NOVEL CYCLOTIDES WITH ANTICANCER ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 61/638,433, filed April 25, 2012, the content of which is incorporated by reference in its entirety into the current disclosure.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. R01 GM090323 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] Since the discovery of its powerful growth suppressive and pro-apoptotic activity, the tumor suppressor p53 has been in the centre of attention of drug hunters (Weber (2010) Expert Opin Ther Pat 20(2): 179-91). The idea of unleashing the destructive powers of p53 inside cancer cells has become even more attractive after the realization that p53 is controlled largely by a single master regulator, Mdm2, which binds the tumor suppressor and negatively modulates its activity and stability. Therefore, Mdm2 antagonists able to release p53 from the inhibitory grip of Mdm2 are expected to stabilize and activate the tumor suppressor, leading to cell cycle arrest or programmed cell death (apoptosis) of cancer cells. Such antagonists could represent a novel modality to treat tumors in which p53 has retained its wild-type structure and function. Targeting the physical interaction between p53 and Mdm2 has been regarded as the most direct of all p53 -activating strategies.

[0004] Human Mdm2 is a 491 -amino acid (aa)-long phosphoprotein that interacts through its NH ₂ terminal domain with an a-helix present in the NH ₂ terminal transactivation domain of p53 (Kussie, P.H. et al. (1996) Science 274:948-953). This entails several negative effects on p53. Mdm2 binding to the NH ₂ terminal transactivation domain of p53 blocks its transcriptional activity directly (Oliner, J.D. et al. (1993) Nature 362:857-860; Chen, J. et al. (1993) Mol Cell Biol 13:4107-4114). More importantly, Mdm2 through the complex with mdmx functions as the E3 ligase that ubiquitinates p53 for proteasome degradation (Haupt, Y. et al. (1997) Nature 387:296-299; Kubbutat, M.H. et al. (1997) Nature 387:299-303; Wang, X. (2011) Cell Cycle 10:4225-4229). The biochemical basis of Mdm2-mediated inhibition of p53 function was further elucidated by crystallographic data that showed that the amino terminal domain of Mdm2 forms a deep hydrophobic cleft into which the

transactivation domain of p53 binds, thereby concealing itself from interaction with the transcriptional machinery (Kussie, P.H. et al. (1996) Science 274:948-953). The direct interaction between the two proteins has been localized to a relatively small (aa 25-109) hydrophobic pocket domain at the NH ₂ terminus of Mdm2 and a 15-aa amphipathic peptide at the NH ₂ terminus of p53 (Kussie, P.H. et al. (1996) Science 274:948-953; Chen, J. et al. (1993) Mol Cell Biol 13:4107-4114). The minimal Mdm2-binding site on the p53 protein was subsequently mapped within residues 18-26 (Chen, J. et al. (1993) Mol Cell Biol 13:4107- 4114; Bottger, A. et al. (1997) J Mol Biol 269:744-756).

[0005] Several peptides based on the N-terminal domain of p53 have shown to inhibit in a very efficient way the interaction between p53 and Mdm2 and Mdmx (Bernal et al. (2010) Cancer Cell 18(5):411-2; Li et al. (2009) Angew Chem Int Ed Engl 48(46): 8712-5; Pazgier et al. (2009) Proc Natl Acad Sci USA 106(12):4665-70; Li et al. (2010) J Mol Biol 398(2):200- 13). Unlike small molecule drugs, peptide and protein-based therapeutics can target with high selectivity and specificity defective protein-protein interactions involved in human disease. Despite their success, however, there are still numerous stability and delivery issues associated with their use as therapeutic agents. For example, monoclonal antibodies (one the most successful protein-based therapeutics with several blockbuster drugs on the market and many more in clinical development) can only target extracellular molecular targets due to their inability to cross biological membranes. They are also extremely expensive to produce and are not bioavailable due to their susceptibility to proteolytic degradation. These issues have led to the exploration of alternative protein scaffolds as a source for novel types of protein-based therapeutics (Gould, A. et al. (2011) Curr Pharm Des 17:4294-4307; Garcia, A.E. et al. (2010) Curr Mol Pharmacol 3:153-163; Henriques, S.T. et al. (2010) Drug Discov Today 15:5764).

SUMMARY OF THE INVENTION

[0006] The activity of the tumor suppressor protein p53 is negatively regulated by the oncoproteins Hdm2 and HdmX. The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. In this context, targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be powerful Hdm2/HdmX antagonists. However, the potential therapeutic use of these peptides is limited by their poor stability and bioavailability. This disclosure provides engineered cyclotide MCoTI-I that efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI binds with low nanomolar affinity to both Hdm2 and HdmX, shows high stability in human serum and is cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

[0007] Applicants have previously utilized cyclotides for the screening and design of biologically relevant peptides (see PCT/US2010/039720, incorporated herein by reference). However, Applicants are unaware of any prior cyclotide that specifically targets and removes p53 tumor suppressor inhibition in vivo.

[0008] Thus, in one aspect, the disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a protease recognition leading signal, fused or grafted to cyclotide comprising a linker and alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx (i..e., "target peptide"), wherein the leading signal is fused or grafted to the cyclotide through a linker that can be cleaved to form a N-terminal cysteine unit and the cyclotide is fused to an engineered intein having a C-terminal thioester.

[0009] In another aspect, the disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a cyclotide fused to a linker and an alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx having a N-terminal cysteine unit and a C-terminal thioester.

[0010] In a yet further aspect, the disclosure provides a cyclic isolated peptide, comprising, or alternatively consisting essentially of, or yet further consisting of a cyclotide comprising a linker and an alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx. [0011] In one aspect, any of the above peptides can further comprise, or alternatively consist essentially of, or yet consist of: one or more of a label such as a detectable fluorescent marker or a discrete marker.

[0012] In one aspect, the target peptide comprises, or alternatively consists essentially of, or yet further consists of the amino acid sequence TSXAEYZNLLSA or a biological equivalent thereof, wherein X is F or A and Z is a naturally occurring amino acid or a chemically modified amino acid. Non-limiting examples of a biological equivalent of the target peptide is a peptide having at least 80% sequence identity to TSXEYWZLLSA, wherein X is F or A, and Z is a naturally occurring amino acid or a chemically modified amino acid or a peptide encoded by a polynucleotide that hybridizes under conditions of high stringency to the coding or non-coding of the target peptide.

[0013] Non-limiting examples of a protease recognition leading signal is selected from the group consisting of TEV, Factor Xa, Met amino peptidase, Entorokinase.

[0014] Non-limiting examples, peptides for use as a linker comprises, or alternatively consists essentially of, or yet further consists of a peptide selected from the group consisting of GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG; GSGAPG; ASKAPG; and ASRAPG.

[0015] Also provided by this disclosure is an isolated polynucleotide encoding a peptide as described above or a complement thereof. The polynucleotides can be incorporated and operatively linked to an expression or replication vector, which can further comprise the appropriate regulatory sequences for expression or duplication of the sequences. The polynucleotides and vectors can further comprise a label or tag, e.g., a fluorescent label or tag.

[0016] The peptides, polynucleotides, vectors can be incorporated into host cells, such as a prokaryotic (E. coli) or eukaryotic cells.

[0017] The peptides, polynucleotides, vectors, host cells can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0018] The peptides and compositions can be used in a method for promoting or enhancing the biological activity of p53 in a cell or tissue by contacting the cell or tissue with an effective amount of the isolated peptide. The contacting can be in vitro, as in a tissue culture. When in vitro, the method is useful to screen new drugs or compositions for the ability to enhance or inhibit the biological or therapeutic activity of the target peptide. In one aspect, the cell or tissue is a cancer cell or tissue. In a further aspect, the cell or tissue has been previously selected for treatment by screening the cell or tissue for p53 dysfunction., e.g., a cancer cell or tissue having diminished or absent p53 protein expression. When the contacting is conducted in vivo, in a human patient or animal model, the method is a therapeutic method.

[0019] In a further aspect, the disclosure provides a method for treating a disease or condition related to p53 misregulation, inhibition and/or dysfunction, comprising

administering to a subject in need of such treatment an effective amount of the isolated cyclotide peptide or a composition containing the cyclotide peptide. In one aspect, the subject or patient is suffering from cancer. In a further aspect, the cancer results from or the cancer cell or tissue isolated from the patient has low or absent p53 protein expression.

[0020] In a yet further aspect, a kit is provided that comprises, or alternatively consists essentially of, or yet further consisting of, any of one or more of the isolated polypeptide as disclosed above, an isolated polynucleotide as disclosed herein, an isolated vector or host cell as described above, or the compositions, and instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 shows the primary and tertiary structure of cyclotides from the plants Momordica cochinchinensis (MCoTI-II) and Oldenlandia affinis (Kalata Bl).

[0022] FIG. 2 shows design of MCoTI-PMI, an MCOTI-based cyclotide with a-helical peptide PMI grafted onto loop 6 of cyclotide MCoTI-I. Binding constants for the different p53-based peptides were taken from Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670. The structure of MCoTI-PMI is based on a model built using the structure MCoTI-II and apamin. The grafted sequence is shown in small letters. The sequence of the polypeptide is shown using a single letter code for every amino acid residue.

[0023] FIG. 3 illustrates chemical synthesis of cyclotide MCoTI-PMI using Fmoc-based solid-phase synthesis on a sulfonamide linker (Contreras et al. (2011) J. Conrol Release 155(2): 134-43). The resulting linear peptide thioester is reacted in PBS in the presence of reduced GSH, this rapidly promotes the backbone cyclization and concomitant folding of the resulting grafted cyclotide MCoTI-PMI.

[0024] FIG. 4 illustrates recombinant expression of cyclotide MCoTI-PMI using a protein splicing unit and a TEV protease recognition leading signal. Once the linear precursor is expressed and purified, the TEV leading signal is cleaved to yield an N-terminal Cys residue that reacts in an intramolecular fashion with the C-terminal thioester generated by the protein splicing unit or intein. The backbone cyclization is induced by the presence of reduced GSH, which also promotes the folding of the resulting cyclic polypeptide into a cyclotide fold (Sancheti, H. et al. (2009) Adv Drug Deliv Rev 61 :908-917) (see Fig. 5).

[0025] FIG. 5A shows ¹⁵ N-HSQC spectra of ¹⁵ N-labeled cyclic MCoTI-I and MCoTI-PMI. FIG 5B shows ¹⁵ N-HSQC spectra of ¹⁵ N-labeled MCoTI-PMI free (grey) and complexed with the p53 binding domain of Mdm2 (black). All spectra were recorded in sodium phosphate buffer at pH 6.5 at 298 K.

[0026] FIGS. 6A and 6B show binding isotherms of the p53 binding domains of Mdm2 and MdmX to cyclotides MCoTI-PMI and MCoTI-PMI-F3A.

[0027] FIG. 7 illustrates competitive inhibition of p53/Mdm2 interaction by nutlin-3, different p53-based peptides, cyclotide MCoTI-1 and PMI-grafted cyclotide MCoTI-PMI. The experiment was perfomed using FRET -reporter fonned by the N-tenninal p53 peptide fused to YPet (YPet-p53) and the p53 binding domain of Mdm2 fused to CyPet (Mdm2- CyPet). Briefly, 20 nM CyPet-Mdm2 and 5 μΜ YPet-p53 were mixed at 23° C in 10 mM sodium phosphate 150 mM NaCl buffer at pH 7.3. Fluorescence was measured at 475 and 525 nm using an excitation wavelength of 414 nm in the presence of different inhibitor concentrations.

[0028] FIG. 8 (eight panels) show cell-based toxicity of cyclotide MCoTI-PMI against different cancer cell lines p53-wt (LNCaP), p53-null (PC3) and p53mutated (DU-145). Noncancerous epithelial cells (HBL-100 and HEK-293T) were also used in the assay. Nutlin-3 and the cyclotide MCoTI-PMI-F3A were used as postive and negative controls, respectively. Cell cytotoxicity was estimated by standard MTT assay after 48 h of treating the cells with the corresponding compounds for 1 h in PBS. [0029] FIG. 9 shows serum stability of cyclotide MCoTI-PMI in human serum at 37° C. Cyclotide MCoTI-I and linearized/reduced MCoTI-PMI were also used as controls.

[0030] FIG. 10 (16 panels) show analytical reverse-phase C18-HPLC traces and elecstrospray mass spectra (deconvo luted) of purified MCo-PMI cyclotides. HPLC analysis was performed using a linear gradient of 0-70% solvent B over 30 minutes.

[0031] FIG. 11 (2 panels) show analytical reverse-phase C18-HPLC traces and

electrospray mass spectra (deconvoluted) of purified ¹⁵ N-labeled MCo-PMI cyclotide. HPLC analysis was performed using a linear gradient of 0-70% solvent B over 30 minutes.

[0032] FIGS. 12A-G show NMR analysis of MCo-PMI. (A) Selected Fi( ¹ H)-F ₃ ( ¹ H ^a )) regions from a 3D 1H- ¹⁵ N NOESY HSQC spectrum of free MCo-PMI in solution. (B)

Selected Fi(1H)- Fs^H") regions from a 3D 1H- ¹⁵ N NOESY HSQC spectrum of MCo-PMI bound to Hdm2 in solution shows a helical pattern characteristics. (C) Overlay of HSQC spectra of MCoTI-I (black) and MCo-PMI (red) in solution. (D) Backbone superposition of ten energy minimized structures of MCo-PMI Hdm2 bound complex. (E) Changes in the backbone protons (Η-Ν ^α (Η') and H ^a ) and N ^a chemical shifts between the common sequence of MCoTI-I and MCo-PMI, residues 1 through 43. (F) Probability of secondary structure formation for the apamin-PMI grafted peptide segment on MCo-PMI (residues 36 through 50). The probabilities are based on the NH backbone chemical shifts (H-N ^a and ¹⁵ N). The positive chemical shift index with respect to random coil implies that PMI loop adopts conformations with alpha helical structure. (G) Changes in the H-N ^a protons and N ^a chemical shifts between Hdm2 -bound and free MCo-PMI.

[0033] FIGS. 13A-B show SDS-PAGE (A) and ES-MS spectra (B) of purified YPet-p53, CyPet-Hdm2 and CyPet-HdmX.

[0034] FIGS. 14A-B show ES-MS spectra of ¹⁵ N- and ¹³ C, ¹⁵ N-labeled Hdm2 used for NMR spectroscopy.

[0035] FIGS. 15A-B show (A) serum stability of cyclotides MCo-PMI and MCoTI-I, and a linearized and S-alkylated version of MCo-PMI at 37°C. (B) Binding kinetics of cyclotide MCo-PMI to human serum proteins. [0036] FIGS. 16A-D show MCo-PMI treatment does not cause systemic toxicity. Cohorts (N = 3) of HCT116 p53 ^+/+ xenografts mice were treated with vehicle (D5W), MCo-PMI (40 mg/kg, 7.6 mmol/kg) or Nutlin-3 (10 mg/kg, 17.2 mmol/kg) daily for N days. (A) Body weight was measured weekly using an electronic balance. Data are mean ± SEM. No significant differences were noted between cohorts. Upon completion of treatment, tumors were excised and processed for histologic characterization. (B) Micrographs showing histological features of H&E stained tumor sections at 5x and 20x magnifications, upper and lower panels, respectively. (C) H&E stained sections of the kidney and liver, upper and lower panels, respectively. (D) The level of Hdm2, p53 and its transcriptional target, p21 were assessed by immunohistochemical staining.

[0037] FIG. 17 shows exemplary sequences of the MCo-PMI cyclotides. The PMI peptide was grafted onto loop 6 of cyclotide MCoTI-I. To facilitate the insertion of this peptide without disturbing its a-helical character or the cyclotide framework, its N-terminus was fused to the apamin-derived linker Ala-Ser-Lys/Arg-Ala-Pro. The mutations F42A and W46Z (Z = 6-chloro-tryptophan) are shown. The sequence common to MCoTI-I is shown in black. Conserved cysteine residues and disulfide connectivities are marked in different shading.. The circular backbone topology is shown with a line.

[0038] FIGS. 18A-B show scheme summarizing the approaches used for the chemical synthesis (A) or recombinant production (B) of MCo-PMI cyclotides. In both cases the cyclization/folding was performed using an intramolecular version of Native Chemical Ligation in the presence of reduced glutathione (GSH) at pH 7.4. Under these conditions the linear MCo-PMI cyclotide precursors were able to efficiently cyclize and fold in 24 h as shown in the analytical HPLC traces for the crude cyclization/folding reaction for MCo-PMI.

[0039] FIGS. 19A-B show binding activities of the MCo-PMI cyclotides. (A) Direct binding of FITC-labeled MCo-PMI peptides to recombinant Hdm2 (17-125) and HdmX (17- 116) was measured by fluorescence polarization anisotropy. Binding experiments were performed in phosphate buffer at pH 7.4 at room temperature. (B) Competition experiments of MCo-PMI peptides and Nutlin-3 with p53 (15-29) for binding to Hdm2 (17-125) and HdmX (17-116). Binding competition experiments were performed by titrating a solution of YPet-p53 (5 μΜ) and CyPet-Hdm2 (20 nM) or CyPet-HdmX (20 nM) in phosphate buffer at pH 7.4 at room temperature with increasing concentrations of unlabeled inhibitor. The decrease in FRET signal was measured at 525 nm (YPet) by excitation at 414 nm (CyPet). Data are mean ± SD for experiments performed in triplicate.

[0040] FIG. 20 show cyclotide MCo-PMI can cross the cell membrane and target both Hdm2 and HmdX in cell culture experiments. LNCaP cells were treated for 30 h with either vehicle, FITC-MCo-PMI-K37R (50 μΜ) or FITC-MCo-PMI-K37R-F42A (50 μΜ). Soluble cell extracts were immunoprecipitated with anti-FITC murine antibody, analyzed by Hdm2 and HdmX western analysis using GAPDH as a total protein loading control. FITC-labeled cyclotides were visualized by epifluorescence.

[0041] FIG. 21 (8 panels) shows cell viability of cancer and normal cells exposed to MCo- PMI cyclotides. Different cancer cell lines expressing different levels of Hdm2 and/or HdmX (HCT116 p53 ^+/+ , LNCaP and JEG3), non-functional p53 (HCT 116 p53-/-, DU145 and PC3) and non-tumor cells (HBL100 and HEK293T) were treated with 0-100 μΜ Nutlin-3, MCo- PMI, MCo-PMI-F42A and/or MCoTI-I for 1 h as described in the supplemental information section. After 48 h, cell viability was assessed by using the MTT assay. Data are mean ± SD for experiments performed in triplicate.

[0042] FIGS. 22A-C show cyclotide MCo-PMI reactivates, in a dose-dependent manner, the p53 tumor suppressor pathway in cancer cells. (A) LNCaP cells were exposed to 50 μΜ of cyclotides MCo-PMI, MCo-PMI-F42A, MCoTI-I, and Nutlin-3 for 1 h. After 48 h, the soluble cell extracts were analyzed by SDS-PAGE and western blotting for p53, Hdm2, HdmX and p21. (B) LNCaP cells were exposed to different concentrations of MCo-PMI (0- 50 μΜ) fro 1 h and after 48 h cell lysates were analyzed for p53, Hdm2, HdmX and p21 as described above. (C) LNCaP cells were treated with MCo-PMI (50μΜ) for 1 h. The amount of p53, Hdm2, HdmX and p21 was evaluated by western after 0-48 h of treatment. In all the cases GAPDH was used as loading control.

[0043] FIGS. 23A-B show cyclotide MCo-PMI triggers cell cycle arrest and apoptosis. (A) LNCaP cells were treated with vehicle, MCo-PMI (0-100 μΜ) or Nutlin-3 (0-50 μΜ) for 1 h. The amount of caspase-3/7 was evaluated after 24 h by exposure to Caspase-Glo 3/7 reagent (Promega). Caspase 3/7 activation was measured by monitoring the luminescence signal upon proteolytic cleavage of a luciferin-containing caspase-3/7 substrate in the presence of luciferase. (B) LNCaP cells were treated with vehicle, MCo-PMI (50 μΜ), MCo-PMI-F42A (50 μΜ) and Nutlin-3 (50 μΜ) for 1 h. Cell cycle progression was monitored 24 h after treatment by propidium iodide staining and fluorescence-activated cell sorting.

[0044] FIGS. 24A-B show cyclotide MCo-PMI activates the p53 tumor suppressor pathway and blocks tumor growth in vivo. (A) Cohorts (N = 3) of HCT116 p53 ^+/+ xenografts mice were treated with vehicle (5% dextrose in water), MCo-PMI (40 mg/kg, 7.6 mmol/kg) or Nutlin-3 (10 mg/kg, 17.2 mmol/kg) by intravenous injection daily for N days. Tumor volume was monitored by caliper measurement. Data are mean ± SEM (day 31 : MCo- PMI/vehicle, p = 0.019, MCo-PMI/Nutlin-3, p = 0.022 and Nutlin-3/vehicle, p = 0.223). (B) Tumors were excised at day 31 (vehicle), day 36 (Nutlin-3) and day 37 (MCo-PMI) and the level of p53 transcriptional targets HDM2 and P21 was measured after RNA extraction by qPvT-PCR. Data are mean ± SEM.

[0045] FIGS. 25A-D show solution structure of the MCo-PMI and Hdm2 (17-125) complex. (A) Ribbon representation of MCo-PMI and Hdm2 complex. The side-chains of Phe42, Trp46 and Leu49 in MCo-PMI and the Hdm2 residues shaping the hydrophobic binding pocket are shown as sticks. (B) Close-up view of the binding interface within the Hdm2-MCo-PMI complex. The electrostatic potential at the molecular surface of Hdm2 is shown as positive in gray, negative in a darker gray and non-charged in light gray. (C)

Ribbon representation of the backbone superposition for the Hdm2 - MCo-PMI and Mdm2 - PMI peptide complexes. The key side chains of Phe, Trp and Leu in the MCo-PMI cyclotide and PMI peptide are shown as sticks. (D) Close up view of the Hdm2 - Mco-PMI complex reveals and additional additional salt bridge interaction between Asp35 (MCo-PMI) and Lys76 (Hdm2).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] Before the compositions and methods are described, it is to be understood that the invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims.

[0047] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 ^rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 ^th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid

Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987)

Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);

Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 ^rd edition (Cold Spring Harbor Laboratory Press (2002)); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Harlow and Lane, eds. (1999) Using Antibodies, A Laboratory Manual; Animal Cell Culture (R.I. Freshney, ed. (1987)); Zigova, Sanberg and Sanchez -Ramos, eds. (2002) Neural Stem Cells.

[0048] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1 where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". The term "about" also includes the exact value "X" in addition to minor increments of "X" such as "X + 0.1 or 1" or "X - 0.1 or 1," where appropriate. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0049] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[0050] Throughout and within this disclosure the patent and technical literature is identified by a bibliographic citation or by a Arabic number. The bibliographic citations for these references are found in this disclosure immediately preceding the claims. All references disclosed here are incorporated by reference to more fully describe the state of the art to which this invention pertains.

Definitions

[0051] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0052] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. A method "consisting essentially of would provide the object of the claim, e.g., restoring p53 function in a cell or tissue.

"Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention. [0053] The term "isolated" as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides is meant to encompass both purified and recombinant polypeptides.

[0054] As used herein, the term "recombinant" as it pertains to polypeptides or

polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.

[0055] A "subject," "individual" or "patient" is used interchangeably herein and refers to a vertebrate, for example a primate, a mammal or preferably a human. Mammals include, but are not limited to equines, canines, bovines, ovines, murines, rats, simians, humans, farm animals, sport animals and pets.

