WO2013176623A1 - Inhibitors for the treatment of cancer - Google Patents

Abstract

An isolated peptide comprising a sequence of the N-terminal end of MDM2 that is capable of inhibiting TCTP interaction with p53 and/or MDM2. Also provided is an isolated peptide that is capable of inhibiting MDM2. Also disclosed are nucleic acids, vector, uses, methods of treatment comprising the peptide of the present disclosure and methods of detecting the peptide.

Description

INHIBITORS FOR THE TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of US provisional application No. 61/649,594, filed 21 May 2012 and 61/664,637, filed 26 June 2012, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention generally relates to the field of biochemistry. In particular, the present invention relates to peptides and/or nucleic acids which can be used for the treatment of proliferative disease such as tumour and/or cancer.

BACKGROUND OF THE INVENTION

[003] Tumour and/or cancer occur when abnormal cells grow out of control. Normal healthy cells rest, divide, differentiate or die as required. In tumour and/or cancer, the delicate balance that controls healthy cells to rest, divide, differentiate or die as required is skewed to allow cells escape from this self-regulating pathway.

[004] One pathway commonly investigated in tumour and/or cancer research is the apoptotic pathway. One gene most commonly associated with apoptosis and tumour/cancer is the p53 gene, which is a tumour suppressor gene that is known to be the checkpoint protein involved in cell-cycle control, maintenance of genetic stability and apoptosis. The protein that is expressed by p53 gene is important in protecting the organism against cellular damage and disorder. Transgenic mice that do not have both copies of the gene will develop cancer by the age of three months.

[005] Another protein that is known to be involved in tumour and/or cancer development is the translationally controlled tumour protein (TCTP), also known as Fortilin/Histamine Releasing Factor (HRF). TCPT is a highly conserved protein present in all eukaryotic organisms and was first discovered over two decades ago as a growth promoting factor in Ehrlich ascites tumour. Since then, a diverse range of biological functions have been attributed to the protein including essential roles in cell proliferation and growth regulation, histamine releasing properties and other 'cytokine-like' activity and anti-apoptotic activity. TCTP is overexpressed in many human cancers including prostate, liver and breast and tumour reversion results in its downregulation. TCTP has also been shown to indirectly control p53 levels in cells.

[006] As balance between anti-apoptotic and apoptotic activity in tumour and/or cancer is clearly skewed to the former, rather than the latter, there is a need to provide a molecule that can reverse this imbalance such that more apoptosis and less anti-apoptotic activity occurs. Accordingly, there is a need to provide molecules that can inhibit an anti-apoptotic protein pathway.

SUMMARY OF THE INVENTION

[007] According to one aspect, there is provided an isolated peptide that may be capable of specifically binding to TCTP (translationally controlled tumour protein). In one example, the specific binding of the isolated peptide of this aspect results in the inhibition of TCTP interaction with p53 and/or Mouse double minute 2 homolog (MDM2). The isolated peptide may comprise a sequence of the N-terminal end of MDM2.

[008] According to another aspect, there is provided an isolated peptide that may be capable of inhibiting MDM2. The isolated peptide may comprise the basic domain 2 region of TCTP.

[009] According to yet another aspect, there is provided a nucleic acid that may encode the peptide as described herein.

[0010] According to yet another aspect, there is provided a vector that may comprise the nucleic acid as described herein.

[0011] According to yet another aspect, there is provided the use of the peptide as described herein in the manufacture of a medicament for treating cancer.

[0012] According to yet another aspect, there is provided a method of treating cancer. The method of treating cancer may comprise the administration of at least one peptide as described herein to a patient in need thereof.

[0013] According to yet another aspect, there is provided a pharmaceutical composition. The pharmaceutical composition may comprise at least one peptide as described herein.

[0014] According to yet another aspect, there is provided a method of detecting the presence or absence of TCTP in a sample. The method of detection as described herein may comprise (a) contacting the sample with a peptide as described herein. The method of detection as described herein may further comprise the step of analyzing the sample resulting from (a) to identify the presence or absence of the binding product of TCTP and the peptide as described herein.

[0015] According to yet another aspect, there is provided a microarray that may comprise a peptide as described herein for detection of TCTP in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0017] Fig. 1 is a collective image of immunoprecipitation and Western blot results of in vitro pull-down assays of protein interaction site(s) between TCTP and HDM2. A, in vitro pull-down of TCTP by C-terminally truncated HDM2 variants. Upper panel, Western blot of pulled down TCTP (anti-FLAG antibody). Lower panel, input levels of respective HDM2 variants (anti-HA antibody, arrowed). B, in vitro precipitation of TCTP by HDM2 (110-491). Upper panel, Western blot of pulled down TCTP. Lower panel, input levels of respective HDM2 variants (arrowed). C, in vitro precipitation by HDM2 N-terminal domain deletion mutants. Upper panel, Western blot of pulled down TCTP. Lower panel, input levels of respective HD 2 variants (arrowed). Control lanes indicate TCTP pull-down in the absence of HDM2. D, in vitro precipitation of TCTP by HDM2 with point mutations in a2 helix. Upper panel, Western blot of pulled down TCTP (anti-FLAG antibody) by indicated mutants. Lower panel, input levels of respective HDM2 point mutants. Thus, Fig. 1 demonstrates the interaction of TCTP with the N-terminal region of HDM2.

[0018] Fig. 2 shows immunoprecipitation and Western blot results of the investigation of whether TCTP binding site of HDM2 overlap with the p53 binding site. A, In vitro translated HDM2 was immobilised on beads and pre-treated with indicated amounts of Nutlin-3, p53 peptide and p53 control peptide (ImM). Bound TCTP detected by Western blot (anti-FLAG antibody). Control lanes indicate TCTP pull-down in the absence of HDM2. B, Recombinant HDM2 (residues 17-125) or control peptide was immobilised on beads and incubated with recombinant TCTP either in presence or absence of 200μΜ Nutlin-3. Thus illustrating the inhibition of TCTP-HDM2 interaction by Nutlin-3 and p53 peptide.

[0019] Fig. 3 shows Western blot image of the investigation of HDM2-M62A mutant to see if it retained the capacity to bind p53. vitro translated HDM2 or HDM2-M62A was immobilised on beads and pre-treated with 0/100/200/400 μΜ Nutlin-3. Bound p53 detected by Western blot using DOl anti-p53 antibody. Control lane indicates p53 pull-down in the absence of HDM2. Accordingly, Fig. 3 demonstrates Nutlin-3 does not inhibit p53 binding to HDM2-M62A.

[0020] Fig. 4 is the immunoprecipitation and Western blot images of C-terrninal deletion analysis of TCTP. This analysis was carried out to map TCTP interaction site with HDM2. A, in vitro precipitation of C-terminally deleted TCTP mutants by HDM2. Upper panel, Western blot of pulled down TCTP variants (anti-FLAG antibody). Lower panel, input levels of respective TCTP variants (arrowed). Control lanes indicate pull-down of TCTP variants in absence of HDM2. B, Reciprocal IP of HDM2 by C-terminally deleted TCTP mutants. Upper panel, Western blot of pulled down HDM2 (anti-HA antibody). Lower panel, input levels of respective TCTP variants (arrowed). Thus, Fig. 4 illustrates the interaction interface within amino acids 80-133 of TCTP which comprises a helix-loop-helix motif.

[0021] Fig. 5 is the results of a series of pull-down experiments using 2 synthetic peptides spanning the TCTP regions of TH2: residues 81-110; TH3 residues 107-133 along with a peptide spanning the HDM2 a2 helix (residues 43-65). A, Western blot showing binding of recombinant HDM2(1 - 125) or TCTP to beads pre-coated with TCTP peptides (TH2, TH3) or HDM2 peptide (a2) respectively. Control pull-down (CON) was performed using non- specfic peptide (tandem GSSS 20-mer peptide). Positive control (DOl) carried out using DOl peptide to pull down HDM2(1-125). Lower panel shows binding of TCTP to a2 peptide or a2M62A peptide. B, Effect of Nutlin-3 (2/20/200μΜ) on binding of TH2 and TH3 peptides to HDM2(1-125). Lane C indicates HDM2 binding to non-specific peptide. p53 labelled lanes shows positive control binding of HDM2(1-125) to p53peptide in the absence or presence of 200uM Nutlin-3. C, Fluorescence polarization assay measuring displacement of fluorescein labelled p53 peptide (12.1) from HDM2(1-125) by TH2/TH3 peptides (50/200/600 μΜ), control peptide (600μΜ) and Nutlin-3 (200μΜ). Accordingly, Fig. 5 illustrates the interaction of peptides with recombinant HDM2(1-125) and TCTP.

