to stir for 1 hr. Volatiles were removed under reduced pressure, and the mixture dried under high vacuum. The residue was treated with diethyl ether, giving a red solid, was decanted and an additional portion of diethyl ether was added and decanted before drying the residue under hi vacuum overnight. The resulting solid was dissolved in 1: 1 :0.01 acetonitrile, water, trifluoroacetic acid and subjected to reverse phase-preparative HPLC, affording the desired product (67.9 mg, 66.7%) as a yellow solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C32H44CIN8O6: 671.31, found 671.35.

Example 10

Compound CC-1804

2-((2-((4-(4-( 1 -amino-3 ,6,9, 12-tetraoxapentadecan- 15 -oy l)piperazin- 1 -y l)pheny l)amino)-5 - chloropyrimidin-4-yl)amino)benzamide-TFA (20.0 mg, 0.03 mmol) was taken up in 5 ml anhydrous DMF with stirring. To the mixture, AOV-d i i s 0 p ro p y 1 e th y 1 am i n e (22.2 pL, 0.03 mmol) was added, and the reaction allowed to stir for 10 min. NanoBRET™ 590 SE tracer (Promega Corp.; 16.3 mg, 0.038 mmol) was added, and the reaction stirred in the dark for 90 min. Volatiles were removed under reduced pressure giving a blue film that was taken up in 8 mL of 1: 1 :0.01 acetonitrile, water, trifluoroacetic acid and subjected to reverse phase preparative HPLC purification to give the product (24.8 mg, 99.0%) as a blue solid. MS (ESI+) m/z calc’d for [M+H] ⁺ C48H57BC1F2N11O7: 983.31, found 983.42. Example 11

Experiments were conducted during development of embodiments herein to derive a comprehensive analysis of intracellular kinase target engagement using NanoBRET™ tracers derived from CC-1294 versus NanoBRET™ tracers derived from novel inhibitors. In wells of 96-well plates, 20,000 HEK293 cells/well were transfected with pFN3lK and pFN32K plasmids for expression of kinase/NanoLuc® (Nluc) fusion proteins. Transfections were performed using 3: 1 FuGENE HD:plasmid ratios. 24 hours post transfection, cells were treated for 2 hours in the presence of 0.5uM CC-1804, CC-1852, or 1 uM CC-1294. After incubation, NanoBRET™-Target Engagement (TE) substrate/inhibitor solution was added to a final concentration of IX. NanoBRET was measured on a Glomax® Discover plate reader. To determine relative BRET signals, BRET arising from the tracer was divided by BRET in the absence of tracer. As demonstrated in Figure 1 A-B, unexpectedly broad spectrum target engagement/kinase profiling was observed with CC-1804 and CC-1852 compared to CC- 1294.

Example 12

Experiments were conducted during development of embodiments herein to demonstrate novel live cell target engagement assays uniquely enabled by CC-1804, which are not enabled by CC-1294. In wells of 96-well plates, 20,000 HEK293 cells/well were transfected with pFN3lK and pFN32K plasmids for expression of kinase/NanoLuc® (Nluc) fusion proteins. Transfections were performed using 3: 1 FuGENE HD:plasmid ratios. 24 hours post transfection, cells were treated for 2 hours in the presence of varying concentrations of tracer and varying concentrations of control inhibitor as indicated in Figure 2A-C. After incubation, NanoBRET™-TE substrate/inhibitor solution was added to a final concentration of IX, and BRET was measured on a Glomax® Discover plate reader.

Example 13

Experiments were conducted during development of embodiments herein to analyze the broad-spectrum intracellular kinase target engagement of parental molecules CC-1861 versus CTx-0294885 (Figure 3). In wells of 96-well plates, 20,000 HEK293 cells per well were transfected with kinase/Nluc fusions expressed from pFN3lK and pFN32K plasmids.

Transfections were performed using 3: 1 FuGENE HD:plasmid ratios. 24 hours post transfection, cells were treated for 2 hours in the presence of NanoBRET™ tracers (K4, K5, K7, K8, K9, K10, Kl 1) introduced at optimized concentrations in the presence of test compounds 300 nM CC-1861, 300 nM CTx-0294885, or 0.1% DMSO (vehicle). Analysis was also performed in using untagged Nluc to determine zero BRET (full occupancy control sample). After incubation, NanoBRET™-TE substrate/inhibitor solution was added to a final concentration of IX. NanoBRET was measured on a Glomax® Discover plate reader. To generate raw BRET ratio values, the acceptor emission value (e.g. 6l0nm) was divided by the donor emission value (e.g. 450nm) for each sample.

Determine Fractional Occupancy with the following equation;

% Occupancy = [1 - (X - Z)/( Y - Z)]*l00 where X = BRET in the presence of the test compound and tracer, Y = BRET in the presence of tracer only (zero occupancy control), and Z = BRET for the full occupancy control (ie., BRET from the untagged Nluc fusion).

Of the 200 kinases evaluated 71 kinases preferred CC-1861 to CTx; only 4 kinase preferred CTx over CC-1861 (Figure 5).

Example 14

Experiments were conducted during development of embodiments herein to demonstrate the increased kinase affinity of unmodified CC-1861 versus CTx-0294885. In wells of 96-well plates, 20,000 HEK293 cells per well were transfected with kinase/Nluc fusions expressed from pFN31K and pFN32K plasmids. Transfections were performed using 3 : 1 FuGENE HD:plasmid ratios. 24 hours post transfection, cells were treated for 2 hours in the presence of tracer and varying concentration of control inhibitor indicated. After incubation,

NanoBRET™-TE substrate/inhibitor solution was added to a final concentration of IX, and BRET was measured on a Glomax® Discover plate reader. CC-1861 shows increased inhibitor potency compared to CTx (Figure 7). CC-1861 had greater than 0.5 log shift in potency improvement compared to the CTx-0294885 for many of the kinases screened thus demonstrating CC-1861 as an improved broad-spectrum kinase binding agent (Figure 8).

Example 15

2-((5-chloro-2-((4-(piperazin-l-yl)phenyl)amino)pyrimidin-4- yl)amino)benzamide

Compound CC-1861

Tert-butyl 4-(4-((4-((2-carbamoylphenyl)amino)-5-chloropyrimidin-2- yl)amino)phenyl)piperazine-l-carboxylate (90.0 mg, 0.17 mmol) in DCM (2.0 mL) was treated with triisopropylsilane (200 uL, 0.17 mmol) followed by trifluoroacetic acid (2.0 mL). The capped mixture was stirred for 3 hrs, and volatiles removed under reduced pressure to give a yellow oil. The residue was treated with 20 mL diethyl ether giving a white solid. The ether was decanted, and the resulting white solid triturated with a 20 mL portion of fresh diethyl ether. The ether was decanted, and the resulting white solid dried under high vacuum for 20 min. The residue was dissolved in methanol (10 mL) and treated with 45 mg of potassium carbonate. The resulting mixture was stirred for 1 hr, absorbed on celite, and subjected to flash silica gel chromatography using a gradient of methanol in DCM. Product containing fractions were pooled and concentrated to give the product (50.0 mg, 68.7%) as a white solid. MS (ESI+) m/z calc’d for [M+H]+ C21H22C1N70: 424.17, found 424.24.