[0056] "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0057] "Amplify" "amplifying" or "amplification" of a polynucleotide sequence includes methods such as traditional cloning methodologies, PCR, ligation amplification (or ligase chain reaction, LCR) or other amplification methods. These methods are known and practiced in the art. See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202 and Innis et al. (1990) Mol. Cell Biol. 10(11):5977-5982 (for PCR); and Wu et al. (1989) Genomics 4:560- 569 (for LCR). In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0058] Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.

[0059] As used herein, the term "grafted" intends replaced or inserted, e.g., the phrase "grafted between" means that a peptide replaces the amino acid residues between the two indicated amino acids.

[0060] As used herein, the term "fused" intends the attachment of two amino acids to each other by a peptide bond.

[0061] The term "genotype" refers to the specific allelic composition of an entire cell, a certain gene or a specific polynucleotide region of a genome, whereas the term "phenotype' refers to the detectable outward manifestations of a specific genotype.

[0062] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. A gene may also refer to a polymorphic or a mutant form or allele of a gene.

[0063] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present invention.

[0064] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are

BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address:

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, last accessed on May 21, 2008. Biologically equivalent polynucleotides are those having the above -noted specified percent homology and encoding a polypeptide having the same or similar biological activity.

[0065] The term "an equivalent" nucleic acid, polynucleotide or peptide refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homo logs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.

[0066] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0067] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%>; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0068] As used herein, the term "oligonucleotide" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes of clarity, when referring herein to a nucleotide of a nucleic acid, which can be DNA or an RNA, the terms "adenosine", "cytidine", "guanosine", and "thymidine" are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.

[0069] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger R A (mR A), transfer R A, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A

polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a

polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0070] A polynucleotide is composed of a specific sequence of four nucleotide bases:

adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term

"polymorphism" refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene". A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0071] "Overexpression" or "underexpression" refers to increased or decreased expression, or alternatively a differential expression, of a gene in a test sample as compared to the expression level of that gene in the control sample. In one aspect, the test sample is a diseased cell, and the control sample is a normal cell. In another aspect, the test sample is an experimentally manipulated or biologically altered cell, and the control sample is the cell prior to the experimental manipulation or biological alteration. In yet another aspect, the test sample is a sample from a patient, and the control sample is a similar sample from a healthy individual. In a yet further aspect, the test sample is a sample from a patient and the control sample is a similar sample from patient not having the desired clinical outcome. In one aspect, the differential expression is about 1.5 times, or alternatively, about 2.0 times, or alternatively, about 2.0 times, or alternatively, about 3.0 times, or alternatively, about 5 times, or alternatively, about 10 times, or alternatively about 50 times, or yet further alternatively more than about 100 times higher or lower than the expression level detected in the control sample. Alternatively, the gene is referred to as "over expressed" or "under expressed". Alternatively, the gene may also be referred to as "up regulated" or "down regulated".

[0072] The term "likely to respond" intends to mean that the patient of a genotype is relatively more likely to experience a complete response or partial response than patients similarly situated without the genotype. Alternatively, the term "not likely to respond" intends to mean that the patient of a genotype is relatively less likely to experience a complete response or partial response than patients similarly situated without the genotype.

[0073] As used herein, the term "carrier" encompasses any of the standard carriers, such as a phosphate buffered saline solution, buffers, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see

Sambrook and Russell (2001), supra. Those skilled in the art will know many other suitable carriers for binding polynucleotides, or will be able to ascertain the same by use of routine experimentation. In one aspect of the invention, the carrier is a buffered solution such as, but not limited to, a PCR buffer solution.

[0074] A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0075] "Gene delivery," "gene transfer," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection, sometimes called transduction), transfection, transformation or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). Unless otherwise specified, the term transfected, transduced or transformed may be used interchangeably herein to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a cell. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

[0076] A cell that "stably expresses" an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell either after replication if the cell is dividing or for longer than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, for several weeks, up to a month, up to two months, up to three months, for several months, up to a year or more.

[0077] The term "express" refers to the production of a gene product.

[0078] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

[0079] A "gene product" or alternatively a "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0080] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked" intends the polynucleotides are arranged in a manner that allows them to function in a cell. [0081] The term "encode" as it is applied to polynucleotides refers to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the

complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0082] As used herein, a "vector" is a vehicle for transferring genetic material into a cell. Examples of such include, but are not limited to plasmids and viral vectors. A viral vector is a virus that has been modified to transduct genetic material into a cell. A plasmid vector is made by splicing a DNA construct into a plasmid. As is apparent to those of skill in the art, the appropriate regulatory elements are included in the vectors to guide replication and/or expression of the genetic material in the selected host cell.

[0083] A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0084] In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, "retroviral mediated gene transfer" or "retroviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a pro virus. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. A "lentiviral vector" is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral Vectors, New York: Spring- Verlag Berlin Heidelberg.

[0085] In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the

polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470 and

Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0086] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5 ' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 ' of the start codon to enhance expression.

[0087] Gene delivery vehicles also include several non-viral vectors, including

DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., a cell surface marker found on stem cells.

[0088] A "plasmid" is an extra-chromosomal DNA molecule separate from the

chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.

[0089] "Plasmids" used in genetic engineering are called "plasmic vectors". Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.

[0090] "Eukaryotic cells" comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane -bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane -bound structure is the nucleus. A eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples include simian, bovine, ovine, porcine, murine, rats, canine, equine, feline, avian, reptilian and human.

[0091] "Prokaryotic cells" that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells' genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 μιη in diameter and 10 μιη long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to prokaryotic Cyanobacteria, bacillus bacteria, E. coli bacterium, and Salmonella bacterium. [0092] The term "propagate" means to grow a cell or population of cells. The term

"growing" also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.

[0093] The term "culturing" refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0094] A "probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels are described and exemplified herein.

[0095] A "primer" is a short polynucleotide, generally with a free 3' -OH group that binds to a target or "template" potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or a "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are well known in the art, and taught, for example in MacPherson et al. (1991) PCR: A Practical Approach, IRL Press at Oxford University Press. All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication." A primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et al., supra. The primers may optionall contain detectable labels and are exemplified and described herein.

[0096] As used herein, the term "detectable label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluoresecence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0097] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 ^th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0098] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 ^th ed.).

[0099] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to,

isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0100] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to,

antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.

[0101] As used herein, the term "discrete marker" intends an entity or molecule that allows for the selective identification of a peptide of interest. Non-limiting examples of such include, isotope-coded affinity tags, isobaric tags, radioactively labeled amino acids, and bioorthogonial noncanonical amino acid tags. Dieterich et al. (2006) PNAS, 103(25):9482- 9487.

[0102] The phrase "solid support" refers to non-aqueous surfaces such as "culture plates" "gene chips" or "microarrays." Gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are attached and arrayed on a gene chip for determining the DNA sequence by the hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. Polynucleotides and probes can be used for detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659. A probe also can be attached or affixed to an electrode surface for the

electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Patent No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

[0103] Various "gene chips" or "microarrays" and similar technologies are known in the art. Examples of such include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); a low-density array with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput, automated mass spectrometry systems with liquid-phase expression technology (Gene Trace Systems, Inc.); a thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput microarry system that can dispense from 12 to 64 spots onto multiple glass slides (Intelligent Bio-Instruments); Molecular Biology Workstation and NanoChip (Nanogen, Inc.); a microfluidic glass chip (Orchid Biosciences, Inc.); BioChip Arrayer with four PiezoTip piezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet (Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome); ChipMaker 2 and ChipMaker 3

(TeleChem International, Inc.); and GenoSensor (Vysis, Inc.) as identified and described in Heller (2002) Annu. Rev. Biomed. Eng. 4: 129-153. Examples of "gene chips" or a

"microarrays" are also described in U.S. Patent Publication Nos. 2007/0111322;

2007/0099198; 2007/0084997; 2007/0059769 and 2007/0059765 and U.S. Patent Nos.: 7,138,506; 7,070,740 and 6,989,267.

[0104] In one aspect, "gene chips" or "microarrays" containing probes or primers homologous to p53 are prepared. A suitable sample is obtained from the patient, extraction of genomic DNA, RNA, protein or any combination thereof is conducted and amplified if necessary. The sample is contacted to the gene chip or microarray panel under conditions suitable for hybridization of the gene(s) or gene product(s) of interest to the probe(s) or primer(s) contained on the gene chip or microarray. The probes or primers may be detectably labeled thereby identifying the sequence(s) of interest. Alternatively, a chemical or biological reaction may be used to identify the probes or primers which hybridized with the DNA or RNA of the gene(s) of interest. The genotypes or phenotype of the patient is then determined with the aid of the aforementioned apparatus and methods.

[0105] A "composition" is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0106] A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0107] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0108] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of

administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. Studies in animal models generally may be used for guidance regarding effective dosages for treatment of diseases. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Thus, where a compound is found to demonstrate in vitro activity, for example as noted in the Tables discussed below one can extrapolate to an effective dosage for administration in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition and as used herein, the term "therapeutically effective amount" is an amount sufficient to treat a specified disorder or disease or alternatively to obtain a pharmacological response treating a cancer.

[0109] As used herein, "treating" or "treatment" of a disease in a patient refers to (1) preventing the symptoms or disease from occurring in an animal that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its

development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this invention, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Preferred are compounds that are potent and can be administered locally at very low doses, thus minimizing systemic adverse effects.

[0110] "Suppressing" tumor growth indicates a growth state that is curtailed compared to growth without any therapy. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a H-thymidine incorporation assay, or counting tumor cells.

"Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and "suppressing" tumor growth indicates a growth state that is curtailed when stopping tumor growth, as well as tumor shrinkage.

[0111] A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative". For example, where the purpose of the experiment is to determine a correlation of a mutated allele with a particular phenotype, it is generally preferable to use a positive control (a sample from a subject, carrying such mutation and exhibiting the desired phenotype), and a negative control (a subject or a sample from a subject lacking the mutated allele and lacking the phenotype).

[0112] As used herein, the term "intein" intends a segment of a protein that is able to excise itself and rejoin the remaining portions (the exteins) with a peptide bond by protein splicing. Inteins have also been called "protein introns." Inteins are known in the art and sequences are publicly available, see for example, InBase, The Intein Database and Registry, available at the web addresss: www.neb.com/external-links/inbase (accessed on March 5, 2013) and Inteins - protein introns, available at the web address:

http://bioinformatics.weizmann.ac.il/~pietro/inteins/ (accessed on March 5, 2013).

[0113] As used herein, the term "promoting or enhancing the biological activity of p53 in a cell or tissue" intends the enhancement of the tumor suppressor activity or expression of p53 in the cell or tissue. [0114] As used herein, the term "a disease or condition related to p53 misregulation, inhibition and/or dysfunction" intends a disease, such as cancer, that has been correlated with p53 mutation or loss of p53 expression or function in a cell or tissue. The p53 tumor suppressor gene is mutated more often in human cancers than any other gene. Royds and Iacopetta (2006) Cell Death Differ. 13(6):1017-1026. It has been reported to be mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. In addition, Royds and Iacopetta (2006), supra, reports that at least one inherited cancer predisposing syndrome called Li-Fraumeni is caused by p53 mutations.

Descriptive Embodiments

Cyclotides, Peptides and Polynucleotides

[0115] Cyclotides are small globular microproteins (ranging from 28 to 37 amino acids) with a unique head-to-tail cyclized backbone, which is stabilized by three disulfide bonds forming a cystine -knot motif. This cyclic cystine-knot (CCK) framework provides a rigid molecular platform with exceptional stability towards physical, chemical and biological degradation. These micro-proteins can be considered natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization, but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot. Furthermore, naturally-occurring cyclotides have shown to posses various pharmacologically-relevant activities, and have been reported to cross cell membranes.

[0116] The construction of cyclotides is known in the art and has been described previously (see WO 2011/005598). Synthesis of the peptides and cyclotides useful in the methods and compositions of the disclosure are also described in the Examples that follow.

[0117] Reference herein to a "cyclic backbone" includes a molecule comprising a sequence of amino acid residues or analogues thereof without free amino and carboxy termini. The cyclic backbone of the disclosure comprises sufficient disulfide bonds, or chemical equivalents thereof, to confer a knotted topology on the three-dimensional structure of the cyclic backbone. The term "cyclotide" as used herein refers to a peptide comprising a cyclic cystine knot motif defined by a cyclic backbone, at least two but preferably at least three disulfide bonds and associated beta strands in a particular knotted topology. The knotted topology involves an embedded ring formed by at least two backbone disulfide bonds and their connecting backbone segments being threaded by a third disulfide bond. However, a disulfide bond may be replaced or substituted by another form of bonding such as a covalent bond.

[0118] This disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a protease recognition leading signal fused to a cyclotide comprising a linker and alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx (i.e., "target peptide"), wherein the leading signal is fused to the cyclotide through a linker that can be cleaved to form a N-terminal cysteine unit and the cyclotide is fused to an engineered intein having a C-terminal thioester.

[0119] In another aspect, the disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a cyclotide fused to a linker and an alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx and the isolated peptide having a N-terminal cysteine unit and a C-terminal thioester.

[0120] In a yet further aspect, the disclosure provides a cyclic isolated peptide, comprising, or alternatively consisting essentially of, or yet further consisting of a cyclotide comprising a linker and an alpha-helical fragment of a target peptide that binds the p53 binding domain of Hdm2 or Hdmx.

[0121] Unless specifically defined, the peptides can be linear or processed and in their cyclic form.

[0122] In another aspect, the isolated peptide further comprises, or alternatively consists essentially of, or yet further consists of, one or more of a discrete protein binding domain for isolation of the a peptide from the cell, a detectable label, such as a fluorescent marker or a discrete marker. Non-limiting examples of detectable labels comprise a fluorescent label and such as FRET reporters, e.g., CyPet and YPet and equivalents of each thereof. Non-limiting examples of discrete markers include isotope-coded affinity tags, isobaric tags, radioactively labeled amino acids, and bioorthogonial noncanonical amino acid tags.

[0123] In this disclosure, the target peptide can be grafted into any available loop, e.g., loop 1 , or loop 2, or loop 3, or loop 4, or loop 5 or loop 6, of the cyclotide. The cyclotide comprises a molecular framework comprising a sequence of amino acids forming a cyclic backbone wherein the cyclic backbone comprises sufficient disulfide bonds to confer knotted topology on the molecular frameword or part therof. The cyclic backbone comprises the structure: C[X . ..X _a ]C[X ⁿ i . ..X _b ]C[X ^m i . ..X _c ]C[X ^IV i . ..X _d ]C[X ^V i . ..X _e ]C[X ^VI i . ..X _f ] wherein C is cysteine; and each of each of [X\ . ..X _a ], [Χ ^Π ι . ..¾], [X ⁿ \ . ..X _c ], [X^i . ..¾], [X ^V i . ..X _e ], and [X ^VI i . ..X _f ], represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20. When the target peptide is grafted into loop 6 of the cyclotide, the amino acid residues

corresponding to [X ^v \ . ..X _f ] in the cyclotide comprise the target peptide. In some embodiments, the target peptide is grafted into loop 1 of the cyclotide. In this embodiment, the amino acid residues corresponding to . ..X _a ] in the cyclotide comprise the target peptide. In another embodiment, the target peptide is grafted into loop 2 of the cyclotide. In this embodiment, the amino acid residues corresponding to [Χ ^Π ι . ..X _b ] in the cyclotide comprise the target peptide. In a further embodiment, the target peptide is grafted into loop 3 of the cyclotide. In this embodiment, the amino acid residues corresponding to [Χ ^ΠΙ ι . ..X _c ] in the cyclotide comprise the target peptide. In yet a further embodiment, the target peptide is grafted into loop 4 of the cyclotide. In this embodiment, the amino acid residues

corresponding to [X ^W _\ . ..X _c ] in the cyclotide comprise the target peptide. In another embodiment, the target peptide is grafted into loop 5 of the cyclotide. In this embodiment, the amino acid residues corresponding to [X ^V i . ..X _e ] in the cyclotide comprise the taget peptide peptide. In one appect, the target peptide is grafted into loop 6 of the cyclotide, e.g., between residues Ser31 and Gly33 of a MCoTI cyclotide. The resulting grafted cyclotide was called MCo-PMI (Fig. 17).

[0124] Additional cyclotides useful in the peptides, methods, and compositions described herein are known in the art and non-limiting examples include, the cyclotides shown in Table 1, below.

Table 1

Cylcotide Protein Sequence

cycloviolacin Oi l GTLPCGESCVWIPCISAVVGCSCKSKVCYKN kalata_B4 GLPVCGETCVGGTCNTPGCTCSWPVCTRD vodo_M GAPICGESCFTGKCYTVQCSCSWPVCTRN cyclopsychotride_A SIPCGESCVFIPCTVTALLGCSCKSKVCYKN cycloviolacin HI GIPCGESCVYIPCLTSAIGCSCKSKVCYRN cycloviolacin 09 GIPCGESCVWIPCLTSAVGCSCKSKVCYRN vico_A GSIPCAESCVYIPCFTGIAGCSCKNKVCYYN vitri A GIPCGESCVWIPCITSAIGCSCKSKVCYRN kalata S GLPVCGETCVGGTCNTPGCSCSWPVCTRN cycloviolacin 012 GLPICGETCVGGTCNTPGCSCSWPVCTRN vodo_N GLPVCGETCTLGKCYT AGC S C S WPVC YRN vico B GSIPCAESCVYIPCITGIAGCSCKNKVCYYN kalata Bl Ila GLPVCGETCVGGTCNTPGCTCSWPVCTRN

Hypa_A GIPCAESCVYIPCTITALLGCSCKNKVCYN circulin B GVIPCGESCVFIPCISTLLGCSCKNKVCYRN circulin C GIPCGESCVFIPCITSVAGCSCKSKVCYRN circulin D KIPCGESCVWIPCVTSIFNCKCENKVCYHD circulin E KIPCGESCVWIPCLTSVFNCKCENKVCYHD circulin F AIPCGESCVWIPCISAAIGCSCKNKVCYR cycloviolacin 04 GIPCGESCVWIPCISSAIGCSCKNKVCYRN cycloviolacin 03 GIPCGESCVWIPCLTSAIGCSCKSKVCYRN cycloviolacin 05 GTPCGESCVWIPCISSAVGCSCKNKVCYKN cycloviolacin 06 GTLPCGESCVWIPCISAAVGCSCKSKVCYKN cycloviolacin 07 SIPCGESCVWIPCTITALAGCKCKSKVCYN cycloviolacin 010 GIPCGESCVYIPCLTSAVGCSCKSKVCYRN kalata B5 GTPCGESCVYIPCISGVIGCSCTDKVCYLN varv_peptide_B GLPVCGETCFGGTCNTPGCSCDPWPMCSRN varv_peptide_C GVPICGETCVGGTCNTPGCSCSWPVCTRN varv_peptide_D GLPICGETCVGGSCNTPGCSCSWPVCTRN varv_peptide_F GVPICGETCTLGTCYTAGCSCSWPVCTRN varv_peptide_G GVPVCGETCFGGTCNTPGCSCDPWPVCSRN Cylcotide Protein Sequence

varv_peptide_H GLPVCGETCFGGTCNTPGCSCETWPVCSPvN cycloviolin A GVIPCGESCVFIPCISAAIGCSCKNKVCYRN cycloviolin B GTACGESCYVLPCFTVGCTCTSSQCFKN cycloviolin C GIPCGESCVFIPCLTTVAGCSCKNKVCYRN cycloviolin D GFPCGESCVFIPCISAAIGCSCKNKVCYRN violapeptide l GLPVCGETCVGGTCNTPGCSCSRPVCTXN vhl-1 SISCGESCAMISFCFTEVIGCSCKNKVCYLN

Vontr Protein ALETQKPNHLEEALVAFAKKGNLGGLP hcf-1 GIPCGESCHYIPCVTSAIGCSCRNRSCMRN htf-1 GIPCGDSCHYIPCVTSTIGCSCTNGSCMRN

Oantr_protein GVKSSETTLMFLKEMQLKLP

vhl-2 GLPVCGETCFTGTCYTNGCTCDPWPVCTRN cycloviolacin H3 GLPVCGETCFGGTCNTPGCICDPWPVCTRN cycloviolacin H2 SAIACGESCVYIPCFIPGCSCRNRVCYLN

Hyfl_A SISCGESCVYIPCTVTALVGCTCKDKVCYLN

Hyfl B GSPIQCAETCFIGKCYTEELGCTCTAFLCMK

N

Hyfl_C GSPRQCAETCFIGKCYTEELGCTCTAFLCMK

N

Hyfl D GSVPCGESCVYIPCFTGIAGCSCKSKVCYYN

Hyfl_E GEIPCGESCVYLPCFLPNCYCRNHVCYLN

Hyfl_F SISCGETCTTFNCWIPNCKCNHHDKVCYWN

Hyfl GJpartial) CAETCWLPCFIVPGCSCKSSVCYFN

Hyfl HJpartial) CAETCIYIPCFTEAVGCKCKDKVCYKN

Hyfl_I GIPCGESCVFIPCISGVIGCSCKSKVCYRN

Hyfl_J GIACGESCAYFGCWIPGCSCRNKVCYFN

Hyfl_K GTPCGESCVYIPCFTAVVGCTCKDKVCYLN

Hyfl_L GTPCAESCVYLPCFTGVIGCTCKDKVCYLN

Hyfl_M GNIPCGESCIFFPCFNPGCSCKDNLCYYN

Hyfl_N_(partial) CGETCVILPCISAALGCSCKDTVCYKN

Hyfl O (partial) CGETCVIFPCISAAFGCSCKDTVCYKN Cylcotide Protein Sequence

Hyfl P GSVPCGESCVWIPCISGIAGCSCKNKVCYLN

Hymo A (partial) CGETCLFIPCIFSWGCSCSSKVCYRN

Hymo B (partial) CGETCVTGTCYTPGCACDWPVCKPvD

Hyst_A_(partial) CGETCIWGRCYSENIGCHCGFGICTLN

Hyve A (partial) CGETCLFIPCLTSVFGCSCKNRGCYKI

Hyca A (partial) CGETCWDTRCYTKKCSCAWPVCMRN

Hyde A (partial) CVWIPCISAAIGCSCKSKVCYRN

Hyen A (partial) CGESCVYIPCTVTALLGCSCKDKVCYKN

Hyen B (partial) CGETCKVTKRCSGQGCSCLKGRSCYD

Hyep A (partial) CGETCVVLPCFIVPGCSCKSSVCYFN

Hyep B (partial) CGETCIYIPCFTEAVGCKCKDKVCYKN tricyclon B GGTIFDCGESCFLGTCYTKGCSCGEWKLCY

GEN

kalata_B8 GSVLNCGETCLLGTCYTTGCTCNKYRVCTK

D

cycloviolacin H4 GIPCAESCVWIPCTVTALLGCSCSNNVCYN cycloviolacin 013 GIPCGESCVWIPCISAAIGCSCKSKVCYRN violacin A SAISCGETCFKFKCYTPRCSCSYPVCK cycloviolacin 014 GSIPACGESCFKGKCYTPGCSCSKYPLCAKN cycloviolacin 015 GLVPCGETCFTGKCYTPGCSCSYPICKKN cycloviolacin 016 GLPCGETCFTGKCYTPGCSCSYPICKKIN cycloviolacin 017 GIPCGESCVWIPCISAAIGCSCKNKVCYRN cycloviolacin 018 GIPCGESCVYIPCTVTALAGCKCKSKVCYN cycloviolacin 019 GTLPCGESCVWIPCISSVVGCSCKSKVCYKD cycloviolacin 020 GIPCGESCVWIPCLTSAIGCSCKSKVCYRD cycloviolacin 021 GLPVCGETCVTGSCYTPGCTCSWPVCTRN cycloviolacin 022 GLPICGETCVGGTCNTPGCTCSWPVCTRN cycloviolacin 023 GLPTCGETCFGGTCNTPGCTCDS S WPICTHN cycloviolacin 024 GLPTCGETCFGGTCNTPGCTCDPWPVCTHN cycloviolacin 025 DIFCGETCAFIPCITHVPGTCSCKSKVCYFN