[0022] Fig. 6 is the immunoprecipitation and Western blot results of investigation into the effect of Nutlin-3 on the endogenous TCTP-HDM2 interaction in the HCT116 p53^"A cell line. A, Co-IP of endogenous HDM2-TCTP complexes in HCT116 p53^"A cells. Upper panel, Western blot of immunoprecipitated TCTP in absence or presence of Nutlin-3 (10μΜ). Lower panels, input levels of HDM2 and TCTP. B, Co-IP of exogenous HDM2 and HDM2- M62A with endogenous TCTP in H1299 p53^"A cells. Upper panel, Western blot of immunoprecipitated HDM2 variants. Thus, Fig. 6 illustrates Nutlin-3 is capable of disrupting HDM2-TCTP interaction ex vivo.

[0023] Fig. 7 is a model of the putative interactions between the N-terminal domain of HDM2 and TCTP. Model was constructed by docking the structures of the two proteins (for MDM2, the N-terminal domain consisting of residues 25-109, RCSB entry 1YCR; for TCTP, residues 1-172, RCSB entry 1YZ1). The two proteins were aligned in different orientations that suggested the experimentally observed interactions between TH2 and TH3 and the p53- binding cavity of MDM2. Molecular dynamics (MD) simulations were carried out using methods described elsewhere. The simulations yielded only one complex as stable and a snapshot taken from this simulation is shown (details will be published elsewhere) with MDM2 in surface and the residues that were subject to Ala scan in spheres; TCTP is shown in magenta cartoon with TH2 highlighted in brown and TH3 in green. Thus, Fig. 7 demonstrates the interactions between a2 helix of HDM2 with both TH2 and TH3 of TCTP, with M62 closely packed under TH3.

[0024] Fig. 8 is a schematic diagram of a simplified TCTP network.

[0025] Fig. 9 is a diagram depicting amino acid sequence of peptides synthesized with N- terminal HIV- Tat tag. Peptides were synthesized with an N-terminal HIV-Tat tag (for cellular uptake) conjugated to the amino acid sequence corresponding to Hdm2 cc.2 helix (TMl) which was shown to interact with TCTP. A Met to Ala substituted peptide sequence, corresponding to a HDM2-M62A substitution, (bound TCTP significantly weaker) was synthesized as a treatment control peptide (TMC).

[0026] Fig. 10 is a histogram plot of Annexin-V FACS analysis of the effect of TMC or TMl on p53 null small lung carcinoma cell lines (H1299). P53 null H1299 is treated for 24 hour with DMSO, 15 μΜ ΤΜΟ or 15 μΜ ΤΜΙ. Region H-1 shows population gate for annexin-V positive events. Faint-dotted and dark-solid traces represent treatment with TMC and TMl peptides, respectively. Thus, illustrating TMl peptide to induce apoptosis in HI 299 cells.

[0027] Fig. 11 is a histogram plot of Annexin-V FACS analysis of the effect of TMC or TMl on p53 positive breast cancer cell line MCF-7. p53 positive MCF-7 cells were treated for 36 hours with DMSO, TMC or TMl peptides in serum-fed media across a concentration range. Region H-1 shows population gate for annexin-V positive events. Faint-dotted and dark-solid traces represent treatment with TMC and TM1 peptides, respectively. Thus, illustrating TM1 peptide to induce apoptosis in MCF-7 cells.

[0028] Fig. 12 is a histogram plot of Annexin-V FACS analysis of the effect of TMC or TM1 on p53 positive breast cancer cell line MCF-7. p53 positive MCF-7 cells were treated for 36 hours with DMSO, TMC or TM1 peptides in serum-free media across a concentration range. Region H-l shows population gate for annexin-V positive events. Faint-dotted and dark-solid traces represent treatment with TMC and TM1 peptides, respectively. Thus, illustrating TM1 peptide to induce apoptosis in MCF-7 cells. DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0029] Translationally controlled tumour protein (TCTP) has a number of intracellular and extracellular functions including an anti-apoptotic role. TCTP's anti-apoptotic function is attributed in part to interactions with both anti-apoptotic (Mcl-1 and Bcl-xl) and pro-apoptotic (BAX) members of the Bcl-2 family. TCTP has been shown to bind directly to p53, with TCTP overexpression increasing p53 degradation and promoting lurig cancer cell survival. It has recently been demonstrated that TCTP binds to E3 ubiquitin ligase of Human double minute 2 homolog (HDM2).

[0030] HDM2 and/or MDM2 (these terms may be used interchangeably), also known as E3 ubiquitin-protein ligase Mdm2, is a protein that is encoded by the MDM2 gene. HDM2 is an important negative regulator of the p53 tumour suppressor. HDM2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumour suppressor and an inhibitor of p53 transcriptional activation.

[0031] Recent studies have shown that TCTP binding to E3 ubiquitin ligase of HDM2 appears to control p53 levels by inhibiting HDM2 auto-ubiquitination, thereby promoting p53 ubiquitination and degradation. The increase of p53 degradation caused by interaction between TCTP with p53 and HDM2 has been shown to promote lung cancer cell survival. Thus, it appears that the disruption of the interaction between TCTP molecule with p53 and HDM2 may provide a molecule useful for inhibiting anti-apoptotic pathway that may be rampant in proliferative diseases such as tumour or cancer. Accordingly, one aspect of the present disclosure relates to an isolated peptide that is capable of specifically binding to TCTP. In one example, the specific binding results in the inhibition of TCTP interaction with p53 and/or MDM2. [0032] In one example, the isolated peptide comprises a sequence of the N-terminal end of MDM2, wherein the peptide may be capable of specifically binding to TCTP. In one example, the specific binding to TCTP results in the inhibition of TCTP interaction with p53 and/or MDM2. In one example, the peptide as described herein comprises various binding domains of MDM2. In one example, the peptide as described herein comprises the a2 -helix of MDM2. In another example, the peptide as described herein comprises amino acids 1 to 83 of the N-terminal end of MDM2. In one example, the reference amino acid sequence of MDM2 taken from the UNIPROT database is: (SEQ ID NO: 5) and provided as follows: >s I Q00987 |MDM2_HUMAN E3 ubiquitin-protein ligase Mdm2 OS=Homo sapiens GN=MDM2 PE=1 SV=1

10 20 30 40 50 60

MCNT MSVPT DGAVTTSQIP ASEQETLVRP KPLLLKLLKS VGAQKDTYTM KEVLFYLGQY

70 80 90 100 110 120

IMTKRLYDEK QQHIVYCSND LLGDLFGVPS FSVKEHRKIY TMIYRNLWV NQQESSDSGT

130 140 150 160 170 180

SVSENRCHLE GGSDQKDLVQ ELQEEKPSSS HLVSRPSTSS RRRAISETEE NSDELSGERQ

190 200 210 220 230 240

RKRHKSDSIS LSFDESLALC VIREICCERS SSSESTGTPS NPDLDAGVSE HSGDWLDQDS

250 260 270 280 290 300

VSDQFSVEFE VESLDSEDYS LSEEGQELSD EDDEVYQVTV YQAGESDTDS FEEDPEISLA

310 320 330 340 350 360

DYWKCTSCNE M PPLPSHCN RCWALRENWL PEDKGKDKGE ISEKAKLENS TQAEEGFDVP

370 380 390 400 410 420

DC KTIVNDS RESCVEE DD KITQASQSQE SEDYSQPSTS SSIIYSSQED VKEFEREETQ

430 440 450 460 470 480

DKEESVESSL PL AIEPCVI CQGRPK GCI VHGKTGHL A CFTCAKKLKK R KPCPVCRQ

490

PIQMIVLTYF P

[0033] The underlined region represents the a.2 helix region with amino acids 43 to 65, which may interact with TCTP.