[P20D,V21K]- GLPVCGETCVGGTCNTPGCTCSWDKCTRN Cylcotide Protein Sequence

kalata Bl

[W19K,_P20N,_V21K] GLPVCGETCVGGTCNTPGCTCSKNKCTRN -kalata Bl

[Glu(Me)]cy02 GIPCGXSCVWIPCISSAIGCSCKSKVCYRN

[Lys(Ac)]2cy02 GIPCGESCVWIPCISSAIGCSCXSXVCYRN

[Arg(CHD)]cy02 GIPCGESCVWIPCISSAIGCSCKSKVCYXN

([Lys(Ac)]2[Arg(CHD) GIPCGESCVWIPCISSAIGCSCXSXVCYXN ])cy02

kalata Bl oia GLPVCGETCVGGTCNTPGCTCSWPVCTRN kalata Bl nfk GLPVCGETCVGGTCNTPGCTCSWPVCTRN kalata B2 nfk GLPVCGETCFGGTCNTPGCSCTWPICTRD kalata B2 kyn GLPVCGETCFGGTCNTPGCSCTWPICTRD kalata_B9 GSVFNCGETCVLGTCYTPGCTCNTYRVCTK

D

kalata BlO GLPTCGETCFGGTCNTPGCSCSSWPICTRD kalata BIO oia GLPTCGETCFGGTCNTPGCSCSSWPICTRD kalata Bl l GLPVCGETCFGGTCNTPGCSCTDPICTRD kalata B12 GSLCGDTCFVLGCNDSSCSCNYPICVKD kalata_B13 GLPVCGETCFGGTCNTPGCACDPWPVCTRD kalata_B14 GLPVCGESCFGGTCNTPGCACDPWPVCTRD kalata_B15 GLPVCGESCFGGSCYTPGCSCTWPICTRD kalata B16 GIPCAESCVYIPCTITALLGCKCQDKVCYD kalata_B17 GIPCAESCVYIPCTITALLGCKCKDQVCYN

Amrad 5 CGETCVGGTCNTPGCTCSWPVCRRKRRR

Amrad 9 CGETCRRKRRRCNTPGCTCSWPVCTRNGLP

V

Amrad 11 CGETCVGGTCNTRRKRRRGCTCSWPVCTR

NGLPV

Amrad 17 CGETCVGGTCNTPGCTCRRKRRRVCTRNGL

PV

Amrad 7 CGETCVGGTCNTPGCTCRRKRRRCTRNGLP Cylcotide Protein Sequence

V

Amrad 8 CGETCVGGTCRRKRRRCTCSWPVCTRNGLP

V

kalata_B18 GVPCAESCVYIPCISTVLGCSCSNQVCYRN

PS-1 GFIPCGETCIWDKTCHAAGCSCSVANICVRN

CD-I GADGFCGESCYVIPCISYLVGCSCDTIEKVC

KRN

cycloviolacin Yl GGTIFDCGETCFLGTCYTPGCSCGNYGFCYG

TN

cycloviolacin Y2 GGTIFDCGESCFLGTCYTAGCSCGNWGLCY

GTN

cycloviolacin Y3 GGTIFDCGETCFLGTCYTAGCSCGNWGLCY

GTN

cycloviolacin Y4 GVPCGESCVFIPCITGVIGCSCSSNVCYLN cycloviolacin Y5 GIPCAESCVWIPCTVTALVGCSCSDKVCYN vibi_A GLPVCGETCFGGTCNTPGCSCSYPICTRN vibi B GLPVCGETCFGGTCNTPGCTCSYPICTRN vibi_C GLPVCGETCAFGSCYTPGCSCSWPVCTRN vibi D GLPVCGETCFGGRCNTPGCTCSYPICTRN vibi E GIPCAESCVWIPCTVTALIGCGCSNKVCYN vibi F GTIPCGESCVFIPCLTSALGCSCKSKVCYKN vibi_G GTFPCGESCVFIPCLTSAIGCSCKSKVCYKN vibi_H GLLPCAESCVYIPCLTTVIGCSCKSKVCYKN vibi l GIPCGESCVWIPCLTSTVGCSCKSKVCYRN vibi J GTFPCGESCVWIPCISKVIGCACKSKVCYKN vibi_K GIPCGESCVWIPCLTSAVGCPCKSKVCYRN

Viba_2 GIPCGESCVYLPCFTAPLGCSCSSKVCYRN

Viba_5 GIPCGESCVWIPCLTATIGCSCKSKVCYRN

Viba lO GIPCAESCVYLPCVTIVIGCSCKDKVCYN

Viba_12 GIPCAESCVWIPCTVTALLGCSCKDKVCYN

Viba_14 GRLCGERCVIERTRAWCRTVGCICSLHTLEC Cylcotide Protein Sequence

Viba_17 GLPVCGETCVGGTCNTPGCGCSWPVCTRN

Viba_15 GLPVCGETCVGGTCNTPGCACSWPVCTRN mram 1 GSIPCGESCVYIPCISSLLGCSCKSKVCYKN mram 2 GIPCAESCVYIPCLTSAIGCSCKSKVCYRN mram 3 GIPCGESCVYLPCFTTIIGCKCQGKVCYH mram 4 GSIPCGESCVFIPCISSVVGCSCKNKVCYKN mram 5 GTIPCGESCVFIPCLTSAIGCSCKSKVCYKN mram 6 GSIPCGESCVYIPCISSLLGCSCESKVCYKN mram 7 GSIPCGESCVFIPCISSIVGCSCKSKVCYKN mram 8 GIPCGESCVFIPCLTSAIGCSCKSKVCYRN mram 9 GVPCGESCVWIPCLTSIVGCSCKNNVCTLN mram 10 GVIPCGESCVFIPCISSVLGCSCKNKVCYRN mram 11 GHPTCGETCLLGTCYTPGCTCKRPVCYKN mram 12 GSAILCGESCTLGECYTPGCTCSWPICTKN mram 13 GHPICGETCVGNKCYTPGCTCTWPVCYRN mram 14 GSIPCGEGCVFIPCISSIVGCSCKSKVCYKN

Viba l GIPCGEGCVYLPCFTAPLGCSCSSKVCYRN

Viba_3 GIPCGESCVWIPCLTAAIGCSCSSKVCYRN

Viba_4 GVPCGESCVWIPCLTSAIGCSCKSSVCYRN

Viba_6 GIPCGESCVLIPCISSVIGCSCKSKVCYRN

Viba_7 GVIPCGESCVFIPCISSVIGCSCKSKVCYRN

Viba_8 GAGCIETCYTFPCISEMINCSCKNSRCQKN

Viba_9 GIPCGESCVWIPCISSAIGCSCKNKVCYRK

Viba l 1 GIPCGESCVWIPCISGAIGCSCKSKVCYRN

Viba_13 TIPCAESCVWIPCTVTALLGCSCKDKVCYN

Viba_16 GLPICGETCTLGTCYTVGCTCSWPICTRN

[GlA]kalata_Bl ALPVCGETCVGGTCNTPGCTCSWPVCTRN

[L2A]kalata_Bl GAPVCGETCVGGTCNTPGCTCSWPVCTRN

[P3A]kalata_Bl GLAVCGETCVGGTCNTPGCTCSWPVCTRN

[V4A]kalata_Bl GLPACGETCVGGTCNTPGCTCSWPVCTRN Cylcotide Protein Sequence

[G6A]kalata_Bl GLPVCAETCVGGTCNTPGCTCSWPVCTRN

[E7A]kalata_Bl GLPVCGATCVGGTCNTPGCTCSWPVCTRN

[T8A]kalata_Bl GLPVCGEACVGGTCNTPGCTCSWPVCTRN

[V10A]kalata_Bl GLPVCGETCAGGTCNTPGCTCSWPVCTRN

[Gl lA]kalata_Bl GLPVCGETCVAGTCNTPGCTCSWPVCTRN

[G12A]kalata_Bl GLPVCGETCVGATCNTPGCTCSWPVCTRN

[T13A]kalata_Bl GLPVCGETCVGGACNTPGCTCSWPVCTRN

[N15A]kalata_Bl GLPVCGETCVGGTCATPGCTCSWPVCTRN

[T16A]kalata_Bl GLPVCGETCVGGTCNAPGCTCSWPVCTRN

[P17A]kalata_Bl GLPVCGETCVGGTCNTAGCTCSWPVCTRN

[G18A]kalata_Bl GLPVCGETCVGGTCNTPACTCSWPVCTRN

[T20A]kalata_Bl GLPVCGETCVGGTCNTPGCACSWPVCTRN

[S22A]kalata_Bl GLPVCGETCVGGTCNTPGCTCAWPVCTRN

[W23A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSAPVCTRN

[P24A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSWAVCTRN

[V25A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSWPACTRN

[T27A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSWPVCARN

[R28A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSWPVCTAN

[N29A]kalata_Bl GLPVCGETCVGGTCNTPGCTCSWPVCTRA

Cter_A GVIPCGESCVFIPCISTVIGCSCKNKVCYRN

Cter B GVPCAESCVWIPCTVTALLGCSCKDKVCYL

N

hcf-1 variant GIPCGESCHIPCVTSAIGCSCRNRSCMRN

Vpl-1 GSQSCGESCVLIPCISGVIGCSCSSMICYFN

Vpf-1 GIPCGESCVFIPCLTAAIGCSCRSKVCYRN c031 GLPVCGETCVGGTCNTPGCSCSIPVCTRN c028 GLPVCGETCVGGTCNTPGCSCSWPVCFRD c032 GAPVCGETCFGGTCNTPGCTCDPWPVCTND c033 GLPVCGETCVGGTCNTPYCTCSWPVCTRD c034 GLPVCGETCVGGTCNTEYCTCSWPVCTRD c035 GLPVCGETCVGGTCNTPYCFCSWPVCTRD Cylcotide Protein Sequence

c029 GIPCGESCVWIPCISGAIGCSCKSKVCYKN cO30 GIPCGESCVWIPCISSAIGCSCKNKVCFKN c026 GSIPACGESCFRGKCYTPGCSCSKYPLCAKD c027 GSIPACGESCFKGWCYTPGCSCSKYPLCAK

D

Globa F GSFPCGESCVFIPCISAIAGCSCKNKVCYKN

Globa A GIPCGESCVFIPCITAAIGCSCKTKVCYRN

Globa B GVIPCGESCVFIPCISAVLGCSCKSKVCYRN

Globa D GIPCGETCVFMPCISGPMGCSCKHMVCYRN

Globa G GVIPCGESCVFIPCISSVLGCSCKNKVCYRN

Globa E GSAFGCGETCVKGKCNTPGCVCSWPVCKK

N

Globa C APCGESCVFIPCISAVLGCSCKSKVCYRN

Glopa D GVPCGESCVWVPCTVTALMGCSCVREVCR

KD

Glopa E GIPCAESCVWIPCTVTKMLGCSCKDKVCYN

Glopa A GGSIPCIETCVWTGCFLVPGCSCKSDKKCYL

N

Glopa B GGSVPCIETCVWTGCFLVPGCSCKSDKKCY

LN

Glopa C GDIPLCGETCFEGGNCRIPGCTCVWPFCSKN

Co36 GLPTCGETCFGGTCNTPGCTCDPFPVCTHD cycloviolacin Tl GIPVCGETCVGGTCNTPGCSCSWPVCTRN cycloviolacin T2 GLPICGETCVGGTCNTPGCSCSWPVCTRN psyle_A GIACGESCVFLGCFIPGCSCKSKVCYFN psyle_B GIPCGETCVAFGCWIPGCSCKDKLCYYD psyle_C KLCGETCFKFKC YTPGC S C S YFPCK psyle_D GIPCGESCVFIPCTVTALLGCSCQNKVCYRD psyle_E GVIPCGESCVFIPCISSVLGCSCKNKVCYRD psyle_F GVIPCGESCVFIPCITAAVGCSCKNKVCYRD vaby A GLPVCGETCAGGTCNTPGCSCSWPICTRN Cylcotide Protein Sequence

vaby B GLPVCGETCAGGTCNTPGCSCTWPICTRN vaby_C GLPVCGETCAGGRCNTPGCSCSWPVCTRN vaby D GLPVCGETCFGGTCNTPGCTCDPWPVCTRN vaby E GLPVCGETCFGGTCNTPGCSCDPWPVCTRN

Oak6 cyclotide 2 GLPICGETCFGGTCNTPGCICDPWPVCTRD

Oak7_cyclotide GSHCGETCFFFGCYKPGCSCDELRQCYKN

Oak8_cyclotide GVPCGESCVFIPCLTAVVGCSCSNKVCYLN

Oak6_cyclotide_ 1 GLPVCGETCFGGTCNTPGCACDPWPVCTRN

Cter C GVPCAESCVWIPCTVTALLGCSCKDKVCYL

D

Cter D GIPCAESCVWIPCTVTALLGCSCKDKVCYLN

Cter E GIPCAESCVWIPCTVTALLGCSCKDKVCYLD

Cter F GIPCGESCVFIPCISSWGCSCKSKVCYLD

Cter G GLPCGESCVFIPCITTVVGCSCKNKVCY N

Cter H GLPCGESCVFIPCITTVVGCSCKNKVCYND

Cter l GTVPCGESCVFIPCITGIAGCSCKNKVCYIN

Cter_J GTVPCGESCVFIPCITGIAGCSCKNKVCYID

Cter K HEPCGESCVFIPCITTVVGCSCKNKVCYN

Cter L HEPCGESCVFIPCITTVVGCSCKNKVCYD

Cter_M GLPTCGETCTLGTCYVPDCSCSWPICMKN

Cter_N GSAFCGETCVLGTCYTPDCSCTALVCLKN

Cter O GIPCGESCVFIPCITGIAGCSCKSKVCYRN

Cter P GIPCGESCVFIPCITAAIGCSCKSKVCYRN

Cter Q GIPCGESCVFIPCISTVIGCSCKNKVCYRN

Cter R GIPCGESCVFIPCTVTALLGCSCKDKVCYKN vitri B GVPICGESCVGGTCNTPGCSCSWPVCTTN vitri C GLPICGETCVGGTCNTPGCFCTWPVCTRN vitri D GLPVCGETCFTGSCYTPGCSCNWPVCNRN vitri E GLPVCGETCVGGTCNTPGCSCSWPVCFRN vitri F GLTPCGESCVWIPCISSVVGCACKSKVCYKD hedyotide B 1 GTRCGETCFVLPCWSAKFGCYCQKGFCYRN [0125] In some aspects, the peptides comprise a protease recogniation leading signal. Non- limiting examples of protease recognition leading signals include TEV, Factor Xa, Met amino peptidase, Entorokinase, methionine, ubiquitin, modified ubiquitin or an equivalent of each thereof.

[0126] In one aspect, the target peptide that binds the p53 binding domain of Hdm2 or Hdmx comprises, or alternatively consists essentially of, or yet further consists of, the amino acid sequence TSXAEYZNLLSA or a biological equivalent thereof, wherein X is F or A, and Z is a naturally occurring amino acid, such as tryptophan or a chemically modified amino acid such as chemically modified, unnatural amino acid 6-chloro tryptophan. A biological equivalent of the target peptide is a peptide having at least 95%, or alternatively at least 90 %, or alternatively at least 85 %, or alternatively at least 80%, or alternatively at least 75 %, or alternatively at least 70% sequence identity to TSXEYWZLLSA, wherein X is F or A and Z is a naturally occurring amino acid, such as tryptophan or a chemically modified amino acid such as chemically modified, unnatural amino acid 6-chloro tryptophan. Alternatively, a biological equivalent of the target peptide is a peptide encoded by a polynucleotide that hybridizes under stringent conditions to the coding or non-coding of TSXAEYZNLLSA or a biological equivalent thereof, wherein X is F or A, and Z is a naturally occurring amino acid, such as tryptophan or a chemically modified amino acid such as chemically modified, unnatural amino acid 6-chloro tryptophanthe.

[0127] In one aspect where the peptide comprises a linker, any appropriate linker can be used. An example of a linker is based on the N-terminal region of apamin, a bee-venom neurotoxin that adopts a coil-turn-a-helix structure, e.g., a peptide selected from the group consisting of GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG; GSGAPG; ASKAPG; and ASRAPG.

[0128] In another aspect, this disclosure provides an isolated polynucleotide encoding one or more of the isolated peptides described above, alone or in a gene delivery vehicle (e.g., liposome, biocompatible polymers, lipoproteins, artificial viral envelopes, plasmids, viral vectors) a replication or an expression vector, e.g., a viral vector or a plasmid. Also provided are isolated complementary sequences to the isolated polynucleotide encoding one or more of the isolated peptides described above. The polynucleotides can further contain the necessary regulatory element(s) operatively linked to the coding sequences for expression of the polynucleotide in a host cell. In a further aspect, the isolated polynucleotides can further comprise a detectable label, e.g. a radioactive or fluoresencent label.

[0129] The isolated peptides and polynucleotides of this invention can be produced by chemical synthetic methods or by recombinant expression of isolated polynucleotides as described herein or using methods known to those of skill in the art.

Host Cells and Compositions

[0130] This disclosure also provides an isolated host cell comprising one or more of: the peptides as described above, an isolated polynucleotide, a gene delivery vehicle or vector containing the isolated polynucleotdses. The isolated host cell is a prokaryotic or a eukaryotic cell. In one particular aspect, the host cell is an E. coli cell.

[0131] "Host cell" refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0132] Examples of such include, prokaryotic cells such as E. coli cells. Examples of eukaryotic cells include, but are not limited to cells from animals, e.g., murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. The cells can be cultured cells or they can be primary cells. Cultured cell lines can be purchased from vendors such as the American Type Culture Collection (ATCC), U.S.A.

[0133] The host cells can be used for recombinant production of the polynucleotides or peptides of this invention or to provide a screen for therapeutic agents. Further provided is a method for recombinantly producing the peptides of this disclosure by growing an isolated host cell as described above under conditions that favor the expression of the polynucleotide. In one aspect, the peptides are isolated from the host cells. [0134] Any of the above noted compositions can be combined with a carrier, such as a pharmaceutically acceptable carrier.

Methods and Uses

[0135] This disclosure also provides methods for recombinantly producing peptides and polynucleotides of this invention by culturing a host cell the isolated polynucleotides, the plasmid or vector containing the polynucleotide encoding the peptide, under condtions for the expression of the polynucleotide to peptide. In a further aspect, the peptide is isolated from the cell.

[0136] In one aspect, the method can identify leads such as small molecules and additional therapeutic methods. In one aspect, the methods identifies small molecules or biological agents that mimic the cyclotide as described herein. Potential agents or small molecules for screening can come from any source. They can be libraries of natural products,

combinatorial chemical libraries, biological products made by recombinant libraries, etc. The source of the test substances is not critical to the disclosure.

[0137] To practice the screen or assay in vitro, suitable cell cultures or tissue cultures are first provided. The cell can be a cultured cell or a genetically modified cell which

differentially expresses p53. Alternatively, the cells can be from a tissue culture isolated from a patient. The cells are cultured under conditions (temperature, growth or culture medium and gas (C0 ₂ )) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. It also is desirable to maintain an additional separate cell culture; one which does not receive the agent being tested as a control.

[0138] As is apparent to one of skill in the art, suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting genotypic changes, phenotypic changes and/or cell death.

[0139] When the agent is a composition other than a DNA or RNA nucleic acid molecule, the suitable conditions may be by directly added to the cell culture or added to culture medium for addition. As is apparent to those skilled in the art, an "effective" amount must be added which can be empirically determined. [0140] The screen involves contacting the agent with a test cell comprising the cyclotide (or alternatively without) and then assaying the cell its ability to provide a biological response similar to the cyclotide of this disclosure. In yet another aspect, the test cell or tissue sample is isolated from a subject to be treated and one or more potential agents are screened to determine the optimal therapeutic and/or course of treatment for that individual patient.

[0141] For the purposes of this invention, an "agent" is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic small molecule, a peptide, a protein or an oligonucleotide. A vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and

oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in

combination with another agent, having the same or different biological activity as the agents identified by the inventive screen. The agents and methods also are intended to be combined with other therapies. They can be administered concurrently or sequentially.

[0142] This disclosure also provides methods for inhibiting the binding of the target peptide to its ligand (p53) and for promoting or enhancing the biological activity of p53 in a cell or tissue comprising, or alternatively consisting essentially of, or yet further consisting of, contacting the cell or tissue with an effective amount of one or more of: the polypeptide, the isolated polynucleotide, or the compositions as described herein. The contacting can be in vitro or in vivo. When contacted in vitro, the method can be used to screen potential or complementary therapeutics as discussed above.

[0143] In one aspect, the cell or tissue to be contacted is one in which there is low or absent p53 expression in a cell, e.g., a cancer cell such as breast, colon or brain cancer cell. In one apsect, the cell to be contacted is screened for low or absent p53 expression prior to contacting. Cells that have low or absent p53 expression or function are then contacted with the peptides and compositons of this disclosure.

[0144] Thus, in some aspects, the methods further comprise determining expression level of p53 using methods known in the art. Known methods for measuring gene expression include, but are not limited to, immunological assays, nuclease protection assays, northern blots, in situ hybridization, reverse transcriptase Polymerase Chain Reaction (RT-PCR), Real-Time Polymerase Chain Reaction, expressed sequence tag (EST) sequencing, cDNA microarray hybridization or gene chip analysis, statistical analysis of microarrays (SAM), subtractive cloning, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature

Sequencing (MPSS), and Sequencing-By-Synthesis (SBS). See for example, Carulli et al, (1998) J. Cell. Biochem. 72 (S30-31): 286 - 296; Galante et al, (2007) Bioinformatics, Advance Access (February 3, 2007).

[0145] SAGE, MPSS, and SBS are non-array based assays that determine the expression level of genes by measuring the frequency of sequence tags derived from polyadenylated transcripts. SAGE allows for the analysis of overall gene expression patterns with digital analysis. SAGE does not require a preexisting clone and can used to identify and quantitate new genes as well as known genes. Velculescu et al, (1995) Science 270(5235):484 - 487; Velculescu (1997) Cell 88(2):243-251.

[0146] MPSS technology allows for analyses of the expression level of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene. As with SAGE, MPSS does not require that genes be identified and characterized prior to conducting an experiment. MPSS has a sensitivity that allows for detection of a few molecules of mRNA per cell. Brenner et al. (2000) Nat. Biotechnol. 18:630-634; Reinartz et al, (2002) Brief Funct. Genomic Proteomic 1 : 95-104.

[0147] SBS allows analysis of gene expression by determining the differential expression of gene products present in sample by detection of nucleotide incorporation during a primer- directed polymerase extension reaction.

[0148] SAGE, MPSS, and SBS allow for generation of datasets in a digital format that simplifies management and analysis of the data. The data generated from these analyses can be analyzed using publicly available databases such as Sage Genie (Boon et al, (2002) PNAS 99: 11287-92), SAGEmap (Lash et al.,(2000) Genome Res 10: 1051-1060), and Automatic Correspondence of Tags and Genes (ACTG) (Galante (2007), supra). The data can also be analyzed using databases constructed using in house computers (Blackshaw et al. (2004) PLoS Biol, 2:E247; Silva et al. (2004) Nucleic Acids Res 32:6104-6110)). [0149] In addition, knowledge of the identity of the expression level of a gene in an individual (the gene profile) allows customization of therapy for a particular disease to the individual's genetic profile, the goal of "pharmacogenomics". For example, an individual's genetic profile can enable a doctor: 1) to more effectively prescribe a drug that will address the molecular basis of the disease or condition; 2) to better determine the appropriate dosage of a particular drug and 3) to identify novel targets for drug development. The identity of the genotype or expression patterns of individual patients can then be compared to the genotype or expression profile of the disease to determine the appropriate drug and dose to administer to the patient.

[0150] The ability to target populations expected to show the highest clinical benefit, based on the normal or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical

development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling.

[0151] The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits, such as those described below, comprising at least one probe or primer nucleic acid described herein, which may be conveniently used to determine p53 expression level.

[0152] Diagnostic procedures can also be performed in situ directly upon tissue sections (fixed and/or frozen) of primary tissue such as biopsies obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents can be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J. (1992) PCR IN SITU HYBRIDIZATION: PROTOCOLS AND APPLICATIONS, RAVEN PRESS, NY).

[0153] In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles can also be assessed in such detection schemes. Fingerprint profiles can be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.

[0154] Antibodies directed against p53 proteins may also be used in disease diagnostics and prognostics. Such diagnostic methods may be used to detect abnormalities in the level of expression of the peptide, or abnormalities in the structure and/or tissue, cellular, or subcellular location of the peptide. Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to one of skill in the art, including but not limited to Western blot analysis. For a detailed explanation of methods for carrying out Western blot analysis, see Sambrook and Russell (2001) supra. The protein detection and isolation methods employed herein can also be such as those described in Harlow and Lane, (1999) supra. This can be accomplished, for example, by

immunofluorescence techniques employing a fiuorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. The antibodies (or fragments thereof) useful in the present disclosure may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of the peptides or their allelic variants. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present disclosure. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the subject polypeptide, but also its

distribution in the examined tissue. Using the present disclosure, one of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[0155] Probes can be affixed to surfaces for use as "gene chips." Such gene chips can be used to detect genetic variations by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence of a by the sequencing by hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The probes of the disclosure also can be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659. A probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Patent No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

[0156] This disclosure also provides for a prognostic panel of genetic markers selected from, but not limited to the probes and/or primers to determine gene expression of p53 as identified herein. The probes or primers can be attached or supported by a solid phase support such as, but not limited to a gene chip or microarray.

[0157] When contacting is performed in vivo by administration of the peptides and/or polynucleotides to a subject other than a human patient such as a mouse, the method provides an animal model for use in discovering alternative agents, compositions and therapies. In a human patient, the method treats pathologies as described above or as characterized by hyperproliferative cells, e.g., cancer, breast cancer, colon cancer, colorectal cancer, brain cancer. Methods for detecting clinical and sub-clinical evidence of effective therapy are known in the art and described in U.S. Patent Appl. No. 2004/0087651, (published May 6, 2004), Balassiano et al. (2002) Intern. J. Mol. Med. 10:785-788; Thorne et al. (2004)

Neuroscience 127:481-496; Fernandes et al. (2005) Oncology Reports 13:943-947; da Fonseca et al. (2006) 66:611-615; da Fonseca et al. (2008) Surgical Neurology 70:259-267; da Fonseca et al. (2008) Arch. Immunol. Ther. Exp. 56:267-276 and Hashizume et al. (2008) Neuroncology 10: 112-120. In each of these methods, an effective amount of a peptide, polynucleotides and compositions are delivered or administered to the subject, e.g., mouse or human patient.

[0158] Further provided is a method for treating a disease or condition that can be treated by inhibiting the binding of a target peptide to its ligand, e.g., a disease or condition related to p53 misregulation or dysfunction, comprising, or alternatively consisting essentially of, or yet further consisting of, administering to a subject in need of such treatment an effective amount of one or more of: the polypeptide, the isolated polynucleotide, the host cell or the

compositions as described herein, thereby treating the disease or condition. In one aspect, the disease or conditions is cancer, e.g., breast cancer, colon cancer, colorectal cancer, brain cancer. In a further aspect, the patient has been pre-selected for the therapy as described above.

[0159] The compositions can be administered to an animal or mammal by a treating veterinarian or to a human patient by a treating physician.

[0160] The invention further provides methods of treating subjects such as cancer patients, in one aspect, patients pre-identified as having a cancer with low or absent p53 expression or p53 dysfunction. In one embodiment, the method comprises (a) determining the gene expression level of p53; and (b) administering to the subject an effective amount of a cyclotide. This therapy can be combined with other suitable therapies or treatments.

[0161] The cyclotide is administered in an effective amount to treat the condition or disease and by any suitable means and with any suitable formulation as a composition and therefore includes a carrier such as a pharmaceutically acceptable carrier. Accordingly, a formulation comprising the necessary cyclotide is further provided herein. The formulation can further comprise one or more preservatives or stabilizers. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, O.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, and 1.0%). The term carrier can includes a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-. quadrature. - cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0162] Exemplary combination therapies include chemotherapeutic regimens, such as combinations of platinum compounds and taxanes, e.g. carboplatin/paclitaxel,

capecitabine/docetaxel, the "Cooper regimen", fluorouracil-levamisole, fluorouracil- leucovorin, fluorouracil/oxaliplatin, methotrexate-leucovorin, and the like. Additional combination therapies include biologic therapies and radiation therapies; including therapies such as trastuzumab plus paclitaxel, alone or in further combination with platinum compounds such as oxaliplatin, for certain breast cancers, and many other such regimens for other cancers; and the "Dublin regimen" 5-fluorouracil IV over 16 hours on days 1-5 and 75 mg/m cisplatin IV or oxaliplatin over 8 hours on day 7, with repetition at 6 weeks, in combination with 40 Gy radiotherapy in 15 fractions over the first 3 weeks) and the

"Michigan regimen" (fluorouracil plus cisplatin or oxaliplatin plus vinblastine plus radiotherapy), both for esophageal cancer, and many other such regimens for other cancers, including colorectal cancer.

[0163] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

[0164] The invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of the cyclotide as described herein and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36,40, 48, 54, 60, 66, 72 hours or greater. The invention further comprises an article of manufacture, comprising packaging material, a first vial comprising the cyclotide and/or a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a physician, technician or patient to reconstitute the therapeutic in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.

[0165] Various delivery systems are known and can be used to administer a cyclotide of the disclosure, e.g., direct delivery, encapsulation in liposomes, microparticles, microcapsules. Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal and oral routes. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection or by means of a catheter.

[0166] The agents identified herein as effective for their intended purpose can be administered to subjects or individuals identified by the methods herein as suitable for the therapy. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the cyclotide or therapy.

Kits

[0167] In a yet further aspect, a kit is provided that comprises, or alternatively consists essentially of, or yet further consisting of, any of one or more of the isolated polypeptide as disclosed above, an isolated polynucleotide as disclosed herein, an isolated vector or host cell as described above, or the compositions, and instructions for use.

[0168] Having described the general concepts of this invention, the following illustrative examples are provided.

EXPERIMENTAL

Experiment No. 1

Cyclotides, a novel type of peptide-based therapeutics

[0169] Special attention has been recently given to the use of highly constrained peptides, also known as micro- or miniproteins, as extremely stable and versatile scaffolds for the production of high affinity ligands for specific protein capture and/or development of therapeutics (Sancheti, H. et al. (2009) Adv Drug Deliv Rev 61 :908-917; Craik, D.J. et al. (2007) Expert Opin Investig Drugs 16:595-604), Cyclotides are fascinating micro-proteins (-30 aa long) present in plants from the Violaceae, Rubiaceae, Cucurbitaceae and more recently Fabaceae (Poth, A.G. et al. (2011) Proc Natl Acad Sci USA 108: 1027-1032) and featuring various biological actions such as protease inhibitory, anti-microbial, insecticidal, cytotoxic, anti-HIV or hormone-like activity (Gould, A. et al. (2011) Curr Pharm Des 17:4294-4307). They share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology (Fig. 1). Cyclotides belong to the family of knottins, a group of microproteins that also includes conotoxins (389 sequences) and spider toxins (257 sequences). Cyclotides can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot. The main features of cyclotides are therefore a remarkable stability due to the cystine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. For example, the first cyclotide to be discovered, kalata Bl, is an orally effective uterotonic (Saether, O. et al. (1995) Biochemistry 34:4147- 4158), and other cyclotides have been shown to cross the cell membrane (Cascales, L. et al. (2011) J Biol Chem; Contreras, J. et al. (2011) J. Control Release). Cyclotides thus appear as promising leads or frameworks for peptide drug design (Henriques, S.T. et al. (2010) Drug Discov Today 15:5764; Garcia, A.E. et al. (2011) Biochemistry).

MCoTI-based cyclotides that induce programmed cell death

[0170] Applicants have developed cyclotides that activate programmed cellular death, a promising target for cancer therapy. Specifically, Applicants engineered a cyclotide (MCoTI- PMI) able to inhibit the interaction between p53 and Mdm2/MdmX. This was accomplished by engineering one the loops of the cyclotide MCoTI-I to display a small helical peptide derived from the natural Hdm2 -binding sequence of p53 (Li, C. et al. (2010) J Mol Biol 398:200-213). The a-helical segment was engineered using the peptide apamin (Li, C. et al. (2009) Agew Chem Int Ed Engl 48:8712-8715), a component of Apis melifera venom, to engineer the p53-based a-helical peptide PMI a-helix (Li, C. et al. (2010) J Mol Biol 398:200-213) and a turn into loop 6 of cyclotide MCoTI-L. Engineered cyclotide MCoTI- PMI was chemically produced (Fig. 3) or recombinantly expressed using intein-mediated cyclization with good yields (Fig. 4). MCoTI-PMI was also shown to fold adopting a native cyclotide fold by 2D-heteronuclear NMR (Fig. 5) and bind with high affinity to both Hdm2 and HdmX with a IQ value of 2.3 ± 0.1 nM and 9 ± InM, respectively as determined by fluorescence anisotropy. As expected, the cyclotide MCoTI-PMI-F3 A mutant did not bind neither the p53 binding domains of Mdm2 nor MdmX. This cyclotide contains a mutation (F5A) in the PMI peptide, which has been show to disable the interaction between PMI and Mdm2 or MdmX. The sequences of the peptides are shown in Table 2. Table 2

Sequences of MCoTI-PMI peptides

MCoT!-P !-2

MCoTi-PMi-3

here X*6~cNoro tryptophan

MCoTi-PMi-FM

[0171] Competitive inhibition experiments carried out in vitro with a FRET-based reporter formed by the p53 peptide (p53, aa 15-29) and the p53 binding domains of Mdm2 or Mdmx fused to the fluorescent protein YPet or CyPet, respectively also showed that MCoTI-PMI was able to inhibit the p53/Mdm2 and p53/MdmX interaction in a dose-dependent fashion with IC50 values of 41±1 nM and 171±1 nM, respectively. The IC50 value for nutlin-3 (a small molecule designed to inhibit the p53/Mdm2) (Vassilev, L.T. et al. (2004) Science 303:844-848) was around 3 times larger than of the cyclotide MCoTI-PMI (Fig. 7).

Interestingly, the cyclotide MCoTI-PMI was also able to inhibit the interaction between p53 and MdmX while nutlin-3 completely failed to inhibit it. As shown in Fig. 7, the empty scaffold, i.e. cyclotide MCoTI-I did not present any activity against either Mdm2 or MdmX.

[0172] More importantly, cyclotide MCoTI-PMI was shown to induce cytoxicity in p53-wt human cancer cells in a p53 dependent pathway with an IC50 value of ~10 μΜ, which similar to that of the small molecule nutlin-3 recently developed to antagonize p53/Mdm2 (Vassilev, L.T. et al. (2004) Science 303:844-848) (Fig. 8). In contrast to nutlin-3, the cyclotide MCoTI- PMI showed very little toxicity to normal epithelial cell lines (Fig. 8). Importantly, the cyclotide MCoTI-PMI also showed a remarkable resistance to degradation in human serum at 37°C (τι/2 ~ 32 h), while the linear and reduced form of MCoTI-PMI was rapidly degraded under the same conditions (τι _/2 ~ 0.5 h). It is worth noting that the chemical reduction of MCoTI-PMI required extreme conditions involving the use of 8 M urea in the presence of 100 mM OTT at pH 8.0. Remarkably, these results demonstrate that MCoTI-based engineered cyclotides can cross cell membranes to target cytosolic protein/protein

interactions, such as p53/Mdm2 and p53/MdmX and highlight the extraordinary resistance of this scaffold to chemical, physical and biological degradation.

[0173] Analytical HPLC was performed on a HP 1100 series instrument with 220 nm and 280 nm detection using a Vydac CI 8 column (5 μιη, 4.6 x 150 mm) at a flow rate of 1 mL/min. Semipreparative HP1C was performed on a Waters Delta Prep system fitted with a Waters 2487 Ultraviolet- Visible (UV-vis) detector using a Vydac C18 column (15-20 μιη, 10 x 250 mm) at a flow rate of 5 mL/Min. All runs used linear gradients of 0.1% aqueous trif uoroacetic acid (TFA, solvent A) vs. 0.1% TFA, 90% acetonitrile in H ₂ 0 (solvent B). UV- vis spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer, and fluorescence analysis on a Jobin Yvon Flurolog-3 spectrof urometer. Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all cyclized peptides. ES-MS was performed on a Sciex API-150EX single quadrupole electrospray mass spectrometer, MS/MS was performed on an Applied Biosystems API 3000 triple quadrupole mass spectrometer. Calculated masses were obtained by using ProMac vl .5.3. Protein samples were analyzed by SDS-PAGE. Samples were run on Invitrogen (Carlsbad, CA) 4-20% Tris-Glycine Gels. The gels were then stained with Pierce (Rockford, IL) Gelcode Blue, photographed/digitized using a Kodak (Rochester, NY) ED AS 290, and quantified using NIH Image-J software (http://rsb.info.nih.gov/ij/). DNA sequencing was performed by Davis Sequencing (Davis, CA) or DNA Sequencing and Genetic Analysis Core Facility at the University of Southern California using an ABI 3730 DNA sequencer, and the sequence data was analyzed with DNAStar (Madison, WI) Lasergene v5.5.2. All chemicals were obtained from Sigma-Aldrich (Milwaukee, WI) unless otherwise indicated.

Preparation of Fmoc-Tyr(tBu)-F

[0174] Fmoc-Tyr(tBu)-F was prepared using diethylaminosulfur trifluoride DAST and quickly used afterwards. To a stirred solution of the Fmoc-amino acid (10 mmol) in 60 ml of dry dichloromethane (DCM), under an argon atmosphere, 805 (10 mmol) of pyridine (dry) were added at room temperature followed by dropwise addition of 1.57 ml (12 mmol) of DAST. After stirring for 20 min, the mixture was extracted three times with 150 ml of ice water and the combined organic layers were dried over MgS04 and molecular sieves (10 A). The solvent was removed in vacuo at room temperature. Recrystallization or precipitation from DCM/n-hexane gave the Fmoc-amino acid fluoride.

Loading of 4-sulfamylbutyryl AM resin with Fmoc-Tyr(tBu)-F

[0175] Loading of the first residue was accomplished using Fmoc-Tyr(tBu)-F according to standard protocol (Ingenito, R. et al. (2002) Org. Lett. 4:1187-1188). Briefly, 4

sulfamylbutyryl AM resin (420 mg, 0.33 mmol) (Novabiochem) was swollen for 20 minutes with dry DCM and then drained. A solution of Fmoc-Tyr(tBu)-F (-461 mg, 1 mmol) in dry DCM (2 mL) and di-isopropylethylamine (DIEA) (180 μί, 1 mmol) was added to the drained resin and reacted at 25° C for 1 h. The resin was washed with dry DCM (5 x 5 mL), dried and kept at -20°C until use.

Chemical synthesis of MCoTI-PMI cyclotides

[0176] Solid-phase synthesis was carried out on an automatic peptide synthesizer ABI433A (Applied Biosystems) using the Fast-Fmoc chemistry with 2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU) activation protocol at 0.1 mmole scale on a FmocTyr(tBu)-sulfamylbutyryl AM resin. Side-chain protection was employed as previously described for the synthesis of peptide a-thiesters by the Fmoc-protocol (Camarero, J.A. et al. (2005) Protein Pept Lett 12:723-728), except for the N-terminal Cys residue, which was introduced as Boc-Cys(Trt)-OH. After chain assembly, the alkylation, thiolytic cleavage and deprotection were performed as previously described (Camarero, J.A. et al. (2005) Protein Pept Lett 12:723-728; Contreras, J. et al. (2011) J Control Release 155: 134-143). Briefly, -100 mg of protected peptide resin were first alkylated two times with ICH ₂ CN (174 μί, 2.4 mmol; previously filtered through basic silica) and DIEA (82 μί, 0.46 mmol) in N- methylpyrrolidone (NMP) (2.2 mL) for 12 h. The resin was then washed with NMP (3 x 5 mL) and OCM (3 x 5 mL). The alkylated peptide resin was cleaved with HSCH ₂ CH ₂ C0 ₂ Et (200 μΐ _^ , 1.8 mmol) in the presence of a catalytic amount of sodium thiophenolate (NaSPh, 3 mg, 22 μιηοΐ) in dimethylformamide (DMF):DCM (3:4 v/v, 1.4 mL) for 24 h. The resin was then dried at reduced pressure. The side-chain protecting groups were removed by treating the dried resin with trifluoro acetic acid (TFA):H ₂ 0:tri-isopropylsilane (TIS) (95:2:3 v/v, 5 mL) for 3-4 h at room temperature. The resin was filtered and the linear peptide thioester was precipitated in cold Et ₂ 0. The crude material was dissolved in the minimal amount of H ₂ 0:MeCN (4: 1) containing 0.1% TFA and characterized by HPLC and ES-MS as the desired MCoTI-I PMI linear precursor a-thioester [Expected mass (average isotopic composition) =5388.186 Da; measured =5388.26 ± 0.2 Da] and MCoTI-I PMI mutant linear precursor a-thioester [Expected mass (average isotopic composition) = 5312.08 Da; measured = 5312.85 ± 0.5 Da]. Cyclization and folding was accomplished by flash dilution of the MCoTI-I-PMI-linear α-thioester TFA crude to a final concentration of ~50μΜ into freshly degassed 2 mM reduced glutathione (GSH), 50mM sodium phosphate buffer at pH 7.5 for 18 h. Folded peptides were purified by semi-preparative HPLC using a linear gradient of 25- 45% solvent B over 30 min. Pure peptides were characterized by HPLC and ES-MS

[Expected mass for MCoTI-PMI (average isotopic composition) = 5262.04 Da; found= 5262.90 ± 0.4 Da; Expected mass for MCoTI-PMI-F3A (average isotopic composition) = 5185.10 Da; measured = 5185.80 ± 0.4 Da].

Construction of cyclotide expressing plasmids

[0177] Plasmids expressing the MCoTI-I precursors were constructed using the pTXBl expression plasmids (New England Biolabs), which contain an engineered Mxe Gyrase intein, respectively, and a chitin-binding domain (CBD). Oligonucleotides coding for the grafted MCoTI-I variants (Table 2) were synthesized, phosphorylated and PAGE purified by IDT DNA (Coralville, IA). Complementary strands were annealed in 0.3 M NaCl and the resulting double stranded DNA (dsDNA) was purified using Qiagen's (Valencia, CA) miniprep column and buffer PN. pTXBl plasmids was double digested with Ndel and Sapl (NEB). The linearized vectors and the MCoTI-I encoding dsDNA fragments were ligated at 15°C overnight using T4 DNA Ligase (New England Biolabs). The ligated plasmids were transformed into DH5a cells (Invitrogen) and plated on Luria Broth (LB)-agar containing ampicillin. Positive colonies were grown in 5 mL LB containing ampicillin at 37°C overnight and the corresponding plasmids purified using a Miniprep Kit (Qiagen). Plasmids were initially screened by EcoRI digestion, as this restriction site is removed during cloning.

Plasmids expressing the MCoTI-I precursors with an N-terminal TEV recognition sequence were cloned as follows. The DNA encoding TEV N-terminal recognition sequence was generated by PCR using the corresponding MCoTI-pTXBl plasmid. The 5' primer (5'- AAA CAT ATG GAA AAC CTG TAC TTC CAG TGC GGT TCT GGT TCT GG-3 ^* ) encoded a Ndel restriction site. The 3 ^* oligonucleotide (5 ^* -GAT TGC CAT GCC GGT CAA GG-3 ^* ) introduced a Spel restriction site during the PCR reaction. The PCR amplified product was purified, digested simultaneously with Ndel and Spel and then ligated into a Ndel- and Spel- treated plasmid pTXB-1 (New England Biolabs). The linearized vectors and the TEV- MCoTI-I encoding dsDNA fragments were ligated at 15°C overnight as described above. The ligated plasmids were transformed into DH5a cells and screened as described above. The DNA sequence of all the plasmids was confirmed by sequencing.

Table 3

Forward (p5) and reverse (p3) 5'-phosphorylated oligonucleotides used to clone the different MCo-PMI linear precursors into the pTXBl expression plasmid.