[0034] In one example, the peptide as described herein comprises amino acids from 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 to 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or any combination of range thereof of the N-terminal end of MDM2. In yet another example, the peptide as described herein comprises amino acids 10-73, 20-70, 30-68, 40-65, or 43-65 of the N-terminal end of MDM2 as set out in SEQ ID NO: 5. In one example, as demonstrated by the Experimental Section, the peptide as described herein comprises 43-65 of the N-terminal end of MDM2. In one example, the peptide as described herein includes, but is not limited to, amino acid sequence (SEQ ID NO: 1): N-AQKDTYTMKEVLFYLGQYIMTKR-C.

[0035] In one example, the peptide as described herein may be a mutant or derivative of a portion or the entire N-terminal end of MDM2 which still retains the capability of specifically binding to TCTP. In one example, the binding to TCTP results in the inhibition of TCTP interaction with p53 and/or MDM2. In one example, the peptide comprises at least one, at least two, at least three, at least four or all mutations including, but is not limited to Y48A, L54A, Y56A, Y60A and M62A of the TCTP protein as set out in SEQ ID NO: 5. In one example, as used in the Experimental Section below, the peptide comprises M62A mutation. In one example, the peptide may comprise the mutation M62A.

[0036] In one example, the peptides as described herein comprises amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acids 1 to 83 of the N-terminal end of MDM2 as set out in SEQ ID NO: 5.

[0037] The inventors of the present disclosure also envisaged inhibiting anti-apoptotic HDM2 and/or MDM2 activity directly and/or indirectly. Accordingly, in another aspect, there is provided an isolated peptide that is capable of inhibiting MDM2, wherein the isolated peptide comprises the basic domain 2 region of TCTP (translationally controlled tumour protein). In one example, the peptide as described herein comprises the helix-loop-helix motif of the basic domain 2 region of TCTP.

[0038] The helix-loop-helix motif is a protein structural motif that characterises a family of transcription factors. This motif is characterised by two a-helices connected by a loop and is typically associated with protein-DNA binding. However, in the present disclosure, helix- loop-helix motif may be associated with protein-protein interaction, in particular MDM2 and TCTP interaction.

[0039] In one example, TCTP as described herein comprises (SEQ ID NO: 6) as follows: MIIYRDLISH DEMFSDIYKI REIADGLCLE VEGKMVSRTE GNIDDSLIGG NASAEGPEGE

70 80 Ψ 90 100 Ψ 110 120 GTESTVITGV DIVMNHHLQE TSFTKEAYKK YIKDYMKSIK GKLEEQRPER VKPFMTGAAE

130 140 150 160 170

QIKHILANFK ATYQFFIGE M NPDGMVALLD YREDGVTPY IFFKDGLEME KC

>s I P13693 I CTP_HUMAN Translationally-controlled tumour protein OS=Homo sapiens GN=TPT1 PE=1 SV=1

[0040] The basic domain 2 region of TCTP as presented herein is underlined in SEQ ID NO: 6. An isolated peptide as disclosed herein comprises TH2 peptide sequence, which is represented between two arrows starting from amino acid 81 to 107. In one example, the isolated peptide comprises TH3 sequence, which is in italic. Both peptides share the RPER sequence. In one example, a peptide of the present disclosure may span from residues 100 to 120.

[0041] In one example, the peptide as described herein comprises amino acids 60 to 150, 70 to 140, 80 to 135, 80 to 133 or 100 to 120 of TCTP. In one example, the peptide as described herein comprises amino acids 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 to 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 or 100, or any combination of range thereof. In one example, the peptide as described herein includes, but is not limited to, any one of the following sequences N- TSFTKEAYKKYIKDYMKSIKGKLEEQRPER-C (SEQ ID NO: 2), N- RPERVKPFMTGAAEQIKHILANFKNYQ-C (SEQ ID NO: 3) or N- GKLEEORPER VKPFMTGAAE-C (SEQ ID NO: 4).

[0042] In one example, the peptide as described herein may be a mutant or derivative of a portion or the entire basic domain 2 region of TCTP, which still retains the capability of inhibiting MDM2.

[0043] In one example, the peptides as described herein is useful in inhibiting wild type p53-HDM2 interaction, inhibiting p53 degradation and activating cell cycle arrest/apoptosis mediated by the p53 network. In one example, the peptides as described herein are useful in inhibiting the TCTP-HDM2 interaction both in vitro and ex vivo. In one example, the peptides as described herein may comprise amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%», at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the basic domain 2 region of TCTP as set out in SEQ ID NO: 6.

[0044] As used herein, the term "isolated" refers to, e.g., a peptide, DNA, or RNA separated from other peptides, DNAs, or RNAs, respectively, and being found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a biochemical solution of the same. An "isolated" peptide, DNA or RNA does not encompass peptide, DNA or RNA that are present in a human and/or animal body and does not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.

[0045] In one example, the peptides as described herein may comprise at least one amino acid modification. In one example, the amino acid modification may be chemical modification. The chemical modification may be for protection against enzymatic degradation and may include, but is not limited to, amidation, acetylation, stapling, replacing at least one L-amino acid with a corresponding D-amino acid, introducing and replacing at least one amino acid with a non-natural amino acid. In one example, the peptide may be a mutant or derivative of a portion or the entire N-terminal end of MDM2 which still retains the capability of specifically binding to TCTP. In one example, the mutant or derivative of a portion or the entire N-terminal end of MDM2 still retains its binding capability that results in the inhibition of TCTP interaction with p53 and/or MDM2. In one example, the peptide is a mutant or derivative thereof of a portion or the entire basic domain 2 region of TCTP which still retains the capability of specifically binding to MDM2. In one example, the specific binding to MDM2 results in the inhibition of TCTP interaction with p53 and/or MDM2.

[0046] Examples on how to staple peptides are described for example in Synthesis of all- hydrocarbon stapled a-helical peptides by ring-closing olefin metathesis, Kim YW, Grossmann TN, Verdine GL. Nat Protoc. 2011 Jun;6(6):761-71. doi: 10.1038/nprot.2011.324. Epub 2011 May 12. See also US 2002016010. [0047] Non-natural amino acid as described herein may include, but is not limited to, a β- amino acid (β and β ), a homo-amino acid, a proline and pyruvic acid derivative, a 3- substituted alanine derivative, a glycine derivative, a ring-substituted phenylalanine and tyrosine derivative, a linear core amino acid, a diamino acid and a N-methyl amino acid.

[0048] An isolated peptide, in some example, may be substantially pure and free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use. In particular, the peptide may be sufficiently pure and may be sufficiently free from other biological constituents of host cells so as to be useful in, for example, producing pharmaceutical preparations, for sequencing applications, as a laboratory reagent, etc. In some cases, an isolated peptide as disclosed herein may be admixed with other agents in a preparation such that the peptide may comprise only a small percentage by weight of the preparation. The peptide is nonetheless substantially pure or isolated in that it has been substantially separated from the substances with which it may be associated in living systems. As an example, a peptide sequence as discussed herein may be prepared in cells then isolated from the cells. See, e.g., the examples herein.

[0049] In addition to isolated peptides described herein which comprise or consist of naturally-occurring amino acids, peptidomimetics or peptide analogs are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm peptide (i.e., a peptide that has a biological or pharmacological activity), such as naturally-occurring receptor-binding peptide, but have one or more peptide linkages optionally replaced by a linkage that may include, but is not limited to : -CH₂NH-, -CH₂S-, - CH₂=CH₂-, -CH=CH- (cis and trans), -COCH₂-, -CH(OH)CH -, and. -CH₂SO-, by methods known in the art. In one example, the non-peptide linkage is -CH₂NH-. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad- spectrum of biological activities), reduced antigenicity, and others. Labelling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which the peptidomimetic binds to produce the therapeutic effect. Derivatization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. Generally, peptidomimetics of receptor- binding peptides bind to the target with high affinity and possess detectable biological activity.

[0050] In yet another aspect, there is provided a nucleic acid that may encode the peptides as described herein. The terms "polynucleotide," "oligonucleotide" and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g.^ cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double- stranded. In one example, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a peptide as described herein, or a fragment, derivative, mutein, or variant thereof.