Cyclotide Name Oligonucleotide sequence

MCo-PMI p5 5' -

TATGTGCGGTTCTGGTTCTGGTGCTTCTAAAGCTCCGACCTCTTTCGCTGAATACTG G

AACCTGCTGTCTGCTGGTGGTGTTTGCCCGAAAATCCTGCAGCGTTGCCGTCGTGAC T

CTGACTGCCCGGGTGCTTGCATCTGCCGTGGTAACGGTTAC-3' p3

5'-

GCAGTAACCGTTACCACGGCAGATGCAAGCACCCGGGCAGTCAGAGTCACGACGG

CAACGCTGCAGGATTTTCGGGCAAACACCACCAGCAGACAGCAGGTTCCAGTATTCA

GCGAAAGAGGTCGGAGCTTTAGAAGCACCAGAACCAGAACCGCACA-3'

MCO-PMI-F42A p5 5'-

TATGTGCGGTTCTGGTTCTGGTGCTTCTAAAGCTCCGACCTCTGCTGCTGAATAC

TGGAACCTGCTGTCTGCTGGTGGTGTTTGCCCGAAAATCCTGCAGCGTTGCCGTCGT G

ACTCTGACTGCCCGGGTGCTTGCATCTGCCGTGGTAACGGTTAC-3' p3 5' -

GCAGTAACCGTTACCACGGCAGATGCAAGCACCCGGGCAGTCAGAGTCACGACGG CAACGCTGCAGGATTTTCGGGCAAACACCACCAGCAGACAGCAGGTTCCAGTATTCA GCAGCAGAGGTCGGAGCTTTAGAAGCACCAGAACCAGAACCGCACA -3 '

Expression and purification of recombinant MCoTI-PMI cyclotide

[0178] BL21 (DE3), Origami(DE3) or Origami2(DE3) cells (Novagen, San Diego, CA) were transformed with the MCoTI-I plasmids (see above). Expression was carried out in LB medium (1-2 L) containing ampicillin at room temperature or 30°C for 2 h or overnight (20 h), respectively. Briefly, 5 mL of an overnight starter culture derived from either a single clone or single plate were used to inoculate 1 L of LB media. Cells were grown to an OD at 600 nm of -0.6 at 37°C, and expression was induced by the addition of isopropyl-P-D- thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM at 30° C for 3 to 4 h. The cells were then harvested by centrifugation. For fusion protein purification, the cells were resuspended in 30 mL of lysis buffer (0.1 mM EDTA, 1 mM PMSF, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2 containing 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 min. The clarified supernatant was incubated with chitin-beads (2 mL beads/L cells, New England Biolabs), previously equilibrated with column buffer (0.1 mM EDTA, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2) at 4°C for 1 h with gentle rocking. The beads were extensively washed with 50 bead- volumes of column buffer containing 0.1% Triton XI 00 and then rinsed and equilibrated with 50 bead- volumes of column buffer. For the purification of TEV-MCoTI-intein-CBD fusion proteins, the beads were washed with 50 bead-volumes of TEV reaction buffer (50mM Tris-HCI, 0.5mM EDTA pH 8.0). Proteolytic cleavage of the TEV sequence was performed on the column by complementing the buffer with 3 mM reduced GSH and adding TEV protease to a final concentration of ~0.1 mg/mL. The proteolytic reaction was kept at 4° C overnight with gentle rocking. Once the proteolytic step was completed, the column was then washed with 50-bead volumes of column buffer. Chitin beads containing the different purified MCoTI-lntein-CBD fusion proteins were cleaved with 50 mM GSH in degassed column buffer. The cleavage reactions were kept for up to 1-2 days at 25°C with gentle rocking. Once the cleavage reaction was complete, the supernatant of the cleavage reaction was separated by filtration and the beads were washed with additional column buffer to reach a final concentration of 5 mM GSH, and the folding was allow to proceed with gently rocking at 4° C for 18 h. The folded MCoTI peptides were purified by semipreparative HPLC using a linear gradient of 25-45% solvent B over 30 min. Purified MCoTI-PMI cyclotides were characterized by C18-RP-HPLC and ES-MS; and quantified by UV-vis spectroscopy using an extinction coefficient at 280nm of 8,855 M cm ^_1 .

Refolding of TEV-MCoTI-PMI-intein-CBD from inclusion bodies

[0179] Inclusion bodies were first washed with column buffer containing 0.2%> tween 20 (3 x 50 mL) and then just column buffer (3 x 50 mL). The pellet was dissolved in 8M urea, 50 mM sodium phosphate and 250 mM NaCl, 8 M urea buffer at pH 7.2 (10 mL). After centrifugation at 15,000 rpm in a Sorval SS-34 rotor the supernatant was slowly flash diluted in 0. ImM EDTA, 50 mM sodium phosphate and 250 mM NaCl, 0.5 M Arg-HCI buffer at pH 7.2. This solution was dialyzed against column buffer (2 L) at 4° C for 2 days. The dialyzed solution was centrifuged at 15,000 rpm for 20 min in a Sorval SS-34 rotor and the

supernatant was purified by affinity chromatography on chitin beads. The fusion TEV- MCoTI-PMI-intein-CBD was treated as described above to remove the TEV-leading signal and induce backbone cyclization/folding of the MCoTI-PMI.

Expression of ¹⁵ N-labeled MCoTI-PMI

[0180] Expression was carried out using BL21(DE3) cells as described above except grown in M9 minimal medium containing 0.1% ¹⁵ NH ₄ C1 as the nitrogen source. Cyclization and folding was performed in solution and from the inclusion bodies as described above. The ¹⁵ N- labeled MCoTI-PMI was purified by semipreparative HPLC as before. Purified products were characterized by HPLC and ES-MS.

Preparation of FITC-labeled MCoTI-PMI-3

[0181] MCoTI-PMI-3 was prepared either by chemical synthesis or recombinant expression as described above. The pTXBl-MCoTI-PMI-3 plasmid was prepared by mutagenesis using pTXBl-TEV-MCoTI-PMI plasmid as template, forward primer (5 ^* -TGG TGC TTC TCG TGC TCC GAC CTC-3 ^* ) and reverse primer (5 ^* -AGG TCG GAG CAC GAG AAG CAC CAG-3'). MCoTI-PMI-3 was purified and characterized as described before. [Expected mass for MCoTI-I PMI-3 (average isotopic composition) = 5288.9 Da; found= 5289.3 ± 0.9 Da].

[0182] MCoTI-PMI-3 (100 μg) was mixed with 5 times excess FITC (molar ratio) in 0.1 M sodium bicarbonate buffer at pH 9.0. Reaction was carried out at room temperature in the dark for 2 h. The labeling reaction was quenched with diluted AcOH and the labeled cyclotide purified by CI 8 Semi-prep HPLC using a linear gradient of 10-45%) solvent B over 30 min. Pure labeled peptide was characterized by HPLC and ES-MS [Expected mass for FITC-MCoTI-PMI-3 (average isotopic composition) = 5678.3; found= 5678.0 ± 1.0 Da]. TEV protease expression and purification

[0183] BL21(DE3) cells were transformed plasmid pR 793, which encodes His-tagged TEV protease (Addgene).

[0184] Expression was carried out in 1 L of LB medium containing ampicillin (100 mg/L) and chloramphenicol (34 mg/L) at 30°C for 4 h. Briefly, 5 mL of an overnight starter culture derived from a single clone was used to inoculate 1 L of LB media. Cells were grown to an OD at 600 nm of ~ 0.6 at 37°C, and expression was induced by the addition of IPTG to a final concentration of 1 mM at 30° C overnight. The cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer (0.1 mM PMSF, 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl buffer at pH 8.0 containing 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 minutes. The clarified supernatant was incubated with 1 mL of Ni-NTA agarose beads (Qiagen) previously equilibrated with Ni-NTA column buffer (20 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl buffer at pH 8.0) at 4°C for 1 hour with gentle rocking. The Ni-NTA agarose beads were washed sequentially with Ni-NTA column buffer (2 x 100 mL). The fusion protein was eluted with Ni-NTA elution buffer (50 mM sodium phosphate, 250mM imidazole, 300 mM NaCl, buffer at pH 8) and immediately dialyzed in TEV-protease storage buffer (1 mM EDTA, 5 mM DTT, 50mM Tris-HCI buffer at pH 7.5 containing 50% (v/v) glycerol and 0.1% (w/v) Triton X-100 ). The purity of the TEV protease was checked by SDS-PAGE.

Cloning and expression of fluorescent protein YPet-p53

[0185] The DNA encoding the fluorescent protein YPet was isolated by PCR using the plasmid pBAD-6 (Kimura, R.H. et al. (2007) Anal Biochem 369:60-70) as a template. The forward (5 ^* -AAA AGG ATC CGA TGT CTA AAG GTG-3') and reverse (5 ^* -TTT TGA GCT CTT TGT ACA ATT CAT TC-3') primers contained a BamHI and Sacl restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into BamHI- and Sacl-treated plasmid pRSF-DUET-1 (Novagen) to give T7 expression vector pRSF-YPet. The resulting plasmid was sequenced and shown to be free of mutations. 5'-Phosphorylated synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) (5 ^* -pC GGT GGT TCT GGT GGT TCT GGT GGT TCT GGT GGT TCT GGT GGT TCT CTG CAG AGT CAG GAA ACA TTT TCA GAC CTA TGG AAA CTA CTT CCT GAA AAC TAA G-3 ^* and 5 ^* -pTC GAC TTA GTT TTC AGG AAG TAG TTT CCA TAG GTC TGA AAA TGT TTC CTG ACT CTG CAG AGA ACC ACC AGA ACC ACC AGA ACC ACC AGA ACC ACC AGA ACC ACC GAG CT-3 ^* ) encoding the flexible linker (Gly-Gly-Ser) ₆ fused in frame to the DNA encoding human p53 (15-29 aa) were annealed and ligated into pRSF-YPet using the Sacl and Sail restriction sites to give pRSF- YPet-p53. The resulting plasmid was sequenced and shown to be free of mutations.

BL21(DE3) cells (Novagen) (1 L) transformed with pRSF-YPet-p53 plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 mg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were harvested, lysed and YPet-p53 purified by Ni-affinity chromatography as described above. YPet-p53 protein was eluted with 50 mM sodium phosphate, 250 mM imidazole, 300 mM NaCl buffer at pH 8.0 containing 30% glycerol. The purified proteins were immediately dialyzed against the same buffer without imidazole and stored at -20° C until use. The purified protein was

characterized by SDS-PAGE and ES-MS.

Cloning and expression of CyPet-Mdm2

[0186] The DNA encoding the fluorescent protein CyPet was isolated by PCR using the plasmid pBAD-6 (Kimura, R.H. et al. (2007) Anal Biochem 369:60-70) as a template. The forward (5 ^* -AAA AGG ATC CAA TGT CTA AAG GTG AAG-3') and reverse (5 ^* - TTT TGA GCT CTT TGT ACA ATT CAT -3') primers contained a BamHI and Sacl restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into BamHI- and Sacl-treated plasmid pRSF-DUET-1 (Novagen) to give T7 expression vector pRSF-CyPet. The resulting plasmid was sequenced and shown to be free of mutations. The DNA encoding the p53 binding domain of the human homolog of Mdm2 (residues 17-125) was isolated by PCR using the cDNA for human Mdm2 (accession number: BT007258) as template. The forward primer (5 ^* -T GCA CTG CAG TCA CAG ATT CCA GCT TCG GAA C-3') encoded a Pstl restriction site. The reverse primer (5 - GC GTCGAC TTA GTT CTC ACT CAC AGA TGT ACC TGA-3') encoded a Sail site and a stop codon. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into Pstl- and Sall-treated plasmid pRSF-CyPet to give T7 expression vector pRSF-CyPet-Mdm2. The resulting plasmid was sequenced and shown to be free of mutations.

[0187] BL21 (DE3) cells (Novagen) (1 L) transformed with pRSF-CyPet-Mdm2 plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 mg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were harvested, lysed and CyPet-Mdm2 purified by Ni-affinity chromatography and stored as described before for YPet-p53. Purified CyPet-Mdm2 was characterized by SDS-PAGE and ES-MS.

Cloning and expression of CyPet-MdmX

[0188] The DNA encoding the p53 binding domain of the human homolog of MdmX (residues 17-116) was isolated by PCR using the cDNA for human MdmX (accession number: BC 105106) as a template. The forward primer (5 ^* - T GCA CTGCAG TGC AGG ATC TCT C-3 ^* ) encoded a Pstl restriction site. The reverse primer (5 ^* - GC GTC GAC TTA AGC ATC TGT AGT AGC AGT-3') encoded a Sail site and a stop codon. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into Pstl- and Sall-treated plasmid pRSF-CyPet to give T7 expression vector pRSF-CyPet-MdmX.

Rosetaa(DE3) cells (Novagen) (1 L) transformed with pRSF-CyPet-MdmX plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 mg/L) and chlorophenical (34 μg/mL) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were harvested, lysed and CyPet-MdmX purified by Ni-affinity

chromatography and stored as described above for YPet-pS3. Purified CyPet-Mdm2 was characterized by SDS-PAGE and ES-MS.

Human Mdm2 p53 biding domain cloning and expression

[0189] The DNA encoding the p53 binding domain of the human homolog of Mdm2 (residues 17-125) was isolated by PCR using the cDNA for human Mdm2 (accession number: BT007258) as template. The forward (5 ^* -AAA ACA TAT GTC ACA GAT TCC AGC TTC G-3 ^* ) and reverse (5 ^* -AAA AGG ATC CTT AGT TCT CAC TCA CAG ATG -3 ^* ) primers contained a Ndel and BamHI restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit,digested and ligated into Ndel- and BamHI- treated plasmid pET28a (Novagen) to give T7 expression vector pET28-Mdm2. The resulting plasmid was sequenced and shown to be free of mutations. BL21(DE3) cells (1L) transformed with pET28-Mdm2 plasmid were grown to mid-log phase (OD ₆ oo ~ 0.6) in LB medium containing kanamycin (34 mg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were lysed and the protein purified by Ni-affinity chromatography as described above. Mdm2 protein was dialyzed against PBS buffer (50mM sodium phosphate and 150mM NaCl pH 7.2) and stored until use.

Human mdmX p53 binding domain cloning and expression

[0190] The DNA encoding the p53 binding domain of the human homolog of MdmX (residues 17-116) was isolated by PCR using the cDNA for human MdmX (accession number: BC 105106) as a template. The forward (5 ^* -ATT AGG ATC CTG CAG GAT CTC TCC TGG ACA AAT C-3 ^* ) and reverse (5 ^* - ATT AAA GCT TCT ACT AAG CAT CTG TAG TAG CAG TGG CTA AAG TG - 3 ^* ) primers contained a BamHI and Hindlll restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into BamHI- and Hindlll-treated plasmid pET28a (Novagen) to give T7 expression vector pET28-MdmX. The resulting plasmid was sequenced and shown to be free of mutations. BL21(DE3) cells (1L) transformed with pET28-MdmX plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 mg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were lysed and the protein purified by Ni-affinity chromatography as described above.

MdmX protein was dialyzed against PBS buffer (50mM sodium phosphate and 150mM NaCl pH 7.2) and further purified gel filtration chromatography on a Superdex-75 column in PBS buffer. Pure fractions were pooled, re-concentrated and stored until use.

Expression of ¹⁵ N, ¹³ C-labeled Mdm2 p53 binding domain

[0191] Expression was carried out using BL21(DE3) cells as described above except grown

15 13

in M9 minimal medium containing 0.1% NH ₄ C1 and C6-D-glucose as the nitrogen and carbon sources, respectively. Protein purification was performed as described above.

NMR spectroscopy

[0192] NMR samples were prepared by dissolving ¹⁵ N-labeled peptides/proteins into 80 mM potassium phosphate in 90% H ₂ O/10% H ₂ 0 (v/v) or 100% D ₂ 0 to a concentration of approximately 0.2 mM with the pH adjusted to 6.5 by addition of dilute HCI. All 1H NMR data were recorded on a Bruker Avance II 700 MHz spectrometer equipped with a cryoprobe. Data were acquired at 27°C, and 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, was used as an internal reference. All 3D experiments, ¹ H{ ¹⁵ N}-TOCSY-HSQC and ¹ H{ ¹⁵ N}-NOESY, were performed according to standard procedures (Cavanagh, J. et al. (1992) J. Mag. Res. 96:670-678) with spectral widths of 12 ppm in proton dimensions and 35 ppm in nitrogen dimension. The carrier frequency was centered on the water signal, and the solvent was suppressed by using WATERGATE pulse sequence. TOCSY (spin lock time 80 ms) and NOESY (mixing time 150 ms) spectra were collected using 1024 t ₃ points, 256 t ₂ anf 128 ti blocks of 16 transients. Spectra were processed using Topspin 1.3 (Bruker). Each 3D-data set was apodized by 90°-shifted sinebell-squared in all dimensions, and zero filled to 1024 x 512 x 256 points prior to Fourier transformation.

Fluorescence polarization assay for MCoTI-PMI and Mdm2/MdmX interaction

[0193] Fluorescence polarization of FITC-Iabeled MCoTI-PMI-3 upon addition of either Mdm2 or MdmX was measured at 22°C using a Jobin Yvon/Spex Fluorolog 3

spectrofluorometer (Instrument S.A., Inc., Edison, NJ) with the excitation bandwidth set at 1 nm and emission at 5 nm. The excitation wavelength for Fluorescein was set at 495 nm and emission was monitored at 521 nm. The equilibrium dissociation constant (Kv) for the Mdm2/MCoTI-PMI or MdmX/MCoTI-PMI was obtained by titrating a fixed concentration of FITC-Iabeled MCoTI-PMI-3 (5 nM) with increasing concentrations of either Mdm2 or MdmX in 50 mM sodium phosphate, 150 mM NaCl buffer at pH 7.2 by assuming formation of a 1 : 1 complex and using the Prism (GraphPad) software package.

In vitro inhibition competition experiments

[0194] In vitro IC ₅ o values were measured by inhibition competition experiments using the FRET -based reporter formed by CyPet-Mdm2/CyPet-MdmX and YPet-p53. Briefly, a solution of CyPet-Mdm2/MdmX (20 nM) and 5 μΜΥΡεί-ρ53 in 10 mM phosphate buffer, 150 mM NaCl buffer at pH 7.2 was titrated with increasing amounts of inhibitor (ranging from 0 to 100 μΜ. The decrease in fluorescence signal at 525 nm (excited at 414 nm) was measured and plotted against the concentration of free inhibitor. The resulting plot was fitted to a single binding site competition curve using the Prism (GraphPad) software package. Cell culture

[0195] LnCaP, DU145, PC3, HEK293T and HBL100 cell lines were cultured in PRIM 1640 medium supplemented with 10% fetal calf serum, 50 IU/mL penicillin and 50 μg/mL streptomycin at 37° C in 5% C02 until confluent. HeLa and U20S cells were grown in Dulbecco's modified Eagle's medium (DMEM) medium complemented as before and under same conditions in temperature and C0 ₂ content.

MTT assay

[0196] The cytotoxicity of selected cyclotides will be tested against a panel of p53 wt versus p53 mutant or null cancer and normal cell lines using the MTT assay. Briefly, ~ 2 x 10 cells are plated in 96-well microtiter plates in 100 \xL DMEM in the presence of 10% calf serum. After 24 h incubation at 37°C in a humidified C0 ₂ - cotrol atmosphere the cells are washed with PBS and treated with 30 of PBS containing different concentrations (ranging from 0 to 100 μΜ) of peptides or nutlin-3 for 1 h at 37°C in 5% C0 ₂ . After 1 h, 150μΕ of full complemented media was added and the cells were grown for 48 h and then treated with 20 μΕ of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) for 2 h. The medium was discarded and DMSO (100 μΙΕΛνεΙΙ) was added to each well and incubated with gentle shaking for 20 min at room temperature. The OD at 595 nm of the solution was analyzed using a Tecan Genios Multifunctional Microplate Reader (Tecan System Inc., San Jose, CA) and the background at 670 nm subtracted.

Cell Cycle Assay

[0197] LnCaP cells were plated at density of 8 x 10 ⁴ cells per well in 12-well plates and grown in DMEM in the presence of 10% calf serum for 24 h. Cells were then washed with PBS and treated with 300 μΕΛνεΙΙ of PBS containing different concentrations (ranging from 0 to 100 μΜ) of peptides or nutlin-3 for 1 h at 37°C in 5% C0 ₂ . After 1 h, 3 mL of full complemented media was added and the cells were grown for 24 h, washed twice with ice- cold PBS, trypsinized and resuspended in ice-cold PBS at a density of ~ 2 x 10 ⁶ cells/mL. To this solution 9 ml of 70%> EtOH was added dropwise with gentle vortexing and stored at 4° C overnight. Cells were washed twice with ice-cold PBS, resuspended in PBS containing 0.1 % Triton X-100propidium iodide (40 μg/mL) and DNAse-free RNAse A (100 μg/mL); and incubated at 37°C for 15 min. The suspension was then analyzed in a FACSAria II (BD Biosciences, San Jose, CA).

Serum stability

[0198] Peptides (150 μg dissolved in 50 μΐ PBS) were mixed with 500 μΐ human serum and incubated at 37°C in water bath. Aliquot samples (50 μί) were taken at different time points (0-120 h) and precipitated with 20% trichloroacetic acid. After centrifugation the pellet was dissolved in 200 of 8 M GdmCl. Both the supernatant and solubilized pellet fractions were analyzed by HPLC-MS/MS.

Experiment No. 2

[0199] The following experiment was conducted as an extension of Experiment No. 1. Materials and Methods

[0200] Analytical HPLC was performed on a HP 1100 series instrument with 220 nm and 280 nm detection using a Vydac CI 8 column (5 μιη, 4.6 x 150 mm) at a flow rate of 1 mL/min. Semipreparative HPLC was performed on a Waters Delta Prep system fitted with a Waters 2487 Ultraviolet- Visible (UV-vis) detector using a Vydac C18 column (15-20 μιη, 10 x 250 mm) at a flow rate of 5 mL/min. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA, solvent A) vs. 0.1% TFA, 90% acetonitrile in H ₂ 0 (solvent B). UV-vis spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer, and fluorescence analysis on a Flurolog-3 spectroflurometer (Horiba Scientific). Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all cyclized peptides. ES-MS was performed on an Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer using Analyst 1.4.2. LC-MS were performed on a HP 1100 HPLC/API-3000 syste using a multiple reaction monitoring (MRM) mode. Calculated masses were obtained by using ProMac vl .5.3. Protein samples were analyzed by SDS-PAGE. Samples were run on Invitrogen (Carlsbad) 4-20% Tris-Glycine Gels. The gels were then stained with Pierce (Rockford) Gelcode Blue, photographed/digitized using a Kodak (Rochester) ED AS 290, and quantified using NIH Image-J software (http://rsb.info.nih.gov/ij/). DNA sequencing was performed by the DNA Sequencing and Genetic Analysis Core Facility at the University of Southern California using an ABI 3730 DNA sequencer, and the sequence data was analyzed with DNAStar Lasergene v5.5.2. All chemicals were obtained from Sigma-Aldrich unless otherwise indicated.

[0201] Preparation of Fmoc-Tyr(tBu)-F. Fmoc-Tyr(tBu)-F was prepared using diethylaminosulfur trifluoride (DAST) (Kaduk, C. et al. (1997) Lett. Pept. Sci. 2(5):285-288) and quickly used afterwards. To a stirred solution of the Fmoc-amino acid (10 mmol) in 60 ml of dry dichloromethane (DCM), under an argon atmosphere, 805 (10 mmol) of pyridine (dry) were added at room temperature followed by dropwise addition of 1.57 ml (12 mmol) of DAST. After stirring for 20 min, the mixture was extracted three times with 150 ml of ice water and the combined organic layers were dried over MgS04 and molecular sieves (10 A). The solvent was removed in vacuo at room temperature. Recrystallization or precipitation from DCM/n-hexane gave the Fmoc-amino acid fluoride.

[0202] Loading of 4-sulfamylbutyryl AM resin with Fmoc-Tyr(tBu)-F. Loading of the first residue was accomplished using Fmoc-Tyr(tBu)-F according to standard protocol (Ingenito, R. et al. (2002) Org. Lett. 4(7): 1187-1188). Briefly, 4-sulfamylbutyryl AM resin (420 mg, 0.33 mmol) (Novabiochem) was swollen for 20 minutes with dry DCM and then drained. A solution of Fmoc-Tyr(tBu)-F (-461 mg, 1 mmol) in dry DCM (2 mL) and di- isopropylethylamine (DIEA) (180 μί, 1 mmol) was added to the drained resin and reacted at 25° C for 1 h. The resin was washed with dry DCM (5 x 5 mL), dried and kept at -20°C until use.