[0051] Each of the peptides as described herein (or portions thereof) may optionally be associated with a delivery system or vector, according to one aspect of the present disclosure. In its broadest sense, a "vector" is any vehicle capable of facilitating: (1) delivery of the peptides as described herein to a target cell or (2) uptake of the peptides as described herein by a target cell, if uptake is important. Optionally, a "targeting ligand" (in addition to, or the same as, the plasma membrane targeting molecule) may be attached to the vector to selectively deliver the vector to a cell which expresses on its surface the cognate receptor for the targeting ligand. In this manner, the vector (containing one or more of the peptides as described herein) may be selectively delivered to a cell in, e.g., a tumour. In general, the vectors as described herein may be divided into two classes: colloidal dispersion systems and biological vectors. Other example compositions that may be used to facilitate uptake by a target cell of compositions of the invention include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, and electroporation. [0052] In general, the vectors useful may include, but are not limited to, plasmids, phagemids, viruses, or other vehicles derived from viral or bacterial sources. Viral vectors include, but are sot limited to, nucleic acid sequences from any of the following viruses: retrovirus, such as molorsey murine leukemia vims, harvey murine sarcoma virus, murine mammary tumour virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SY40- type viruses; polyoma viruses; Epstein-Barr viruses: papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named above but known to the art.

[0053] In some cases, the viral vectors may be based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent pro viral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful may be those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid; transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in the literature.

[0054] A vims useful for certain applications may be an adeno-associated virus, which is a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication- deficient and may be capable of infecting a wide range of cell types and species in many cases. It further has certain advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition, thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0055] Other suitable vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See e.g., Sambrook, el at., Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, 1989. Plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC 18, pUC 19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and/or ligation reactions to remove and add specific fragments of DNA.

[0056] In yet another aspect, there is provided the use of the peptide as described herein in the manufacture of a medicament for treating cancer. In one example, the medicament as described herein may further comprise at least one excipients or at least one diluent.

[0057] In yet another aspect, there is provided a method of treating cancer that may comprise the administration of at least one peptide as described herein to a patient in need thereof. In one example, the peptide may further comprise at least one excipients or at least one diluent.

[0058] The term "cancer," as used herein, may include, but is not limited to: biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma: ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumours such as seminoma, non-seminoma, teratomas, choriocarcinomas; stromal tumours and germ cell tumours; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumour. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer. In general, an effective amount of the one or more compositions of the invention for treating cancer will be that amount necessary to inhibit mammalian cancer cell proliferation in situ. Those of ordinary skill in the art are well-schooled in the art of evaluating effective amounts of anti-cancer agents. In one example, the cancer may include, but is not limited to prostate cancer, liver cancer and breast cancer.

[0059] In yet another aspect, there is provided a pharmaceutical composition comprising at least one peptide as described herein. In one aspect, the present disclosure provides a method of administering any of the compositions described herein to a subject. When administered, the compositions are applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation. As used herein, the term "pharmaceutically acceptable" is given its ordinary meaning. Pharmaceutically acceptable compositions are generally compatible with other materials of the formulation and are not generally deleterious to the subject. Any of the compositions of the present invention may be administered to the subject in a therapeutically effective dose. The dose to the subject may be such that a therapeutically effective amount of one or more active compounds reaches the active site(s) within the subject. A "therapeutically effective" or an "effective" dose, as used herein, means that amount necessary to delay the onset of, inhibit the progression of, halt altogether the onset or progression of, diagnose a particular condition being treated, or otherwise achieve a medically desirable result, i.e., that amount which is capable of at least partially preventing, reversing, reducing, decreasing, ameliorating, or otherwise suppressing the particular condition being treated. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of mammal, the mammal's age, sex, size, and health; the composition used, the type of delivery system used; the time of administration relative to the severity of the disease; and whether a single, multiple, or controlled-release dose regiment is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation. [0060] In administering the systems and methods of the invention to a subject, dosing amounts, dosing schedules, routes of administration, and the like may be selected so as to affect known activities of these systems and methods. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. The doses may be given in one or several administrations per day.

[0061 ] Administration of a composition of the invention to a subj ect may be accomplished by any medically acceptable method which allows the composition to reach its target. The particular mode selected will depend of course, upon factors such as those previously described, for example, the particular composition, the severity of the state of the subject being treated, the dosage required for therapeutic efficacy, etc. As used herein, a "medically acceptable" mode of treatment is a mode able to produce effective levels of the active compound(s) of the composition within the subject without causing clinically unacceptable adverse effects. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated. For example, the composition may be administered orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, through parenteral injection or implantation, via surgical administration, or any other method of administration where suitable access to a target is achieved. Examples of parenteral modalities that can be used with the invention include intravenous, intradermal, subcutaneous, intracavity, intramuscular, intraperitoneal, epidural, or intrathecal. Examples of implantation modalities include any implantable or injectable drug delivery system. Oral administration may be preferred in some embodiments because of the convenience to the subject as well as the dosing schedule. Compositions suitable for oral administration may be presented as discrete units such as hard or soft capsules, pills, cachettes, tablets, troches, or lozenges, each containing a predetermined amount of the composition. Other oral compositions suitable for use with the invention include solutions or suspensions in aqueous or non-aqueous liquids such as a syrup, an elixir, or an emulsion. In one example, the composition may be used to fortify a food or a beverage. Injections can be e.g., intravenous, intradermal, subcutaneous, intramuscular, or interperitoneal. The systems and methods as described herein can be administered by any method which allows the composition of the invention to reach the target cells, e.g., tumour cells. These methods include, e.g., injection, infusion, deposition, implantation, anal or vaginal supposition, oral ingestion, inhalation, topical administration, etc. [0062] Other delivery systems suitable for use with the present disclosure include time- release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters. polyhydroxybutyric acid, and/or combinations of these.

[0063] In one example, a composition may include a suitable pharmaceutically acceptable carrier, for example, as incorporated into a liposome, incorporated into a polymer release system, or suspended in a liquid, e.g., in a dissolved form or a colloidal form, such as in a colloidal dispersion system. As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic material that does not significantly interfere with the effectiveness of the biological activity of the active compound(s) to be administered, but is used as a formulation ingredient, for example, to stabilize or protect the active compound(s) within the composition before use. The term "carrier" denotes an organic or inorganic ingredient, which may be natural or synthetic, with which one or more active compounds of the present disclosure are combined to facilitate the application of the composition. The carrier may be co-mingled or otherwise mixed with one or more active compounds of the present disclosure, and with each other. In a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The carrier may be either soluble or insoluble, depending on the application. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble. Those skilled in the art will know of other suitable carriers, or will be able to ascertain such, using only routine experimentation.

[0064] In one example, the compositions of the present disclosure may include pharmaceutically acceptable carriers with formulation. Ingredients such as salts, carriers, buffering agents, emulsifiers, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, or stabilizers. For example, if the formulation is a liquid, the carrier may be a solvent, partial solvent, or non-solvent, and may be aqueous or organically based. Examples of suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate: granulating and disintegrating agents such as corn starch or * algenic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time- delay materials such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally-occurring phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents such as acetic acid and salts thereof, citric acid and salts thereof, boric acid and salts thereof, or phosphoric acid and salts thereof; or preservatives such as benzalkonium chloride, chlorobutanol, parabens or thimerosal. Suitable carrier concentrations can be determined by those of ordinary skill in the art, using no more than routine experimentation. The compositions of the present disclosure may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, elixirs, powders, granules, ointments, solutions, depositories, inhalants or injectables. Those of ordinary skill in the art will know of other suitable formulation ingredients, or will be able to ascertain such, using only routine experimentation.