[0203] Chemical synthesis of MCoTI-PMI cyclotides. Solid-phase synthesis was carried out on an automatic peptide synthesizer ABI433A (Applied Biosystems) using the Fast-Fmoc chemistry with 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) activation protocol at 0.1 mmole scale on a Fmoc-Tyr(tBu)-sulfamylbutyryl AM resin. Side-chain protection was employed as previously described for the synthesis of peptide a-thiesters by the Fmoc-protocol (Camarero, J.A. et al. (2005) Protein Pept Lett. 12(8):723-728), except for the N-terminal Cys residue, which was introduced as Boc- Cys(Trt)-OH. After chain assembly, the alkylation, thiolytic cleavage and deprotection were performed as previously described (Camarero, J.A. et al. (2005) Protein Pept Lett. 12(8):723- 728; Contreras, J. et al, (2011) J Control Release 155(2): 134-43). Briefly, -100 mg of protected peptide resin were first alkylated two times with ICH ₂ CN (174 μί, 2.4 mmol; previously filtered through basic silica) and DIEA (82 μΐ _^ , 0.46 mmol) in N- methylpyrrolidone (NMP) (2.2 mL) for 12 h. The resin was then washed with NMP (3 x 5 mL) and DCM (3 x 5 mL). The alkylated peptide resin was cleaved with HSCH ₂ CH ₂ C0 ₂ Et (200 μί, 1.8 mmol) in the presence of a catalytic amount of sodium thiophenolate (NaSPh, 3 mg, 22 μιηοΐ) in dimethylformamide (DMF):DCM (3:4 v/v, 1.4 mL) for 24 h. The resin was then dried at reduced pressure. The side-chain protecting groups were removed by treating the dried resin with trifluoro acetic acid (TFA):H ₂ 0:tri-isopropylsilane (TIS) (95:2:3 v/v, 5 mL) for 3-4 h at room temperature. The resin was filtered and the linear peptide thioester was precipitated in cold Et ₂ 0. The crude material was dissolved in the minimal amount of H ₂ 0:MeCN (4: 1) containing 0.1% TFA and characterized by HPLC and ES-MS. Cyclization and folding was accomplished by flash dilution of the MCoTI-I-PMI-I linear a-thioester TFA crude to a final concentration of ~ 50 μΜ into freshly degassed 2 mM reduced glutathione (GSH), 50 mM sodium phosphate buffer at pH 7.5 for 18 h. Folded peptides were purified by semi-preparative HPLC using a linear gradient of 25-45% solvent B over 30 min. Pure peptides were characterized by HPLC and ES-MS (Figure 10 and Table 4).

Table 4. Molecular weights and molar absortivities for the folded cyclotides.

Peptide Name Molecular weight (Da) Molar extinction coefficient (M ¹ cm ^"1 )

Expected Found

MCo-PMI 5262.0 5262.9 ± 0.4 8855 ^a

¹⁵ N-MCo-PMI 5329.9 5329.9 ± 0.2 8855 ^a

MCo-PMI-K37R 5291.3 5290.8 ± 1.0 8855 ^a

MCo-PMI-F42A 5185.1 5185.8 ± 0.4 8855 ^a

MC0-PMI-6CIW 5324.2 5324.8 ± 0.5 8855 ^a

FITC-MCo-PMI-K37R-F42A 5604.0 5603.4 ± 0.2 70,000 ^b

FITC-MCo-PMI-K37R 5678.3 5679 ± 1 70,000 ^b

FITC-MC0-6CIW 5713.6 5713.3 ± 0.5 70,000 ^b

a 280 nm, pH 7.4

b 494 nm, pH 8.0

Construction of cyclotide expressing plasmids

[0204] Plasmids expressing the MCo-PMI cyclotides were constructed using the pTXBl expression plasmid (New England Biolabs), which contain an engineered Mxe Gyrase intein, respectively, and a chitin-binding domain (CBD). Oligonucleotides coding for the different MCo-PMI variants (Table 2) were synthesized, phosphorylated and PAGE purified by IDT DNA. Complementary strands were annealed in 20 mM sodium phosphate, 300 mM NaCl and the resulting double stranded DNA (dsDNA) was purified using Qiagen's (Valencia, CA) miniprep column and buffer PN. pTXBl plasmids was double digested with Ndel and Sapl (NEB). The linearized vectors and the MCoTI-I encoding dsDNA fragments were ligated at 16° C overnight using T4 DNA Ligase (New England Biolabs). The ligated plasmids were transformed into DH5 cells (Invitrogen) and plated on Luria Broth (LB)-agar containing ampicillin. Positive colonies were grown in 5 mL LB containing ampicillin at 37°C overnight and the corresponding plasmids purified using a Miniprep Kit (Qiagen). Plasmids expressing the MCo-PMI cyclotide precursors with an N-terminal TEV recognition sequence were cloned as follows. The DNA encoding TEV N-terminal recognition sequence was generated by PCR using the corresponding MCo-PMI-pTXBl plasmid. The 5' primer (5'- AAA CAT ATG GAA AAC CTG TAC TTC CAG TGC GGT TCT GGT TCT GG-3') encoded an Nde I restriction site. The 3 ^* oligonucleotide (5 ^* -GAT TGC CAT GCC GGT CAA GG-3') introduced a Spe I restriction site during the PCR reaction. The PCR amplified product was purified, digested simultaneously with Nde I and Spe I and then ligated into an Nde I- and Spe I-treated plasmid pTXB-1 (New England Biolabs). The linearized vectors and the TEV-MCo-PMI encoding dsDNA fragments were ligated at 15°C overnight as described above. The ligated plasmids were transformed into DH5a cells and screened as described above.

Expression and purification of recombinant MCo-PMI cyclotides

[0205] BL21(DE3) (Novagen) were transformed with MCo-PMI encoding plasmids (see above). Expression was carried out in LB medium (1 L) containing ampicillin (100 μg/mL) at 30°C for 4 h respectively. Briefly, 5 mL of an overnight starter culture derived from either a single clone or single plate were used to inoculate 1 L of LB media. Cells were grown to an OD at 600 nm of ~ 0.6 at 37° C, and expression was induced by the addition of isopropyl-β- D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM at 30° C for 4 h. The cells were then harvested by centrifugation. For fusion protein purification, the cells were resuspended in 30 mL of lysis buffer (0.1 mM EDTA, 1 mM PMSF, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2 containing 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 min. The clarified supernatant was incubated with chitin-beads (2 mL beads/L cells) (New

England Biolabs), previously equilibrated with column buffer (0.1 mM EDTA, 50 mM sodium phosphate, 250 mM NaCl buffer at pH 7.2) at 4°C for 1 h with gentle rocking. The beads were extensively washed with 50 bead- volumes of column buffer containing 0.1% Triton XI 00 and then rinsed and equilibrated with 50 bead- volumes of column buffer. For the purification of TEV-MCo-PMI-intein-CBD fusion proteins, the beads were washed with 50 bead-volumes of TEV reaction buffer (50mM Tris'HCl, 0.5mM EDTA pH 8.0). Proteolytic cleavage of the TEV sequence was performed on the column by complementing the buffer with 3 mM reduced GSH and adding TEV protease to a final concentration of ~ 0.1 mg/mL. The proteolytic reaction was kept at 4° C overnight with gentle rocking. Once the proteolytic step was completed, the column was then washed with 50-bead volumes of column buffer. Chitin beads containing the different purified MCo-PMI-Intein-CBD fusion proteins were cleaved with 50 mM GSH in degassed column buffer. The cleavage reactions were kept for up to 1-2 days at 25°C with gentle rocking. Once the cleavage reaction was complete, the supernatant of the cleavage reaction was separated by filtration and the beads were washed with additional column buffer to reach a final concentration of 5 mM GSH, and the folding was allow to proceed with gently rocking at 4° C for 48 h. Folded MCo-PMI cyclotides were purified by semipreparative HPLC using a linear gradient of 25-45% solvent B over 30 min. Purified MCoTI-PMI cyclotides were characterized by C18-RP-HPLC and ES-MS (Fig. 10); and quantified by UV-vis spectroscopy. (Table 4)

Refolding of TEV-MCo-PMI-intein-CBD constructs from inclusion bodies

[0206] Inclusion bodies were first washed with column buffer containing 0.2% Triton X (50 mL) and then just column buffer (3 x 50 mL). The pellet was dissolved in 50 mM sodium phosphate and 250 mM NaCl, 8 M urea buffer at pH 7.2 (10 mL). After centrifugation at 15,000 rpm in a Sorval SS-34 rotor the supernatant was slowly flash diluted in O.lmM EDTA, 50 mM sodium phosphate and 250 mM NaCl, 0.5 M Arg ^» HCl buffer at pH 7.2. This solution was dialyzed against column buffer (2 L) at 4° C for 2 days. The dialyzed solution was centrifuged at 15,000 rpm for 20 min in a Sorval SS-34 rotor and the supernatant was purified by affinity chromatography on chitin beads. TEV-MCo-PMI-intein-CBD constructs were treated as described above to remove the TEV-leading signal and induce backbone cyclization/folding of the MCo-PMI cyclotides.

Expression of ¹⁵ N-labeled MCo-PMI

[0207] Expression was carried out using BL21(DE3) cells as described above except grown in M9 minimal medium containing 0.1% ¹⁵ NH ₄ C1 as the nitrogen source. Cyclization and folding was performed in solution as described above. ¹⁵ N-labeled MCo-PMI was purified by semipreparative HPLC as before. Purified products were characterized by HPLC and ES-MS (Fig. 11).

Preparation of FITC-labeled MCo-PMI cyclotides

[0208] MCO-PMI-K37R and MCo-PMI-K37R-F42A were prepared either by chemical synthesis or recombinant expression as described above. The pTXBl-TEV-MCo-PMI-K37R and pTXBl-TEV-MCo-PMI-K37R-F42A plasmids were prepared by mutagenesis using pTXBl-TEV-MCo-PMI or pTXBl-TEV-MCo-PMI- F42A plasmids as template, respectively, and the forward primer (5' - T GGT GCT TCT CGT GCT CCG ACC TC - 3') and reverse primer (5'- G AGG TCG GAG CAC GAG AAG CAC CA - 3') in both cases. MCoTI-PMI-K37R and MCo-PMI-K37R-F42A were purified and characterized as described before (Fig. 10 and Table 4).

[0209] MCo-PMI cyclotides (100 μg) were mixed with 5 times excess FITC (molar ratio) in 0.1 M sodium bicarbonate buffer at pH 9.0. Reaction was curried out at room temperature in the dark for 2 h. The labeling reaction was quenched with diluted AcOH and the labeled cyclotide purified by CI 8 Semi-prep HPLC using a linear gradient of 10-45% solvent B over 30 min. Pure labeled peptide was characterized by HPLC and ES-MS (Fig. 10 and Table 4).

TEV protease expression and purification

[0210] BL21(DE3) cells were transformed plasmid pRK793, which encodes His-tagged TEV protease (Addgene). Expression was carried out in 1 L of LB medium containing ampicillin (100 μg/L) and chloramphenicol (34 μg/L) at 30°C for 4 h. Briefly, 5 mL of an overnight starter culture derived from a single clone was used to inoculate 1 L of LB media. Cells were grown to an OD at 600 nm of ~ 0.6 at 37°C, and expression was induced by the addition of IPTG to a final concentration of 1 mM at 30° C overnight. The cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer (0.1 mM PMSF, 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl buffer at pH 8.0 containing 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 minutes. The clarified supernatant was incubated with 1 mL of Ni-NTA agarose beads (Qiagen) previously equilibrated with Ni-NTA column buffer (20 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl buffer at pH 8.0) at 4°C for 1 hour with gentle rocking. The Ni-NTA agarose beads were washed sequentially with Ni-NTA column buffer (2 x 100 mL). The fusion protein was eluted with Ni-NTA elution buffer (50 mM sodium phosphate, 250mM imidazole, 300 mM NaCl, buffer at pH 8) and immediately dialyzed in TEV-protease storage buffer (1 mM EDTA, 5 mM DTT, 50mM Tris'HCl buffer at pH7.5 containing 50% (v/v) glycerol and 0.1 % (w/v) Triton X-100). The purity of the TEV protease was checked by SDS-PAGE.

Cloning and expression of fluorescent protein YPet-p53

[0211] The DNA encoding the fluorescent protein YPet was isolated by PCR using the plasmid pBAD-6 (Kimura, R.H. et al. (2007) Anal Biochem 36(l):60-70) as a template. The forward (5 '-AAA AGG ATC CGA TGT CTA AAG GTG-3') and reverse (5'-TTT TGA GCT CTT TGT ACA ATT CAT TC-3') primers contained a BamHI and Sad restriction site, respectively. The resulting amplicon was purified using Qiagen' s PCR purification kit, digested and ligated into BamH - and Sacl-treated plasmid pRSF-DUET-1 (Novagen) to give T7 expression vector pRSF-YPet. The resulting plasmid was sequenced and shown to be free of mutations. 5'-Phosphorylated synthetic DNA oligos (IDT) (5'-C GGT GGT TCT GGT GGT TCT GGT GGT TCT GGT GGT TCT GGT GGT TCT CTG CAG AGT CAG GAA ACA TTT TCA GAC CTA TGG AAA CTA CTT CCT GAA AAC TAA G-3' and 5 '-TC GAC TTA GTT TTC AGG AAG TAG TTT CCA TAG GTC TGA AAA TGT TTC CTG ACT CTG CAG AGA ACC ACC AGA ACC ACC AGA ACC ACC AGA ACC ACC AGA ACC ACC GAG CT-3') encoding a flexible linker (Gly-Gly-Ser) ₆ fused in frame to the DNA encoding human p53 (15-29 aa) were annealed and ligated into pRSF-YPet using the Sac I and Sal I restriction sites to give pRSF-YPet-p53. The resulting plasmid was sequenced and shown to be free of mutations. BL21(DE3) cells (Novagen) (1L) transformed with pRSF- YPet-p53 plasmid were grown to mid- log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 μg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were harvested, lysed and YPet-p53 purified by Ni-affinity chromatography as described above. YPet-p53 protein was eluted with 50 mM sodium phosphate, 250 mM imidazole, 300 mM NaCl buffer at pH 8.0 containing 30% glycerol. The purified proteins were immediately dialyzed against the same buffer with no imidazole and stored at -20° C until use. The purified protein was characterized by SDS-PAGE and ES-MS (Fig. 13).

Cloning and expression of CyPet-Hdm2

[0212] The DNA encoding the fluorescent protein CyPet was isolated by PCR using the plasmid pBAD-6 (Kimura, R.H. et al. (2007) Anal Biochem 36(l):60-70) as a template. The forward (5 '-AAA AGG ATC CAA TGT CTA AAG GTG AAG-3') and reverse (5'- TTT TGA GCT CTT TGT ACA ATT CAT -3') primers contained a BamH I and Sac I restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into BamH I- and Sac I-treated plasmid pRSF-DUET-1 (Novagen) to give T7 expression vector pRSF-CyPet. The resulting plasmid was sequenced and shown to be free of mutations. The DNA encoding the p53 binding domain of the human homo log of Mdm2 (Hdm2, residues 17-125) was isolated by PCR using the cDNA for Hdm2 (accession number: BT007258) as template. The forward primer (5'-T GCA CTG CAG TCA CAG ATT CCA GCT TCG GAA C-3') encoded a Pst I restriction site. The reverse primer (5'- GC GTCGAC TTA GTT CTC ACT CAC AGA TGT ACC TGA-3') encoded a Sal I site and a stop codon. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into Pst I-and Sal I-treated plasmid pRSF-CyPet to give T7 expression vector pRSF-CyPet-Hdm2. The resulting plasmid was sequenced and shown to be free of mutations. BL21(DE3) cells (Novagen) (1L) transformed with pRSF-CyPet-Hdm2 plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 μ^) at 37° C and then induced with 1 mM IPTG at 30°C for 4 h. Cells were harvested, lysed and CyPet-Hdm2 purified by Ni-affinity chromatography and stored as described before for YPet-p53. Purified CyPet-Hdm2 was characterized by SDS-PAGE and ES-MS (Fig. 13).

Cloning and expression of CyPet-HdmX

[0213] The DNA encoding the p53 binding domain of the human homolog of MdmX (HdmX, residues 17-116) was isolated by PCR using the cDNA for HdmX (accession number: BC 105106) as a template. The forward primer (5 '- T GCA CTGCAG TGC AGG ATC TCT C-3 ') encoded a Pst I restriction site. The reverse primer (5 '- GC GTC GAC TTA AGC ATC TGT AGT AGC AGT-3') encoded a Sal I site and a stop codon. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into Pstl- and iSa/I-treated plasmid pRSF-CyPet to give T7 expression vector pRSF-CyPet-HdmX.

[0214] Rosetta(DE3) cells (Novagen) (1L) transformed with pRSF-CyPet-HdmX plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 mg/L) and chlorophenical (34 μ^) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were harvested, lysed and CyPet-HdmX purified by Ni-affinity

chromatography and stored as described above for YPet-p53. Purified CyPet-Hdm2 was characterized by SDS-PAGE and ES-MS (Fig. 13).

Cloning and expression of Hdm2 (17-125)

[0215] The DNA encoding the p53 binding domain of Hdm2 (residues 17-125) was isolated by PCR using the cDNA for Hdm2 (accession number: BT007258) as template. The forward (5 '-AAA ACA TAT GTC ACA GAT TCC AGC TTC G-3 ') and reverse (5 '-AAA AGG ATC CTT AGT TCT CAC TCA CAG ATG -3 ') primers contained an Nde I and BamH I restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into Nde I- and BamH I-treated plasmid pET28a (Novagen) to give T7 expression vector pET28-Hdm2. The resulting plasmid was sequenced and shown to be free of mutations. BL21 (DE3) cells (1L) transformed with pET28-Hdm2 plasmid were grown to mid-log phase (OD ₆ oo ~ 0.6) in LB medium containing kanamycin (34 μg/L) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were lysed and the protein purified by Ni-affinity chromatography as described above. Hdm2 (17-125) was dialyzed against PBS buffer (50mM sodium phosphate and 150mM NaCl pH 7.2 containing 30% glycerol) and immediately used.

Cloning and expression of HdmX (17-116)

[0216] The DNA encoding the p53 binding domain of the human homolog of MdmX (HdmX, residues 17-1 16) was isolated by PCR using the cDNA for HdmX (accession number: BC 105106) as a template. The forward (5 ' -ATT AGG ATC CTG CAG GAT CTC TCC TGG ACA AAT C-3 ') and reverse (5 '- ATT AAA GCT TCT ACT AAG CAT CTG TAG TAG CAG TGG CTA AAG TG -3 ') primers contained a BamH I and Hind III restriction site, respectively. The resulting amplicon was purified using Qiagen's PCR purification kit, digested and ligated into BamH I- and Hind Ill-treated plasmid pET28a (Novagen) to give T7 expression vector pET28-HdmX. The resulting plasmid was sequenced and shown to be free of mutations. BL21(DE3) cells (1L) transformed with pET28-HdmX plasmid were grown to mid-log phase (OD at 600 nm ~ 0.6) in LB medium containing kanamycin (34 μ^) at 37° C and then induced with 1 mM IPTG at 30°C for 4 hours. Cells were lysed and the protein purified by Ni-affinity chromatography as described above. HdmX (17-116) was dialyzed against PBS buffer (50mM sodium phosphate and 150mM NaCl pH 7.2) and further purified by gel filtration chromatography on a Superdex-75 column in PBS buffer. Pure fractions were pooled, re-concentrated in 50mM sodium phosphate and 150mM NaCl pH 7.2 and immediately used.

Expression of ¹⁵ N, ¹³ C-labeled Hdm2 (17-125)

[0217] Expression was carried out using BL21(DE3) cells as described above except grown

15 13

in M9 minimal medium containing 0.1% NH ₄ C1 and C6-D-glucose as the nitrogen and carbon sources, respectively. Protein purification was performed as described above. Protein was characterized by ES-MS. (Fig. 14)

NMR spectroscopy

13 15

[0218] NMR samples were prepared by dissolving C- and/or N-labeled

peptides/proteins into 80 mM potassium phosphate in 90% Η ₂ Ο/10%> H ₂ 0 (v/v) or 100%) D ₂ 0 to a concentration of approximately 0.2 mM and molar ratio of 1 : 1 between MCo-PMI and Hdm2 with the pH adjusted to 6.5 by addition of dilute HC1. All NMR data were recorded on a Bruker Avance II 700 MHz spectrometer equipped with a cryoprobe. Data were acquired at 27 °C, and 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, was used as an internal reference. All 3D experiments, HNCA, HNCACB, CBCACONH, HBHACONH, 1H{ ¹⁵ N}-TOCSY-HSQC 1H{ ¹³ C}-NOESY-HSQC and 1H{ ¹⁵ N}-NOESY-HSQC (Cavanagh, J. et al. (1996) Protein NMR Spectroscopy: Principles and Practice, San Diego: Academic Press, 587), were performed according to standard procedures (Cavanagh, J. et al. (1992) J. Magn. Res. 96:670-678) with spectral widths of 12 ppm in proton dimensions, 70 or 35 ppm in carbon dimension, and 35 ppm in nitrogen dimension. The carrier frequency was centered on the water signal, and the solvent was suppressed by using WATERGATE pulse sequence. TOCSY (spin lock time 80 ms) and NOESY (mixing time 150 ms) spectra were collected using 1024 t ₃ points, 256 t ₂ anf 128 ti blocks of 16 transients. Spectra were processed using Topspin 2.1 (Bruker). Each 3D-data set was apodized by 90°-shifted sinebell-squared in all dimensions, and zero filled to 1024 x 512 x 256 or 1024x256x128 points prior to Fourier transformation. Chemical shifts were assigned by using CARA software (Masse, J.E. et al. (2005) J. Mag Reson 174(1): 133-151).

Structure determination

[0219] Structural calculations were carried out with Cyana 2.1 (Guntert, P. (2004) Methods Mol Biol 278:353-378) using 986 distance restraints derived from ¹ H- ¹ H-NOESY, ¹³ C-edited NOESY and ¹⁵ N-edited NOESY spectra, 150 pairs of backbone torsion angle restraints derived from TALOS (Cornilescu, G.F. et al. (1999) J Biomol NMR 13(3):289-302), 38 restraints for hydrogen bonds, and the restraints from three disulfide bond between Cys25 and Cys42, Cys32 and Cys44, and Cys38 and Cys50 of MCo-PMI. To account for cyclic nature of MCo-PMI, amide nitrogen of Glyl and carbonyl carbon of Cys51 were linked and upper and lower distance restraints between amide proton and nitrogen of Glyl and carbonyl carbon and oxygen of Cys51 were added (Guntert, P. (2004) Methods Mol Biol 278:353-378). nOes were converted to upper limit distances using the CALIBA module in CYANA (Guntert, P. (2004) Methods Mol Biol 278:353-378). The reference volume determined by CALIBA was increased 2 times before conversion in order to loosen the distance restraints. All upper limit distances for intermolecular nOes were set to 5 A. Backbone torsion angle restraints for MCo-PMI-Hdm2 were estimated by using the TALOS (Cornilescu, G.F. et al. (1999) J Biomol NMR 13(3):289-302). These experimental restraints are summarized in Table 5. To perform CYANA calculations, a single polypeptide chain was constructed for the MCo-PMI and Hdm2 molecules. Table 5. NMR and refinement statistics for MCo-PMI Hdm2 complex

MCo-PMI Hdm2

NMR distance and dihedral constraints

Distance restraints

Total NOE 257 433

Intra-residue 28 65

Inter-residue 229 368

Sequential (\i -j\ = 1) 103 166

Nonsequential (\i -j\ > 1 ) 126 202

Hydrogen bonds 22 61

MCo-PMI Hdm2 intermolecular 33

Total dihedral angle restraints 91 159

φ 45 80

ψ 46 79

Structure statistics

Violations (mean and s.d.)

Distance constraints (A) 0.0153 ± 0.0005 0.0175 ± 0.0024

Dihedral angle constraints (°) 0.5494 ± 0.3491

Max. dihedral angle violation (°) 3.81 ± 0.22

Max. distance constraint violation (A) 0.17 ± 0.01

Deviations from idealized geometry

Bond lengths (A) 0.001 0.001

Bond angles (°) 0.2 0.2

Average pairwise r.m.s. deviation* (A)

Heavy 1.26 ± 0.21 1.26 ± 0.13

Backbone 0.64 ± 0.14 0.62 ± 0.14

* Compared to first structure r.m.s deviation was calculated for 10 refined structures.

[0220] The CYANA-generated structures were subjected to minimization in explicit water by using CHRMM (Brooks, B.R. et al. (2009) J Comput Chem 30(10): 1545-1614) and further analysis by PROCHECK NMR (Laskowski, R.A. et al. (1996) J Biomol NMR 8(4):477-486). 86.5% of the V domain residues were in the most favorable regions of Ramachandran plot, 13.5% were in the additional allowed regions. There were no residues in generously allowed or the disallowed regions of the Ramachandran plot. The structural statistics of the 10 best structures are reported in Table 5. Fluorescence polarization binding assays

[0221] Fluorescence polarization of FITC-labeled MCo-PMI cyclotides upon addition of either Hdm2 (17-125) or HdmX (17-1 16) was measured at 22°C using a Spex Fluorolog 3 spectrofluorometer (Horiba Scientific) with the excitation bandwidth set at 1 nm and emission at 5 nm. The excitation wavelength for fluorescein was set at 495 nm and emission was monitored at 521 nm. The equilibrium dissociation constant (Kv) were obtained by titrating a fixed concentration of FITC-labeled MCo-PMI cyclotide (5 nM) with increasing concentrations of either Hdm2 (17-125) or HdmX (17-1 16) in 50 mM sodium phosphate, 150 mM NaCl buffer at pH7.2 by assuming formation of a 1 : 1 complex and using the Prism (GraphPad) software package.

In vitro inhibition p53-Hdm2/HdmX competition experiments

[0222] In vitro IC ₅ o values were measured by inhibition competition experiments using the FRET -based reporter formed by CyPet-Hdm2/CyPet-HdmX and YPet-p53. Briefly, a solution of CyPet-Hdm2/HdmX (20 nM) and YPet-p53 (5 μΜ) in 10 mM phosphate buffer, 150 mM NaCl buffer at pH 7.2 was titrated with increasing amounts of inhibitor (ranging from 0 to 100 μΜ). The decrease in fluorescence signal at 525 nm (excited at 414 nm) was measured and plotted against the concentration of free inhibitor. The resulting plot was fitted to a single binding site competition curve using the Prism (GraphPad) software package.

Cell viability assay

[0223] LnCaP, HCT1 16 p53 ^+/+ , HCT1 16 p53 ^_/" , JEG3, DU145, PC3, HEK293T and HBL100 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (50 IU/mL) and streptomycin (50 μg/mL) at 37° C in 5% C0 ₂ . Cell viability was performed using the MTT assay. Briefly, - 2 x 10 cells were seeded in 96-well microtiter plates in 100 μΐ _^ RPMI 1640 in the presence of 10% calf serum. After 24 h incubation the cells were washed with PBS and treated with 30 μΕΛνεΙΙ of PBS or RPMI 1640 media containing the peptides or Nutlin-3 at the indicated doses for 1 h at 37° C in 5% C0 ₂ . After 1 h, 210μΕΛνε11 of full complemented media was added and the cells were grown for 48 h and then treated with 20 μΐ _^ of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, 5 mg/mL) for 2 h. The medium was discarded and DMSO (100 μΙ,ΛνεΙΙ) was added to each well and incubated with gentle shaking for 20 min at room temperature. The absorbance at 595 nm of the solution was analyzed using a Tecan Genios Multifunctional Microplate Reader (Tecan System Inc) and the background at 670 nm subtracted.

Cell Cycle Assay

[0224] LnCaP and HCT116 p53 ^+/+ cells were seeded at a density of 8 x 10 ⁴ cells per well in 12-well plates and grown in RPMI 1640 in the presence of 10% calf serum for 24 h. Cells were washed with PBS and incubated with 300 μΙ _^ ΛνεΙΙ of PBS or RPMI 1640 containing the peptides or Nutlin-3 at the indicated doses for 1 h at 37°C in 5% C0 ₂ . After 1 h, 2.1 ml of full complemented media was added per well and the cells were grown for 24 h, washed twice with ice-cold PBS, trypsinized and resuspended in 0.3ml of ice-cold PBS at a density of ~ 2 x 10 ⁵ cells/mL. To this solution 0.7 ml of 100% EtOH was added dropwise with gentle vortexing and stored at 4° C overnight. Cells were washed once with ice-cold PBS, resuspended in PBS containing propidium iodide (10 μg/mL) and DNAse-free RNAse A (100 μg/mL); and incubated at 37° C for 15 min. The suspension was then analyzed in a FACSAria II (BD Biosciences).

Caspase-3/7 activation assay

[0225] LNCaP or HCT116 p53 ^+/+ cells were seeded in 96-well plates at a density of ~ 2 x 10 ³ cells and treated with vehicle, Nutlin 3 (EMD Chemicals) (20 μΜ), MCo-PMI (50 μΜ) or MCo-PMI-F43A (50 μΜ) as described before for the cell cycle analysis assay. After 24 h of treatment, caspase-3/7 activity was measured by addition of Caspase-Glo 3/7

chemiluminsicence reagent (Promega) according to the manufacturer's protocol and the luminescence was measured using a Synergy HI Hybrid Multi-Mode Microplate Reader (BioTek).

Co-immunoprecipitation analysis

[0226] LNCaP or HCT 116 p53 ^+/+ cells (~ l x l 0 ⁵ ) were incubated with FITC-labeled MCo- PMI peptides as described above for 1 h. After 30 h of treatment, cells were lysed using RIPA buffer (Sigma) containing 1 mM PMSF and EDTA-free Halt protease Inhibitor cocktail (Thermo Scientific). The cell lysate was incubated with 50 μΐ _^ of protein A/G high capacity agarose beads (Thermo Scientific) for 30 min at 4° C. The supernatant was collected and the proteins were precipitated with mouse-anti-FITC monoclonal IgG antibody (Invitrogen). Western analysis of electrophoresed proteins was performed using mouse anti-Hdni2 (Santacruz), rabbit anti-HdmX (Bethyl Laboratories) and mouse anti-GAPDH (Cell signaling). Western blots were visualized by fluorescence imaging using either a Storm 860 (Molecular Dynamics) or a Typhoon 8600 (Molecular Dynamics) imaging system.

Mice xenografts studies

[0227] HCT116 p53 ^+/+ xenografts were established by injecting 100 uL suspension of basal RPMI containing 0.5 x 10 ⁶ cells into the rear right flanks of female nude mice (nu/nu) mice (Simonsen Laboratories). When tumors reached an average volume of -100 mm , cohorts (n=3) were treated with vehicle (5% dextrose in water, D5W), MCo-PMI (40 mg/kg, 7.6 mmol/kg), or Nutlin-3 (10 mg/kg, 17.2 mmol/kg) (EMD Chemicals) once daily for up to 36 days by intravenous injection (MCo-PMI and vehicle) or by intraperitoneal administration (Nutlin-3). Compounds were prepared in a D5W at a final volume of 50 (peptide) or 100

(Nutlin-3). Health checks were performed daily to observe parameters such as body conditioning score, overall appearance and cleanliness, strength of grip, skin color and tone, mobility, gait and activity level as indicators of potential drug related toxicities. Individual weights were recorded thrice weekly (Fig. S7), comparing the control and treatment groups as an additional indicator of tolerance of drug treatment as well as providing the average weight for calculation of drug dosing. Tumor size was measured with calipers. Tumor volume was calculated by measuring tumor size in two dimensions and applying those measurements to the calculation V = d x D/2, where d and D equal to the smaller and larger of the two measurements, respectively. Mice bearing tumors larger than 2.4 cm were removed from the study, sacrificed and necropsies performed to gather tumor and organ samples for histological analysis. Tumor and tissue samples were perfused with PBS, after which portions were either fixed in 10% formalin overnight then transferred to 100% EtOH or snap-frozen in liquid nitrogen and stored at -70° C until analysis.

Quantitative RT-PCR

[0228] HCT116 p53 ^+/+ subcutaneous tumors were excised, flash frozen and the RNA extracted using the RNeasy Mini kit (QIAGEN). Total RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase (Promega). The generated cDNA was amplified with power SYBR Green PCR master mix (Applied Biosystems) on a 96 well plate and measured the relative transciprt levels by qRT-PCR on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Specific primers for HDM2, p21 and the β-actin control were used. The amplification reactions were done in triplicate in 96-well optical plates. Threshold- cycle (Ct) values were automatically calculated for each replicate and used to determine the relative expression of the gene of interest relative to β-actin.

Immunohistochemistry

[0229] Formalin-fixed, paraffin-embedded (FFPE) tumor samples were sectioned at a thickness of 5 μιη and mounted on pre-cleaned, charged glass slides. For

immunohistochemisty, tumor sections were de-paraffmized in 3 x 5 min changes of clear-rite 3 (Microm International). Samples were then rehydrated by treatment with 100% ethanol (EtOH) (2 x 10 min) followed by treatment with 95% EtOH in H ₂ 0 (2 x 10 min) and then washed with pure H ₂ 0. For antigen unmasking, slides were incubated at 95° C in 10 mM sodium citrate buffer containing 0.05 % tween-20 at pH 6.0 for 10 min. The slides were cooled to room temperature and washed 3 x 2 min in phosphate-buffered saline without calcium and magnesium salts (PBS) (3 x 2 min). Immunohistochemical staining was carried out using the Ultravision ONE Detection System (Thermo Scientific) containing horseradish- peroxidase polymer (Thermo Scientific) and DAB Plus Chromagen (Thermo Scientific) according to the manufacturer's directions. Briefly, tissues were incubated with hydrogen peroxide blocking (Thermo Scientific) reagent to quench endogenous peroxides, and then washed with PBS (4 x 2 min). Ultra V blocking agent (Thermo Scientific) was applied to prevent non-specific binding. The corresponding primary antibodies were prepared at a concentration of 1 μg/mL in PBS containing 1.5 % normal goat serum. Following overnight incubation at 4° C in the presence of antibody, tissue sections were washed as before and incubated with HRP polymer prior to application of DAB chromagen/substrate. Tissue sections were incubated with DAB for 3 min to allow color to develop. Slides were washed with pure H ₂ 0 (4 x 5 min), counterstained with hematoxylin solution (Microm International) for 1 min and immediately washed with pure H ₂ 0. Slides were dehydrated with successive washes of 95 % and 100 % EtOH, and clearite-3, then mounted with clarion mouting media (Sigma-Aldrich) and glass coverslips. Slides were air-dried overnight and visualized using Ziess Axioskop light microscope equipped with 5x and 20x objectives and motic digital camera.

Human serum stability

[0230] Peptides (150 μg dissolved in 50 μΐ, PBS) were mixed with 500 μΐ, human serum and incubated at 37° C. Aliquot samples (50 μί) were taken at different time points (0-120 h) and precipitated with 20% trichloroacetic acid. After centrifugation the pellet was dissolved in 200 μί of 8 M GdmCl. Both the supernatant and solubilized pellet fractions were analyzed by HPLC and LC-MS/MS. Each experiment was done in triplicate.

Human serum binding kinetics

[0231] Binding kinetics were carried out at 25 °C on a BLItz™ instrument (ForteBIO), using biotinylated MCo-PMI immobilized onto a streptavidin coated biosensor tip. MCo-PMI (1 mg, 190 nmol) was conjugated with three-fold molar excess of NHS-PEG4-Biotin in 0.1 M sodium phosphate buffer (1.9 mL) at pH 7.4 for 1 h. The reaction was quenched by adding 2% TFA until pH ~ 4. Purification and desalting of biotinylated MCo-PMI was performed on a Zeba spin desalting columns (Thermo Scientific). Binding of MCo-PMI to human serum proteins was performed at 1/100 and 1/200 serum dilutions in 20 mM sodium phosphate, 100 mM NaCl buffer at pH 7.2, which correspond to a concentration of 25 μΜ and 50 μΜ human serum albumin, respectively. Serum proteins were allowed to bind to the MCo-PMI coated biosensor tip for 2 minutes followed by a dissociation step of 2 minutes. Nonlinear regression analysis was performed using Prism (GraphPad Software) to calculate the association (k _on ) and dissociation {k ₀ fj) rates, and corresponding Κ _Ό value.

Results

Engineering cyclotide MCoTI-I to target p53-Hdm2/HdmX

[0232] Cyclotides MCoTI-I/II are powerful trypsin inhibitors (K, - 20 - 30 pM) which have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of the cucurbitaceae family (Hernandez, J.F. et al. (2000) Biochemistry 40:7973- 7983). Although MCoTI cyclotides do not share significant sequence homology with other cyclotides beyond the presence of the three cystine bridges, structural analysis by NMR has shown that they adopt a similar backbone-cyclic cystine-knot topology. (Heitz, A. et al. (2001) Biochemistry 40:7973-7983; Felizmenio-Quimio, M.E. et al. (2001) J Biol Chem 276:22875-22882) MCoTI cyclotides, however, show high sequence homology with related cystine-knot squash trypsin inhibitors (STIs) ⁴¹ and therefore represent interesting molecular scaffolds for drug design. (Reiss, S. et al. (2006) Platelets 17:153-157; Werle, M. et al. (2007) Int J Pharm 332:72-79).

[0233] To engineer the cyclotide MCoTI-I for antagonizing protein-protein interactions between p53 and Hdm2 or HdmX, Applicants used the phage-selected a-helical peptide PMI (Fig. 16). (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670) This peptide conserves the residues Phel9, Trp23 and Leu26 of p53 required for the interaction with Hdm2 and HdmX, and is able to bind the p53 binding domains of Hdm2 and Hdmx with low nM affinity (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670). The PMI peptide was grafted onto the cyclotide scaffold using loop 6. This loop has been shown previously to be more disordered in solution (Puttamadappa, S.S. et al. (2010) Angew Chem Int Ed Engl 49:7030-7034) and amenable to sequence variation. (Chan, L.Y. et al. (2011) Blood 118:6709-6717; Thongyoo, P. et al. (2009) J Med Chem 52:6197-6200; Austin, J. et al. (2009) Chembiochem 10:2663-2670) Interestingly, loop 6 is missing in the STI family suggesting the possibility that long polypeptide sequences could be grafted in this location without disturbing the overall fold of the MCoTI-I scaffold. To facilitate the grafting of the PMI peptide into loop 6 without disturbing its α-helical character or the cyclotide framework, the N-terminus of the PMI peptide was fused to the linker Ala-Ser-Lys/Arg- Ala-Pro (Fig. 17). This linker is based on the N-terminal region of apamin, a bee-venom neurotoxin that adopts a coil-turn-a-helix structure. (Li, C. et al. (2009) Angew Chem Int Ed Engl 48:8712- 8715). Without being bound by theory, Applicants hypothesized that grafting of this chimeric peptide will make possible the displaying of the PMI peptide in the correct biologically active conformation while minimizing the disruption of the cyclotide scaffold. The engineered apamin-PMI hybrid peptide was grafted between residues Ser31and Gly33 to minimize any possible steric hindrance between the grafted peptide and the MCoTI-I scaffold. The resulting grafted cyclotide was called MCo-PMI (Fig. 17). Applicants also explored the substitution of the tryptophan residue in the grafted sequence of MCo-PMI by the unnatural amino acid 6-chloro tryptophan to provide the cyclotide MC0-PMI-6W (Fig. 17). Replacement of p53 Trp23 by 6-subtituted tryptophan residues has been shown to improve the binding efficiency of p53 -derived peptides to Hdm2. (Garcia-Echevarria, C. et al. (2000) J. Med. Chem. 43:3205-3208) Substitution of the residue Phe3 in the peptide PMI, which is critical for the interaction with Hdm2 and HdmX (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670), by Ala yielded a negative control for biological experiments.

Production and characterization of MCo-PMI cyclotides

[0234] MCoTI-based folded cyclotides were produced either by chemical synthesis or bacterial recombinant expression. Chemical synthesis allowed the introduction of unnatural amino acids, while recombinant expression made possible the production of cyclotides

15 13

labeled with NMR active isotopes like N and C to facilitate their structural analysis by heteronuclear NMR. In both cases the backbone cyclization was performed by an

intramolecular native chemical ligation (NCL) ⁴⁹ using the native Cys located on the beginning of loop 6 to facilitate the cyclization. This ligation site has been shown to give very good cyclization yields (Austin, J. et al. (2009) Chembiochem 10:2663-2670; Camarero, J.A. et al. (2007) Chembiochem 8:1363-1366). Intramolecular NCL requires the presence of an N-terminal Cys residue and C-terminal a-thioester group in the same linear precursor (Camarero, J.A. et al. (1998) Angew. Chem. Int. Ed. 37:347-349; Camarero, J.A. et al. (1999) J. Am. Chem. Soc. 121 :5597-5598).

[0235] Recombinant expression of MCo-based cyclotides was performed by fusing the corresponding linear precursors in frame at the C- and N-terminus to a modified Mxe gyrase A intein and a TEV protease recognition sequence, respectively. Once the intein precursor protein was expressed and purified, the N-terminal TEV protease recognition peptide was proteolytically removed. Backbone cyclization and oxidative folding was performed with reduced glutathione (GSH) at physiological pH in one single step (Fig. 18). Chemical synthesis of the linear precursor peptide thioesters was accomplished using Fmoc-based solid-phase peptide synthesis on a sulfonamide resin. After activation and cleavage of the peptide-resin, the thioester precursors were cyclized and oxidatively folded in one single step with GSH as described above. As shown in Fig. 18, the cyclization and oxidative folding of MCo-cyclotides was remarkably efficient yielding in both cases the peptide as the major product (Fig. 18). MCo-cyclotides were purified by preparative reversed-phase (RP)-HPLC and purity determined by analytical RP-HPLC and electrospray mass spectrometry (ES-MS, Figs. 10 and 11). [0236] Heteronuclear NMR spectroscopy was used to characterize free MCo-PMI (Fig. 12). Chemical shifts are exquisitively sensitive probes of the three-dimensional structure of proteins. Comparison between NMR spectra of MCo-PMI and MCoTI-I showed that the cyclotide fold within MCo-PMI is mostly preserved. Changes in chemical shifts are concentrated around loop 6, which accommodates the PMI peptide segment required for the interaction with the p53-binding domains of Hdm2 and HdmX. The differences in chemical shifts between MCo-PMI and MCoTI-I backbone amide protons from loops 1 through 5 are well within 0.2 ppm, indicative of only minor changes in the backbone conformation (Fig. 12E). These results are remarkable given the size of the peptide grafted in loop 6 (25 residues versus the original loop sequence containing only 8 residues) and highlight the robustness of this scaffold. The NMR analysis of the cyclotide MCo-PMI segment corresponding to the PMI peptide also reveals that although this segment has a predisposition to adopt a-helical conformations as calculated from the NH backbone chemical shifts (Fig. 12F), the absence of a typical a-helical Nuclear Overhauser effect (nOe) pattern clearly indicates that it does not adopt a stable helical structure (Fig. 12A).

Cyclotide MCo-PMI binds with high affinity to the p53-binding domain of Hdm2 and HdmX

[0237] The biological activity of MCo-PMI cyclotides was first tested by fluorescence polarization anisotropy using the p53 binding domains of Hdm2 and HdmX and FITC- labeled derivatives of MCo-PMI-K37R, MC0-PMI-6CIW and MCo-PMI-K37R-F42A (Fig. 19). FITC was site-specifically incorporated into loop 2 by reacting with the ε-ΝΗ2 group of residue Lys6. Cyclotide MCo-PMI-K37R displayed strong affinity for the p53 binding domain of Hdm2 (K _D = 2.3 ± 0.1 nM) and HdmX (K _D = 9.7 ± 0.9 nM). These affinities are similar to those reported for the peptide PMI (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670) thus confirming the PMI peptide segment can adopt a biologically active conformation when grafted onto the cyclotide framework. Intriguingly, the binding affinity of cyclotide MC0-PMI-6W for Hdm2 (K _D = 2.6 ± 0.4 nM) was similar to that of MCo-PMI-K37R suggesting that the replacement of the Trp residue in the PMI peptide is not critical for improving the binding affinity to Hdm2. As expected, cyclotide MCo-PMI-K37R- F42A did not interact with either Hdm2 or HdmX in this dose range (Fig. 19A). [0238] Applicants also performed competition binding assays with unlabeled MCo-PMI cyclotides to test their ability to disrupt the high affinity complexes between the

transactivation domain of p53 and Hdm2 or HdmX (Fig. 19B). This was accomplished by using a FRET-based reporter formed by the fluorescent proteins YPet and CyPet fused to a p53 peptide and the p53 binding domains of Hdm2/HdmX respectively. Cyclotides MCo- PMI and MCo-PMI-K37Pv were able to compete with YPet-p53 for Hdm2 and HdmX binding with similar IC ₅₀ values (Fig. 19B) indicating that as expected the conservative mutation Lys to Arg did not affect the folding or the biological activity of the resulting cyclotides. All wild-type PMI grafted cyclotides showed IC ₅₀ values for the inhibition of the p53/HdmX interaction that were around three times higher than those for the inhibition of p53/Hdm2, which is in agreement with the binding affinities shown for the wild-type PMI grafted cyclotides (Fig. 19A) and the values reported for the PMI peptide (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670). Interestingly, wild-type PMI grafted cyclotides were about three times more effective disrupting the p53-Hdm2 complex than the selective Hdm2-inhibitor Nutlin-3. Without being bound by theory, this could be explained in part by the relatively larger interaction surface of the PMI-grafted cyclotide compared to that of the small molecule Nutlin-3. As expected, cyclotides MCoTI-I and MCo-PMI-F42A did not disrupt the interaction between p53 and Hdm2/HdmX. Taken together, these data demonstrate the PMI-grafted cyclotides target both Hdm2 and HdmX in vitro, and exhibit only a slight binding preference (~ 3-fold) for Hdm2 over HdmX.

Cyclotide MCo-PMI targets both intracellular Hdm2 and HdmX

[0239] Encouraged by the biological activity of MCo-PMI cyclotides in vitro and by the fact that MCoTI-cyclotides have been recently shown to be able to enter human macrophages and breast cancer cell lines (Contreras, J. et al. (2011) J Control Release 155: 134-143;

Cascales, L. et al. (2011) J Biol Chem; Greenwood, K.P. et al. (2007) Int J Biochem Cell Biol 39:2252-2264), Applicants investigated the ability of cyclotide MCo-PMI to target intracellular Hdm2 and HdmX. This was accomplished by using co-immunoprecipitation experiments in LNCaP cells. LNCaP cells express wild-type p53 and therefore have been shown to be sensitive to the Hdm2-inhibitor Nutlin-3 (Logan, I.R. et al. (2007) Prostate 67:900-906). LNCaP cells were treated with FITC-labeled cyclotides MCo-PMI-K37R and MCo-PMI-K37R-F42A for 30h. After cell lysis, FITC-labeled peptides were pulled down using an anti-FITC antibody and the content of proteins Hdm2 and HdmX analyzed by western blotting. As shown in Fig. 20, endogenous Hdm2 and HdmX specifically co- inmmuoprecipitated with FITC-labeled MCo-PMI-K37R but not with the inactive mutant MCo-PMI-K37R-F42A. Fluorescence scanning of the western blot confirmed that in both cases the FITC-labeled cyclotides were able to efficiently penetrate LNCaP cells, therefore confirming that MCo-PMI cyclotide can also target Hdm2 and HdmX within cells.