[0065] Preparations include sterile aqueous or nonaqueous solutions, suspensions and emulsions, which can be isotonic with the blood of the subject in certain examples. Examples of nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oil such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono or di-glycerides. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishes (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. [0066] In some examples, the compositions of the present disclosure may be present as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" includes salts of the composition, prepared in combination with, for example, acids or bases, depending on the particular compounds found within the composition and the treatment modality desired. Pharmaceutically acceptable salts can be prepared as alkaline metal salts, such as lithium, sodium, or potassium salts; or as alkaline earth salts, such as beryllium, magnesium or calcium salts. Examples of suitable bases that may be used to form salts include ammonium, or mineral bases such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and the like. Examples of suitable acids that may be used to form salts include inorganic or mineral acids such as hydrochloric, hydrobromic, hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, phosphorous acids and the like. Other suitable acids include organic acids, for example, acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, j-tolylsulfonic, citric, tartaric, methanesulfonic, glucuronic, galacturonic, salicylic, formic, naphthalene-2-sulfonic, and the like. Still other suitable acids include amino acids such as arginate, aspartate, glutamate, and the like.

[0067] In another aspect, there is provided a method of detecting the presence or absence of TCTP in a sample, wherein the method may comprise contacting the sample with a peptide as described herein. In one example, the method may further comprise the step of analyzing the sample resulting from the contacting step to identify the presence or absence of the binding product of TCTP and the peptide as described herein. In one example of the method of detecting, the peptide may comprise a label. In one example, the label may be an optically identifiable label emitting an optically detectable signal in case of the presence of TCTP. In one example, the method of detecting the presence or absence of TCTP in a sample may be a pull-down method. In one example, the method of detecting the presence or absence of TCTP in a sample may comprise inserting the peptide into a protein scaffold, such as, but is not limited to thioredoxin. The resultant hybrid protein can be used to detect for presence of absence of TCTP by pull-down assay. The generation of hybrid proteins comprising the peptide additionally affords a means to introduce the peptide into cells by introduction of nucleic acids encoding the said hybrid protein. In one example, the peptide may be fused to a protein such as, but is not limited to, green fluorescence protein (GFP) at either terminus and resultant fusion protein introduced to cells by introduction of nucleic acids encoding the said fusion protein.

[0068] In another aspect, there is provided a microarray that may comprise the peptide as described herein for detection of TCTP in a sample.

[0069] While several examples of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, materials kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[0070] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0071] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," ""one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0072] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and SET (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0073] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order In which the steps or acts of the method are recited.

[0074] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but is not limited to. Only the transitional phrases "consisting of* and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 211 1.03.

[0075] Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0076] The invention 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 invention. This includes the generic description of the invention 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.

[0077] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

Materials and Methods

Reagents

[0078] DOl antibody was a kind gift from Dr Borivoj Vojtesek. Anti-FLAG and anti-HA antibodies were from Sigma. Nutlin-3 (IUPAC name: (±)-4-[4,5-Bis(4-chlorophenyl)-2-(2- isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole- 1 -carbonyl]-piperazin-2-one) was from Calbiochem. The following oligonucleotides (FBCO) were used:

1)TCTP-F: 5 ' - ATG ATT ATCT ACCGGG ACCTC A-3 ' (SEQ ID NO: 7)

2) TCTP-R: //5'-TTAACATTTTTCCATTTCTAAACCATCC-3' (SEQ ID NO: 8)

3) TCTPINF-F: 5'- AAGGAGATATACATATGATTATCTACCGGGACCTCATC-3' (SEQ ID NO: 9)

4) TCTPFLAGINFR: 5'- GGTGGTGGTGCTCGAGTTATTTATCATCATCATCTTTATAATCACATTTTTCCATTTCTAA ACCATCC

-3' (SEQ ID NO: 10)

5) TCTPinf3.1HIND-F: 5'-

GCGTTTAAACTTAAGCTTACCATGATTATCTACCGGGACCTCATC-3' (SEQ ID NO: 11) 6) TCTPinf3.1FLAG-R: 5'-

GGCCCTCTAGACTCGAGTCACTTGTCGTCGTCGTCCTTGTAGTCACATTTTTCCATTTCTA AACCATCC -3' (SEQ ID NO: 12)

7) petF2: 5'-CATCGGTGATGTCGGCGAT-3' (SEQ ID NO: 13)

8) petRC: 5 ' -G AT AT AGTTCCTCCTTTC AGC A-3 ' (SEQ ID NO: 14)

9) HDM491HA-R: 5'- CAGTTAAGCGTAATCTGGAACATCGTATGGGTAGGGGAAATAAGTTAGCACAAT-3' (SEQ ID NO: 15)

10) HDM339HA-R: 5'-

CAGTTAAGCGTAATCTGGAACATCGTATGGGTACCCTTTATCTTTCCCTTTATC-3' (SEQ ID NO: 16)

11) HDM302HA-R: 5'-

CAGTTAAGCGTAATCTGGAACATCGTATGGGTAGTCAGCTAAGGAAATTTCAGG-3' (SEQ ID NO: 17)

12) HDM109HA-R: 5'-

CAGTTAAGCGTAATCTGGAACATCGTATGGGTATACTACCAAGTTCCTGTAGAT-3' (SEQ ID NO: 18)

13) HDM65HA-R: 5'-

TTACGC ATAATCCGGC AC ATCATACGGATAGCTTGGC ACGCC AAAC AAATC-3 '(SEQ ID NO: 19)

14) HDM43HA-R: 5'- TTACGCATAATCCGGCACATCATACGGATAATCATATAATCGTTTAGTCAT-3' (SEQ ID NO: 20)

15) TCTPFLAG133-R: 5'-

T ATTT ATC ATC ATC ATCTTT AT AATC AGGTl TACTCTTTCTGGTCTCTG-3 '(SEQ ID NO: 21 )

16) TCTPFLAG79-R: 5'-

TTATTTATCATCATCATCTTTATAATCCTGCAGGTGATGGTTCATG-3' (SEQ ID NO: 22)

17) TCTPFLAG40-R: 5'-

T ATTTATC ATC ATC ATCTTTATAATCTTCTGTCCTACTG ACC ATCTTCC-3 ' (SEQ ID NO: 23)

18) HDML54A-l :5'-CTATGAAAGAGGTTGCGΊTTTATCTTGGCCAG-3^, (SEQ ID NO: 24) 19)HDML54 A-2 : 5 '-CTGGCC AAG ATAAAACGC AACCTCTTTC AT AG ' (SEQ ID NO: 25)

20) HDMY48 A- 1 : 5 '-GC AC AAAAAG AC ACTGCG ACT ATGAAAGAGGT-3 ' (SEQ ID NO: 26)

21) HDMY48A-2:5'-ACCTCTTTCATAGTCGCAGTGTCTTTTTGTGC-3' (SEQ ID NO: 27)

22) HDMY56A-l :5'-GAAAGAGGTTCTTTTTGCGCTTGGCCAGTATATTA-3' (SEQ ID NO: 28)

23) HDMY56 A-2 : 5 ' -TAATATACTGGCCAAGCGC AAAAAG AACCTCTTTC-3 ' (SEQ ID NO: 29) 24)HDM Y60 A- 1 : 5 '-CTTTTTTATCTTGGCC AGGCGATTATGACTAAACG-3 ' (SEQ ID NO: 30)

25) HDM Y60 A-2 : 5 '-CGTTTAGTC ATAATCGCCTGGCC AAG ATAAAAAAG-3 ' (SEQ ID NO: 31 )

26) HDMM62A-l :5'-CTTGGCCAGTATATTGCGACTAAACGATTATATG-3' (SEQ ID NO: 32)

27) HDMM62A-2:5'-CATATAATCGTTTAGTCGCAATATACTGGCCAAG-3' (SEQ ID NO: 33) 28) HDMNtermdel-F: 5'-AAGGACCTTGTACAAGAGCTTCAGG-3' (SEQ ID NO: 34)

29) petATG-R: 5 '-C ATATGTAT ATCTCCTTCTT AAAGTTAAAC-3 ' (SEQ ID NO: 35)

Nucleic acid manipulation

[0079] The TCTP gene was amplified by reverse-transcription PCR on RNA extracted from AGS cells using primers 1 and 2, re-amplified using primers 3 and 4, and cloned into the Ndel-Hindlll sited of pET22-b by infusion cloning (Clontech). The gene was then amplified with primers 5 and 6, and cloned by infusion cloning into the Hindlll-Xhol sites of pcDNA3.1a(+). [0080] Templates for in vitro transcription/translation were prepared by PCR amplification of respective gene cloned in pET22 vector using primers 7 and 8. C-terminal deletion templates were prepared by PCR using primer 7 along with one of primers 9-14 (for HDM2) and primers 15-17 (for TCTP). Primers 9-14 additionally encode a C-terminal HA tag. Primers 15-17 additionally encode a C-terminal FLAG tag. Quickchange mutagenesis (Stratagene) was used to mutate specific residues in TCTP to alanine using primers 18-27. HDM2A1-109 was made by PCR amplification of parental HDM2-pet22 plasmid using primers 28-29 followed by phosphorylation using T4 polynucleotide kinase and intramolecular ligation.

[0081] Vectors for cell culture work were constructed from the parental plasmid HDM2- CMV. HDM2-M62A-CMV was constructed via Quikchange mutagenesis using the primer pairs 26 and 27.

In vitro transcription-translation

[0082] Proteins were synthesised by in vitro transcription translation using the PURESYSTEM kit (NEB). lOng of HDM2 PCR template (-1.7 Kb) was used per 5μΙ. reaction. The amounts of all other templates were appropriately adjusted to maintain same molar concentration. ZnCl₂ was added to a final concentration of 0.5μΜ for expression of MDM2 and p53 proteins. p53 protein was synthesised at 30°C for 1.5 hours. All other proteins were synthesised at 37°C for 1 hour. Completed reactions were incubated on ice until required.

Pull-down assays

[0083] Protein G beads (Invitrogen) were incubated with anti-HA antibody or anti-Flag antibody (^g per 5 Ι beads) for 1 hour in PBST-l%(w/v)BSA and subsequently washed twice in PBST-0.1%(w/v)BSA and once in PBS to remove non-specifically bound protein. In vitro synthesised protein (5μΙ, per 5μΙ, beads) was added directly to beads and incubated on a rotating wheel for 45 minutes. Beads were washed and incubated with in vitro extract containing second protein as before. For competition experiments, beads were incubated with Nutlin-3, 53 peptide/control peptide in PBS for 45 minutes before Washing and addition of second protein. Beads were finally washed as before and bound proteins eluted by resuspension in 20μΙ_, SDS-PAGE loading buffer and incubation at 95°C for 5 minutes.

Where required, blank in vitro extract (no template DNA added) was used as control.

[0084] For pull-downs using peptides the following biotinylated peptides (Mimotopes) were used: ^{' r';} .Ί ■ \ ·:.

TH2 : GGGSTSFTKE AYKKYIKDYMKSIKGKLEEQRPER (SEQ ID NO: 36)

TH3: GGGSRPERVKPFMTGAAEQI KHILANFKNYQ (SEQ ID NO: 37)

TH3-NL: GGGSVKPFMTGAAEQIKHILANFKNYQ (SEQ ID NO: 38)

a2: GGGSAQKDTYTMKEVLFYLGQYIMTKR (SEQ ID NO: 39)

GS-control : GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 40)

p53 peptide: QETFSDLWKLLP (SEQ ID NO: 41)

[0085] Peptides were immobilized on Dynabeads® M-280 Strepavidin beads (Life Technologies) by incubating 10 μg of each peptide with 15 of beads (per sample) in PBST - 3% BSA(w/v) buffer on a rotator for 2 hours at room temperature. Beads were washed twice to remove unbound peptides before incubation with either purified HDM2(1- 125)(15 μΜ) or TCTP (15 or 30 μΜ) proteins for 4 hours at 4°C on a rotator. Captured proteins were eluted as above. A separate pull-down was also performed as described above, but in the presence of either DMSO or Nutlin-3 (2/20/200 μΜ) during the protein-peptide incubation step.

[0086] For pull-down of purified TCTP, cobalt beads were first blocked in PBS- 3%BSA(w/v) for one hour, before coating with 20μΜ recombinant his-tagged HDM2 (residues 17-125) or control peptide (N-HHHHHHYPYDVPDYA-C) on a rotator at 4°C for one hour. Beads were then washed once before a 4-hour incubation at 4°C with 7μΜ of TCTP protein. As a competitor, 200 μΜ of Nutlin-3 was added to the respective supernatant during bead coating and pull-down steps. Finally, beads were washed twice in PBST- 0.1%BSA(w/v), once in PBS and proteins eluted in SDS-PAGE loading buffer prior to Western analysis.

Protein purification

[0087] Both TCTP and Hdm2 (amino acids 1-125) were expressed as GST-fusion proteins using the pGEX-6P-l expression vector. Both proteins were initially passed through a 5 mL GSTrap™ FF (GE life sciences) column and eluted following on-column cleavage with precision protease. Protein fractions were analyzed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). HDM2 protein samples were then dialyzed into a buffer solution containing 20mM Bis-Tris, pH 6.5, 0.05M NaCl with 1 mM DTT and loaded onto a monoS column pre-equilibrated in buffer A (20mM Bis-Tris, pH 6.5, ImMDTT). Bound protein was eluted with a linear gradient of 1M NaCl over 25 column volumes. For TCTP the same protocol was followed but buffers instead contained 20mM Tris at pH 8.0 and the protein was loaded onto a monoQ column before being eluted. Protein fractions were identified using SDS page gel and protein concentration measured using absorbance at A₂₈₀.

Western blot Analysis

[0088] Immunoprecipitated proteins were subjected to electrophoresis, transferred to nitrocellulose membranes and probed for TCTP with horseradish peroxidase conjugated anti- flag antibody (Sigma) or for HDM2 with anti-HA antibody followed by rabbit anti-mouse (Dakocytomation). p53 was probed for using horseradish peroxidise conjugated DOl antibody (Santa Cruz). For peptide-pull-down assays, TCTP was detected with anti-TCTP antibody (ab37506, Abeam) and HDM2 was detected with 4B2a anti-HDM2 antibody.

Fluorescence polarization (FP)

[0089] Fluorescence polarization measurements were performed using purified HDM2 (1- 125) protein and carboxyfluorescein (FAM) labelled 12-1 peptide (FAM-RFMDYWEGL- NH₂) on the En Vision™ Plate Reader (Perkin Elmer). Competition measurements were carried out in triplicate, containing 5 OnM of fluorescence peptide, with or without 250 nM of HDM2 (1-125) and the respective competitors (TH2, TH3, Nutlin-3, p53 peptide or GS- control peptide) in 50 μΐ, of PBS-0.005%(v/v)Tween-20 buffer. Cell culture

[0090] H1299 p53-/- cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. The cells were seeded at 1.4 ^χ 10⁵ cells/well in 6-well plates, 24 hours prior to transfection. A total of 1.37 μg of expression construct DNA was transfected per well with lipofectamine (Invitrogen) according to the manufacturer's instruction. MG132 (Calbiochem) was also added at a final concentration of 20μΜ 4.5 hours post-transfection to prevent proteasomal degradation. 10μΜ Nutlin-3 (Calbiochem) was added to selected wells 4.5 hours post- transfection. HCT116 p53^"A cells were maintained in McCoy's 5A medium with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. The cells were seeded at 2.8 x 10⁶ cells per 10cm² dish and ΙΟμΜ Nutlin-3 was added to selected dishes 24 hours post seeding. Drug treatment was allowed to proceed for 1 hour prior to harvesting.

Immunoprecipitation and western blot analysis

[0091] H1299 p53 ^" cells were harvested 24 hours after transfection and lysed with lysis solution (Applied Biosystems) supplemented with both protease and phosphatase inhibitors. ΙΟμΙ of anti-HA (Sigma) antibody-coated protein G Dynabeads (Invitrogen) was used per reaction. Beads were washed twice in PBS with 0.1% (v/v) Tween-20 and incubated with 150μg of cell lysate on a rotator at 4°C for 3 hours before washing three times with PBS with 0.1% (v/v) Tween-20. The beads were resuspended in 20μΙ. of SDS-PAGE loading buffer and the protein complexes eluted by incubation at 95°C for 5 mins. HCT116 p53 ^" cells were harvested 1 hour post drug treatment and lysed with modified RIPA buffer (50mM Tris-HCl pH 7.4-8.0, 150mM NaCl, 1% NP-40). Beads were prepared as above and incubated with l μg cell lysate at 4°C overnight with 2μg of 2A9 antibody (Abeam). The beads were then washed as described for HI 299 p53^"A cells and the protein complexes eluted by incubation at 95°C for 5 mins. Immunoblotting was carried out with the relevant antibodies and identified by Immun-star™ westernC™ kit (Bio-rad). 5μg of H1299 p53^v" cell lysate and 20μg of HCT116 p53^{" _} cell lysate per reaction was also used to check expression levels of relevant proteins via western blot.