Cyclotide MCo-PMI is cytotoxic to cancer cells with wild-type p53 expressing Hdm2 and/or HdmX

[0240] Applicants then investigated the effect on cell viability by treating a panel of solid tumor cells expressing wild type p53, and different levels of Hdm2 and/or HdmX. The cell lines used in this study included the Hdm2- and HdmX-expressing prostate and colon cancer cell lines LNCaP and HCT116; and the HdmX-overexpressing human choriocarcinoma cell line JEG-3. In order to study the p53 dependence on the cytotoxic activity of MCo-PMI cyclotides, human prostate cancer cell lines PC3 (bearing P274L and V223F p53 mutations) and DU145 (bearing a base pair deletion at codon 138 that generates a stop codon at position 179) (Carroll, A.G. et al. (1993) Prostate 23: 123-134); and a p53 deficient HCT116 cell line (Bunz, F. et al. (1999) J Clin Invest 104:263-269) were used. Applicants also tested breast and kidney epithelial cell lines HBL-100 and HEK293T to evaluate the toxicity of MCo-PMI cyclotides to non-tumorigenic cells.

[0241] Cytotoxicity assays were performed by treating cultured cells during 48 h with serial dilutions of Nutlin-3, MCo-PMI, MCo-PMI-F42A and MCoTI-I. Cell viability was measured at the end of the treatment by the MTT assay (Fig. 21). The cyclotide MCoTI-I, used as scaffold to generate grafted PMI cyclotides, showed no detectable cytotoxicity to any of the cells tested in this study up to 100 μΜ concentration. The lack of cytotoxicity observed in MCoTI-based cyclotides is in agreement with previous reports indicating that these cyclotides are not toxic at concentrations up to 100 μΜ (Contreras, J. et al. (2011) J Control Release 155: 134-143; Greenwood, K.P. et al. (2007) Int J Biochem Cell Biol 39:2252-2264), confirming the preference of this scaffold for the design of peptide-based therapeutics. The cyclotide MCo-PMI showed dose-dependent cytotoxicity in all the three cell lines tested with wild-type p53 phenotypes, suggesting that MCo-PMI can reactivate the p53 pathway efficiently in cells expressing high levels of Hdm2, HdmX or both (Fig. 221). The most sensitive cell line to MCo-PMI was HCTl 16 (EC ₅ o ~ 2 μΜ), while the HdmX-overexpressing LNCaP and JEG3 cells lines were about 10-fold less sensitive (Fig. 21).

[0242] As expected, the PC3 and DU145 prostate cancer cell lines, which both bear inactivating mutations in the p53 gene, were unaffected by MCo-PMI or Nutlin-3 treatments. Genetic deletion of p53 from HCTl 16 cells also had the same effect. Importantly, it was also found that the cyclotide MCo-PMI, showed little cytotoxicity to non-cancerous HBL100 and HEK293T epithelial cells (EC ₅₀ values > 200 μΜ, Fig. 21). In contrast, Nutlin-3 was moderately cytotoxic to the normal breast epithelial HBL-100 cell line (EC ₅₀ = 33 ± 5 μΜ, Fig. 20). It is also worth noting that the mutant cyclotide MCo-PMI-F42A was completely inactive in all the cell lines tested in this work, confirming the specificity and in-cell biological activity of MCo-PMI.

Cyclotide MCo-PMI activates the p53 tumor suppressor pathway

[0243] To investigate whether the in-cell biological activity of MCo-PMI was derived from the stabilization of endogenous p53, LNCaP cells were treated for 48 h with vehicle, Nutlin-3 and cyclotides MCoTI-I, MCo-PMI and MCo-PMI-F42A. The cellular extracts were analyzed by western blotting to visualize the amounts of p53, Hdm2 and HdmX proteins. Applicants also analyzed the intracellular level of p21, which is a regulator of cell-cycle progression at Gi and is tightly controlled by the tumor suppressor protein p53. As shown in Figure 22A, treatment of LNCaP cells with Nutlin-3 (20 μΜ) and cyclotide MCo-PMI (50 μΜ) increased the levels of p53. In contrast, treatment with the parent cyclotide MCoTI-I or the inactive MCo-PMI-F42A at the same concentration had little effect on the level of intracellular p53 when compared to the cells treated with vehicle. As expected, the level of endogeneous Hdm2 also increased in cells treated with Nutlin-3 or cyclotide MCo-PMI. These results are consistent with an intact p53-Hdm2 counter-regulatory mechanism where Hdm2 transcription and expression is under the control of p53 (Juven, T. et al. (1993) Oncogene 8:3411-3416; Barak, Y. et al. (1993) EMBO J 12:461-468). Likewise, MCo-PMI and Nutlin-3 also induced upregulation of the cyclin-dependent kinase inhibitor p21. The levels of Hdm2 and p21 were unchanged in cells treated with the inactive cyclotide MCo- PMI-F42A when compared to vehicle, hence highlighting the specificity of the cyclotide MCo-PMI to modulate the p53-signaling pathway. Interestingly, the intracellular levels of HdmX were downregulated by the cyclotide MCo-PMI, whereas no effect was observed with the inactive mutant MCo-PMI-F42A. This result is consistent with the high affinity of MCo- PMI for both Hdm2 and HdmX, which inhibits binding of endogenous p53 to the Hdm2- HdmX complex preventing its degradation. The stabilization of p53 upregulates the expression of Hdm2, which then can promote ubiquitination and degradation of HdmX (de Graaf, P. et al. (2003) J Biol Chem 278:38315-38324; Pan, Y. et al. (2003) Mol Cell Biol 23:5113-5121).

[0244] Applicants also analyzed by western blotting the intracellular levels of p53, Hdm2, HdmX and p21 in LNCaP cells treated for 48 h with different concentrations of cyclotide MCo-PMI (Fig. 22B). The upregulation of Hdm2 and p53 showed a dose-dependent relationship with EC ₅ o values -20 μΜ (p53) and -15 μΜ (Hdm2), which is consistent with the EC50 values obtained in the cell viability assay for this cell line (Fig. 21). The

upregulation of p21 and down regulation of HdmX was also dose-dependent with EC50 values around 30 μΜ. To investigate the kinetics of p53 activation, LNCaP cells were treated with 50 μΜ MCo-PMI for 8-48 h and monitored the p53 protein levels by western analysis (Fig. 22C). Cells exposed to MCo-PMI demonstrated increased p53 levels that peaked at 36- 48 h post-treatment. A similar trend was also found for the upregulation of Hdm2 and p21, and for the downregulation of HdmX (Fig. 22C).

[0245] To determine whether MCo-PMI mediated stabilization of p53 could inhibit cancer cells by reactivating the apoptotic pathway, Applicants performed a caspase-3/7 assay using LNCaP cells treated with MCo-PMI and MCo-PMI-F42A for 30 h (Fig. 23 A). Analysis of caspase-3 activation showed that cyclotide MCo-PMI induced dose-dependent caspase-3 activation that could be blocked with a specific caspase-3/7 inhibitor. Cells treated with MCo-PMI-F42A, on the other hand, did not show any caspase-3/7 activity.

[0246] Applicants also evaluated cell cycle arrest in LNCaP cells treated with MCo-PMI, MCo-PMI-F42A and Nutlin-3 for 24 h by using the propidium iodide (PI) flow cytometric assay (Riccardi, C. et al. (2006) Nat Protoc 1 : 1458-1461) (Fig. 23B). Both, Nutlin-3 and MCo-PMI impeded cell cycle progression, resulting in a depression of the S-phase fraction. This depression was slightly more marked in the cells treated with Nutlin-3. The reduction in the S-phase was associated with accumulation of cells in the G ₀ /Gi phase suggesting that the treatment in both cases impedes the cell cycle progression at the Gi/S checkpoint, which is in agreement with the upregulation of p21 observed in cells treated with MCo-PMI and Nutlin- 3. Altogether, these data confirm that the in-cell disruption of the p53-Hdm2 and p53-HdmX complexes by MCo-PMI in LNCaP cells leads to the upregulation of p53 transcriptional targets (p21 and Hdm2), caspase-3/7 activation and induction of cell cycle arrest at the Gi/S checkpoint. Interestingly, the upgregulation of Hdm2 combined with the inhibition of the interaction between p53 and Hdm2/HdmX also leads to the downregulation of HdmX likely mediated by the E3-ligase activity of the Hdm2-HdmX complex (Wade, M. et al. (2012) Oncogene).

Stability of native, linear and grafted cyclotides

[0247] To compare the biological stability of the MCo-PMI cyclotides to that of the precursor cyclotide MCoTI-I, Applicants incubated MCo-PMI, linear reduced MCo-PMI and MCoTI-I in human serum at 37° C (Fig. 15 A). The quantitative analysis of undigested polypeptides was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Cyclotide MCoTI-I showed a half-life time of more that 2 days (τι/2 = 55 ± 5 h) under the conditions used in this work. Naturally occurring MCoTI- cyclotides present a very rigid structure (Puttamadappa, S.S. et al. (2010) Anew Chem Int Ed Engl 49:7030-7034), which makes them remarkably stable to proteolytic degradation.

Remarkably, cyclotide MCo-PMI was only slightly less stable (τι _/2 = 30 ± 4 h) than the parent cyclotide. In contrast, a linearized, reduced and alkylated version of MCo-PMI was rapidly degraded under the same conditions (τι/ ₂ = 0.7 ± 0.1 h) indicating the importance of the circular Cys-knot topology for proteolytic stability. Applicants also investigated the ability of MCoTI-based cyclotides to bind to serum proteins. Serum binding has been recently used to extend serum half-life of bioactive peptides (McGregor, D.P. (2008) Curr Opin Pharmacol 8:616-619). Under the conditions used above, the cyclotides MCoTI-I and MCo-PMI were ~ 98% and 99% bound to serum proteins, respectively, under the conditions employed in this study. MCoTI-I and MCo-PMI are almost completely degraded after 120 h of treatment with human serum at 37°C (Fig. 15 A), which suggests that the binding to serum proteins may be reversible. To test this possibility, the association and dissociation constant rates of MCo-PMI to human serum proteins were studied. This was accomplished by biolayer interferometry analysis using the commercially available platform Blitz from ForteBio. The results indicated that MCo-PMI binds serum proteins with an association and dissociation 3 -1 -1 -2 -1

constant rates of 2.4 x 10 M ^" s ^" and 2.2 x 10 ^" s ^" , respectively (Fig. 15B). These results combined provide a relatively weak dissociation constant of ~ 10 μΜ.

Suppression of tumor growth by reactivation of the p53 pathway in vivo

[0248] A murine xenograft model HCT116 p53 ^+/+ xenografts were established by injecting 0.5 x 10 ⁶ cells subcutaneously into the rear right flanks of female nuce mice (nu/nu) mice. When the tumor reached an average volume of -100 mm as determined by caliper measurements, cohorts (n = 3) were treated intravenously with vehicle (5% dextrose in water), MCo-PMI (40 mg/kg, 7.6 mmol/kg), or Nutlin-3 (10 mg/kg, 17.2 mmol/kg) daily for up to 37 days. Treatment with MCo-PMI significantly suppressed tumor growth when compared to animals treated only with vehicle (85% reduction at day 31, p = 0.019) and Nutlin-3 (75% reduction at day 31, p= 0.022) (Fig. 23 A). In contrast, animals treated with Nutlin-3 showed only moderate reduction in tumor growth (40%> reduction at day 31 , p= 0.223) when compared to vehicle (Fig. 24A). At the end of the treatment, the animals were sacrificed and the tumors excised. Snap-frozen tumor samples were analyzed by qRT-PCR using HDM2 and P21 primer sets. Treatment of the tumors with MCo-PMI induced a statistically significant transcriptional activation of HDM2 and P21. Immunohistochemical staining of formalin-fixed paraffin embedded tumor sections was performed using antibodies raised against p53, Hdm2, and p21. Tumors treated with MCoTI-PMI peptide showed a marked increase in p53 expression, as well as a slightly increase in Hdm2 and p21 expression compared to vehicle or Nutlin-treated tumors (Fig. 16). Mice treated with MCo-PMI maintained healthy weight throughout treatment and no gross abnormalities were noted in any of the organs or tissues at the time of necropsy (Fig. 16). In addition, the histological analysis of MCo-PMI treated mice showed no obvious signs of toxicity to normal tissues (Fig. 16), which is agreement with the cytotoxicity results obtained for HBL100 and

HEK293T cells. Remarkably, these results extend our previous in vitro findings by demonstrating that MCo-PMI can also activate the p53 tumor suppressor pathway in vivo, which highlights the pharmacological potential of this cyclotide.

Solution structure of MCo-PMI complexed to the p53-binding domain of Hdm2

[0249] To better understand the molecular interaction between MCo-PMI and

Hdm2/HdmX, Applicants elucidated the three-dimensional structure of the molecular complex between MCo-PMI and Hdm2 (17-116) (Fig. 25). The structure was determined by heteronuclear NMR using ¹⁵ N-labeled MCo-PMI and ¹³ C, ¹⁵ N-labeled Hdm2. The 10 lowest- energy structures (Fig. 12D) are in good agreement with the experimental constraints, with no distance and dihedral angle violations over 0.2 A and 4°, respectively. The solution structure of Hmd2 is in close agreement with the crystal structure of Hdm2 in complex with PMI peptide (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670) with a root-mean- square deviation (RMSD) between the C ^a atoms is 1.3 A (Fig. 25). The overall fold of MCo- PMI conserves the parental Cys-knot topology of MCoTI-I with a more extended loop 6 (Fig. 25). A superimposition of MCoTI-I and MCo-PMI shows RMSD between the C ^a atoms in loops 1 through 5 of ~ 2.0 A confirming the tolerance of loop 6 to accept long polypeptide grafts. As expected, the helical PMI segment in loop 6 of MCo-PMI adopts an amphipathic a-helical conformation (Fig. 12B) allowing the side-chains of Phe43, Trp47 and Leu50 to bury deep in the p53 -binding pocket of Hdm2.

[0250] MCo-PMI binding to Hdm2 is very similar to that of PMI and other p53-like peptide ligands (Fig. 25). The side-chain positions of residues Phe42, Trp46 and Leu49 in MCo-PMI and the equivalent residues in PMI occupy almost identical positions in the complex with Hdm2, and also make identical interactions with the residues of Hdm2. Superimposition of the equivalent C ^a residues of the PMI peptide and PMI segment in MCo-PMI yields an RMSD value of 1.5 A.The total buried surface area (BSA) contributed by these three residues represents around 34% of the total BSA in the complex.

[0251] It is worth noting that loop 2 of MCo-PMI is located within van-der-Waals distance to Hdm2 and may favorably contribute to the observed increase in the binding affinity of MCo-PMI for Hdm2 (Fig. 24C). In 9 out of 10 lowest energy structures, negatively charged Asp35 of loop 2 is located within 2 A from positively charged Lysl 16 of Hdm2 with the potential of forming a salt bridge that stabilized MCo-PMI-Hdm2 complex.

Discussion

[0252] Although small peptides mimicking the N-terminal fragment of p53 have been shown to be powerful Hdm2/HdmX antagonists, the use of peptide -based therapeutics is limited by their poor stability and bioavailability. As shown herein, Applicants have improved the delivery of specific bioactive peptides to specific targets using disulfide rich backbone-cyclized polypeptides.

[0253] The cyclotide MCoTI-I was engineered to display an a-helical peptide derived from the N-terminal fragment of p53 (peptide PMI (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665-4670; Li, C. et al. (2010) J Mol Biol 398:200-213)) to improve its stability and cellular uptake properties. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both p53 -binding domains of Hdm2 and HdmX, showed high stability in human serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway.

[0254] Applicants also used the cyclotide MCoTI-I molecular framework due to its low cytotoxicity and ability to cross mammalian cell membranes (Contreras, J. et al. (2011) J Control Release 155: 134-143; Cascales, L. et al. (2011) J Biol Chem 286:36932-36943). This is a key requirement for targeting intracellular protein-protein interactions. The bioactive a- helical peptide was engineered into loop 6, which has been shown to be more flexible in solution (Puttamadappa, S.S. et al. (2010) Angew Chem Int Ed Engl 49:7030-7034;

Puttamadappa, S.S. et al. (2011) Angew Chem Int Ed Engl 50:6948-6949). To facilitate the introduction of the a-helical segment into this loop without distorting neither the cyclotide framework nor the helical character of the bioactive peptide, a turn-coil peptide segment derived from the bee venom peptide apamin was introduced at the C-terminus of the PMI peptide. The resulting cyclotide was produced by solid-phase peptide synthesis or heterologous expression using a GSH-induced intramolecular native chemical ligation reaction for the cyclization/folding process (Contreras, J. et al. (2011) J Control Release 155: 134-143; Austin, J. et al. (2009) Chembiochem 10:2663-2670). In both cases the cyclization and folding of the cyclotide was remarkably efficient. Characterization of the grafted cyclotides by NMR spectroscopy confirmed that the cyclotide MCo-PMI adopts a cyclotide fold with the p53-derived PMI peptide being flexible without adopting a stable a- helical conformation. Given the length of the new peptide segment grafted into loop 6 (25 residues versus only 8 residues for the original loop) and that the cyclotide was able to adopt a native fold, it is clear that loop 6 can tolerate the grafting of long peptide segments without disturbing the folding of MCoTI-based cyclotides and therefore it is ideal for engineering new biological activities in these cyclic peptides. Chan et al. also recently reported the grafting of smaller peptide segments (6 to 9 residues long) into this loop to produce pro- angiogenic cyclotides (Chan, L.Y. et al. (2011) Blood 118:6709-6717).

[0255] The grafted cyclotide MCo-PMI was biologically active, able to bind with high affinity to both Hdm2 and HdmX to disrupt the interaction between p53 and Hdm2/HdmX in vitro and more importantly also in-cell experiments. These results show that modification of loop 6 does not affect the ability of MCoTI-cylotides to cross mammalian cell membranes (Contreras, J. et al. (2011) J Control Release 155: 134-143; Cascales, L. et al. (2011) J Biol Chem 286:36932-36943), therefore making it an excellent choice for the engineering of cyclotides for targeting intracellular targets. The PMI-grafted cyclotide showed a 3-fold greater binding preference for Hdm2 than for HdmX, which agrees with the same binding preferences of the PMI peptide (Pazgier, M. et al. (2009) Proc Natl Acad Sci USA 106:4665- 4670; Li, C. et al. (2010) J Mol Biol 398:200-213). Given that both Hdm2 and HdmX bind to the transactivation domain of p53, targeting both p53 -binding domains has been shown to be critical for the activation of the p53 tumor suppressor in cancer cells overexpressing Hdm2 and/or HdmX (Wade, M. et al. (2009) Mol Cancer Res 7: 1-11; Bernal, F. et al. (2010) Cancer Cell 18:411-422). In agreement with this, the cyclotide MCo-PMI was able to reactivate the p53 tumor suppressor pathway in different cancer lines expressing different levels of Hdm2 and/or HdmX and was not cytotoxic to non cancer cells or cancer cells with non functional p53. Treatment of p53 wild-type cancer cells with the cyclotide MCo-PMI showed upregulation of p53 transcriptional targets confirming the in cell activation of the p53 pathway upon treatment. Remarkably, intravenous administration of MCo-PMI to mice bearing an HCT116-xenograft cancer tumor also triggered upregulation of p53 transcriptional targets and suppressed tumor growth. It is worth noting that the high biological specificity of MCo-PMI is highlighted by the explicit p53 dependence of its effects and the complete abrogation of its activity by the single point mutation F42A affecting a key residue in the a- helix binding interface. In addition, the mode of inhibition of MCo-PMI was further validated by solving the structure of the complex between MCo-PMI and the p53 binding domain of Hdm2, which confirmed that this cyclotide binds Hdm2 in a similar way to p53 and other p53-like peptide ligands.

[0256] To evaluate the potential therapeutic value of the cyclotide MCo-PMI Applicants also tested its stability in human serum. Evaluation of the biological and chemical stability is critical in the design of novel peptide-based therapeutic leads, since serum stability affects their half-life. The cyclotide MCo-PMI showed a remarkable half-life (30 ± 4 h) in human serum. This result is noteworthy given the length of new peptide segment grafted in loop 6 and shows the high stability of this peptide-scaffold to biological degradation. These results also revealed that the cyclotides MCoTI-I and MCo-PMI were mostly bound (-99%) to serum proteins. Binding to serum proteins is also a key factor to increase the half- life of therapeutics, however this binding has to be reversible (McGregor, D.P. (2008) Curr Opin Pharmacol 8:616-619). Analysis of the binding curve of MCo-PMI to serum revealed that the interaction is weak and reversible. Additional studies can be conducted to evaluate the potential immunogenicity of particular engineered cyclotides; however, this is generally considered not to be a major issue for small-sized and stable microproteins (Craik, D.J. et al. (2007) Expert Opin Investig Drugs 16:595-604; Kolmar, H. (2009) Curr Opin Pharmacol 9:608-614).

[0257] Previous studies have shown that the cyclotides MCoTI-I and kalata Bl can be used for introducing novel biological activities. These reports, however, have been restricted until now to proteases and extracellular targets (Gunasekera, S. et al. (2008) J Med Chem 51 :7697- 7704; Chan, L.Y. et al. (2011) Blood 118:6709-6717; Aboye, T.L. et al. (2012) J Med Chem 55: 10729-10734; Wong, C.T. et al. (2012) Angew Chem Int Ed Engl 51 :5620-5624;

Thongyoo, P. et al. (2008) Org Biomol Chem 6:1462-1470). In this application, it is shown for the first time the engineering of a cyclotide that can effectively and selectively target intracellular protein-protein interactions. This was demonstrated by designing a cyclotide able to target the p53-Hdm2/HdmX complex and activate the p53 tumor suppressor in cancer cells with wild-type p53. Until now, the cyclotide scaffold has been only used to graft small peptide segments (Gunasekera, S. et al. (2008) J Med Chem 51 :7697-7704; Chan, L.Y. et al. (2011) Blood 118:6709-6717; Huang, Y.H. et al. (2010) J Biol Chem 285: 10797-10805) (< 10 residues long) or introduce point mutations (Austin, J. et al. (2009) Chembiochem

10:2663-2670; Thongyoo, P. et al. (2008) Org Biomol Chem 6: 1462-1470; Huang, Y.H. et al. (2010) J Biol Chem 285: 10797-10805) to introduce new biological activities. This application also shows that by careful design, longer peptides containing a-helical segments can be engineered into the cyclotide framework. Protein-protein interactions involving a- helical segments are quite abundant in nature and therefore this approach should also be valuable for other intracellular targets involving this type of interactions. Moreover, the fact that cyclotides have 5 hypervariable loops suggests sequence optimization of neighboring loops using the power of molecular evolution in order to increase the binding surface and consequently affinity. The development of heterologous expression systems for in-cell production of cyclotides makes it possible to generate genetically-encoded cyclotide-based libraries for the rapid selection of optimal sequences (Austin, J. et al. (2009) Chembiochem 10:2663-2670). According to the structure of the complex between MCo-PMI the p53 binding domain of Hdm2, MCo-PMI loop 2 is within van der Waals distance from Hdm2, with Asp35 of MCo-PMI forming a possible salt bridge with Lysl 16 of Hdm2. Therefore, the sequence of loop 2 could be further optimized for optimal binding due to their proximity to the Hdm2 surface. In one aspect, mutating residues or loops in the MCoTI framework can be made to ensure that the cellular uptake capabilities of the resulting cyclotide are not removed.

[0258] It should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0259] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0260] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0261] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.