Annexin-V staining and analysis

[0092] Cells seeded overnight were treated accordingly with either DMSO blank, TM1 or TMC peptides in either serum-fed or -free media for the appropriate duration, washed in PBS and harvested via trypsin/versene treatment. The trypsin/versene solution was removed and cells were then washed and resuspended in annexin-V binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCb, pH 7.4). Cells were then stained with FITC conjugated annexin- V (Life Technologies) for 15 minutes at room temp and FACS analysed using the LSR II flow cytometer (BD Biosciences).

[0093] TM1 peptide used corresponds to wildtype amino acid sequence of Hdm2, which interacts with endogenous TCTP in cancer cells to illicit apoptosis. TMC peptide harbours a single amino-acid, M62A, substitution which was shown to interact weakly with TCTP, and was thus used as a negative control peptide sequence in our experiments.

Results

[0094] Pull-down assays using in vitro expressed proteins were carried out to map the interaction site(s) between TCTP and HDM2. HDM2 (tagged at the C terminus with HA) was bound to protein G beads coated with anti-HA antibody. The beads were subsequently incubated with TCTP (FLAG-tagged). Bound TCTP was identified via Western blot. C- terminal deletion analysis of HDM2 indicated the N-terminal region alone (residues 1-83) was sufficient for interaction with TCTP (Fig. lA). Deletion of residues 1-109 in HDM2 resulted in very minimal interaction with TCTP (Fig. IB), suggesting a predominant N- terminal interaction site. Further deletion analysis highlighted the importance of residues 44- 65 in the interaction (Fig. 1C). This region comprises the a2 helix that forms part of the p53 binding cleft of HDM2. Alanine scanning of this region was carried out to further map the interaction. Residues Y48, L54, Y56, Y60 and M62 were individually mutated to alanine in full length HDM2 and the interaction with TCTP assayed. The results show a progressive reduction in the interaction with TCTP as residues along the a2 helix are mutated, with the M62A mutant showing considerably weaker binding (Fig. ID). The first 25 amino acids in HDM2 comprising the flexible lid region were additionally deleted. Binding to TCTP was unperturbed, further confirming the importance of residues 44-65 in the interaction with TCTP (Fig. ID). M62 forms part of the binding pocket that accommodates the side chain of F19 in the p53 transactivation domain. Accordingly, the possibility that the TCTP binding site of HDM2 overlap with the p53 binding site was investigated. HDM2 was first incubated with a p53 peptide corresponding to residues 19-26 of the p53 transactivation domain that interact with HDM2, followed by TCTP. The HDM2 inhibitor Nutlin-3, which binds to the p53-binding cleft, was also pre-incubated with HDM2. p53 peptide, but not control peptide (p53 peptide with critical contact residues F19, W23, L26 mutated to alanine) diminished TCTP binding (Fig. 2A). Nutlin-3 also showed a dose responsive reduction in TCTP binding. Inhibition by Nutlin-3 was again observed when recombinant HDM2 N-terminal domain was used to pull down recombinant TCTP (Fig. 2B). [0095] The capacity to retain binding to p53 was further investigated in the HDM2-M62A mutant. As shown in Fig. 3, it bound to p53 as strongly as wild-type, indicating the lack of any major structural perturbation due to this mutation. However, whilst Nutlin-3 showed a dose-responsive knock down in the HDM2-p53 interaction, the M62A mutant proved recalcitrant to Nutlin-3 inhibition.

[0096] C-terminal deletion analysis of TCTP was additionally carried out to map its interaction site with HDM2. Whilst full-length TCTP and residues 1-133 bind to HDM2, further truncation to 79 residues completely ablates HDM2 binding (Fig. 4A). The same result was obtained with the reverse configuration of the IP (TCTP captured on beads used to pull down HDM2, Fig. 4B). This indicated a probable interaction interface within amino acids 80-133 of TCTP which comprises a helix-loop-helix motif. A series of pull-down experiments using 2 synthetic peptides spanning this region of TCTP (TH2: residues 81-110 and TH3: residues 107-133) along with a peptide spanning the HDM2 a2 helix (residues 43- 65) was subsequently carried out. The results in Fig. 5A (top panel) indicate that both TH2 and TH3 peptides immobilised on beads can pull down recombinant HDM2 N-terminal domain (residues 1-125), with TH3 showing a stronger binding phenotype. Additionally, immobilised HDM2 a2 helix peptide pulled down recombinant full-length TCTP (bottom left panel). Strikingly, the same peptide with the M62A mutation (a2M62A) showed significantly reduced pull-down of TCTP. Neither recombinant TCTP nor HDM2 (1-125) bound to an immobilised control peptide (CON, top panel). As a positive control, the p53 peptide known to interact with the N-terminal domain of HDM2 was used. Peptides TH2 and TH3 share the sequence RPER comprising the loop region (residues 107 to 110) within the helix-loop-helix motif defining the TCTP basic domain 2 (residues 80-133). TH3 peptide lacking this sequence (TH3-NL) showed significantly reduced pull-down of HDM2 (1-125) (Fig. 5A, bottom right panel), highlighting the important contribution of this region to the interaction. The pull-down experiments with TH2 and TH3 peptides were next repeated in the presence of Nutlin-3 (Fig. 5B). Interaction of TH2 peptide with HDM2 (1-125) was clearly perturbed in a dose-responsive manner. The TH3 peptide interaction was minimally inhibited at the highest concentration of Nutlin-3 used, consistent with its stronger binding phenotype. This was also observed when the interactions were assayed by fluorescence polarisation (Fig. 5C). HDM2 (1-125) was pre-incubated with fluorescently labelled p53 peptide and the ability of the TCTP peptides to displace this was measured. TH3 peptide, but not TH2 was able to displace the p53 peptide, although to a lower extent than the positive controls Nutlin-3 and un-labelled p53 peptide. The effect of Nutlin-3 on the endogenous TCTP-HDM2 interaction in the HCT116 ρ53^'Λ cell line was subsequently investigated. Co-immunoprecipitation was carried out using anti-HDM2 antibody to capture TCTP-HDM2 complexes. The results (Fig. 6 A) indicate disruption of the TCTP-HDM2 interaction by Nutlin-3, consistent with the previous in vitro data (Figures 2, 5B). The same phenotype was seen using exogenously expressed HDM2 in the p53-null H1299 cell line (Fig. 6B). Furthermore, the HDM2-M62A mutant showed very weak interaction with TCTP compared to wild-type, again consistent with the in vitro result (Figure ID).

[0097] To further examine the feasibility of peptide-based cancer treatment based on interacting domains of TCTP and Hdm2, a Tat-conjugated peptide corresponding to the Hdm2-a2 helix was synthesized together with an inactive control peptide which carried the M62A single amino-acid substitution. Both the HI 299 and MCF-7 cells displayed increased Annexin-V positive staining, compared to DMSO control treatments, when treated with the active and not control peptide. This shows an induced apoptotic event, possibly due to both the inactivation of TCTP's anti-apoptotic functions, and, in the case of MCF-7 cells, an abatement of TCTP's inhibition of p53 activity. Accordingly, the results in Figures 9-12 indicate significant increases in apoptosis (as detected by Annexin-V staining) when both HI 200 and MCF-7 cells are treated with TM1 peptide compared to cells treated with TMC peptide.

[0098] It was recently shown that TCTP increased MDM2-mediated ubiquitination of p53 in HCT116 p53^+/+ cells, and that this effect was inhibited by Nutlin-3. In the present disclosure, a possible mechanistic rationale for this observation is provided by showing that TCTP and Nutlin-3 can compete for binding to the p53-binding cleft in the N-terminus of HDM2. The p53-binding cleft consists of 4 a helices, and a pair of β sheets cap each end. Deletion analysis implicated the a2 helix forming one side of the cleft as contributing significantly to the TCTP interaction site. Alanine scanning of the a2 helix further identified critical residues involved in the interaction, with M62 being of particular importance. This residue comprises part of the binding pocket that accommodates F19 of p53 and the ethyl ether moiety of Nutlin-3. Notably, binding of p53 to HDM2-M62A was not inhibited by Nutlin-3, suggesting against mutation-induced structural deformation. Based on these observations, a model was proposed wherein TCTP binds a sub-region of the p53-binding cleft to exert its chaperone-like function on HDM2. TCTP is subsequently displaced by p53 due to its higher affinity for the p53-binding cleft. Additionally, a secondary p53 interaction site within the acidic domain of HDM2 may contribute towards high affinity interaction and TCTP displacement.

[0099] The TCTP interaction site was mapped within residues 80-133 corresponding to the basic domain 2. This region comprises an helix-loop-helix motif and data disclosed herein show residues within the loop to contribute significantly to the interaction with HDM2. Domain 2 has been implicated in TCTP's interaction with tubulin, calcium, and the Na,K- ATPase a subunit. Furthermore, TCTP has recently been shown to interact with p53 through either domain 2 or N- and C-terminal regions. It is noted that an interaction interface between residues 1-68 of TCTP and residues 302-435 of HDM2 has previously been mapped using SPR and recombinant proteins. As described herein, this interaction site was not evident in the results using pull-down assays with in vitro expressed proteins.

[00100] Using molecular simulations, a docked complex of TCTP with HDM2 ( 1 - 125) was derived (Fig. 7). Stable interactions of the TH2 and TH3 helices of TCTP with residues in the HDM2 nutlin-binding pocket were observed in accordance with the alanine scanning data (Fig. ID). The RPER loop region (residues 107-110) connecting TH2 and TH3 is stabilized by intramolecular interactions of R107 and Rl 10 with residues in TCTP, whilst the backbone carbonyl of P108 and the side chain of E109 are stabilized by K51 of HDM2. Additionally, El 04 of TCTP is also stabilized by K45 of MDM2 (Fig. 7A). The loss of affinity seen when the loop region was deleted from peptide TH3 (Fig. 5A) could result from the removal of one salt bridge and/or significant perturbation of the other. It is clear from Fig. 7B that the a2 helix of HDM2 interacts with both TH2 and TH3 of the TCTP, with M62 closely packed under TH3.

[00101] TCTP has been shown to be significantly upregulated in a number of human cancers, with high levels of TCTP correlating with poor prognosis. Nutlin-3 has been shown to be most effective in cancers which express high levels of wild type p53 and high levels of HDM2. It is envisage that Nutlin-3 and/or high levels of TCTP overexpression may be useful in the treatment of cancer in human or animal.

[00102] The value of inhibiting p53-HDM2 interaction as a possible target for cancer therapeutics is currently an area of great activity. The disclosure as described herein that TCTP not only interacts with both these proteins, but has a binding site on HDM2 which overlaps with that of p53, adds further complexity to the p53-HDM2 interaction model. However, it is believed that the disclosure herein provides basis for therapies aimed at inhibiting p53 HDM2 binding.

Claims

Claims

1. An isolated peptide comprising a sequence of the N-terminal end of MDM2, wherein the peptide is capable of specifically binding to TCTP.

2. The peptide of claim 1, wherein the binding to TCTP results in the inhibition of TCTP interaction with p53 and/or MDM2.

The peptide of claim 1 or 2, wherein the peptide comprises the a2 -helix of MDM2.

The peptide of any one of claims 1 to 3, wherein the peptide comprises an amino acid sequence that is at least 80% identical to the amino acids 1 to 83 of the N-terminal end of MDM2 as set out in SEQ ID NO: 5.

The peptide of any one of claims 1 to 4, wherein the peptide comprises amino acids 1 to 83 of the N-terminal end of MDM2 as set out in SEQ ID NO: 5.

The peptide of claim 5, wherein the peptide comprises amino acids 43 to 65 of the N- terminal end of MDM2 as set out in SEQ ID NO: 5.

The peptide of claim 6, wherein the peptide comprises amino acid sequence (SEQ ID NO: 1): N-AQKDTYTMKEVLFYLGQYIMTKR-C.

The peptide of any one of claims 1 to 7, wherein at least one amino acid of the peptide is chemically modified.

The peptide of claim 8, wherein the peptide is chemically modified for protection against enzymatic degradation.

The peptide of claim 8 or 9, wherein the peptide modification is selected from the group consisting of amidation, acetylation, stapling, replacing at least one L-amino acid with a corresponding D-amino acid, introducing or replacing at least one amino acid with a non-natural amino acid.

The peptide of claim 10, wherein the non-natural amino acid is selected from the group consisting of a β-amino acid (β³ and β²), a homo-amino acid, a proline and pyruvic acid derivative, a 3 -substituted alanine derivative, a glycine derivative, a ring- substituted phenylalanine and tyrosine derivative, a linear core amino acid, a diamino acid and a N-methyl amino acid.

The peptide of any one of claims 1 to 11, wherein the peptide is a mutant or derivative of a portion or the entire N-terminal end of MD 2 which still retains the capability of inhibiting TCTP interaction with p53 and/or MDM2.

A nucleic acid encoding a peptide according to any one of claims 1 to 12.

14. A vector comprising a nucleic acid according to claim 13. An isolated peptide comprising the basic domain 2 region of TCTP (translationally controlled tumour protein), wherein the peptide is capable of inhibiting MDM2.

The peptide of claim 15, wherein the peptide comprises the helix-loop-helix motif of the basic domain 2 region of TCTP.

The peptide of claim 15 or 16, wherein the peptide comprises an amino acid sequence that is at least 80% identical to the basic domain 2 region of TCTP as set out in SEQ ID NO: 6.

The peptide of claim 17, comprising amino acids 100 to 120 or 80 to 133 of TCTP as set out in SEQ ID NO: 6.

The peptide of any one of claims 15 to 18, comprising any one of the following sequences N-TSFTKEAYKKYIKDYM SIKGKLEEQRPER-C (SEQ ID NO: 2), N- RPERVKPFMTGAAEQIKHILANFKNYQ-C (SEQ ID NO: 3) or N- KGKLEEORPER VKPFMTGAAE-C (SEQ ID NO: 4).

The peptide of any one of claims 15 to 19, wherein at least one amino acid of the peptide is chemically modified.

The peptide of claim 20, wherein the peptide is chemically modified for protection against enzymatic degradation.

The peptide of claim 20 or 21, wherein the peptide modification is selected from the group consisting of amidation, acetylation, stapling, replacing at least one L-amino acid with a corresponding D-amino acid, introducing or replacing at least one amino acid with a non-natural amino acid.

The peptide of claim 22, wherein the non-natural amino acid is selected from the group consisting of a β-amino acid (β³ and β²), a homo-amino acid, a proline and pyruvic acid derivative, a 3 -substituted alanine derivative, a glycine derivative, a ring- substituted phenylalanine and tyrosine derivative, a linear core amino acid, a diamino acid and a N-methyl amino acid.

The peptide of any one of claims 15 to 23, wherein the peptide is a mutant or derivative of a portion or the entire basic domain 2 region of TCTP which still retains the capability of inhibiting MDM2.

A nucleic acid encoding a peptide according to any one of claims 15 to 24. A vector comprising a nucleic acid according to claim 25.

Use of a peptide of any one of claims 1 to 12 or 15 to 24 in the manufacture of a medicament for treating cancer.

28. The use of claim 27, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer and breast cancer.

29. The use of claim 27 or 28, further comprising at least one excipient or at least one diluent.

30. A method of treating cancer comprising administration of at least one peptide of any one of claims 1 to 12 or 15 to 24 to a patient in need thereof.

31. The method of claim 30, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer and breast cancer.

32. The method of claim 30 or 31 , further comprising at least one excipient or at least one diluent.

33. A pharmaceutical composition comprising at least one peptide of any one of claims 1 to 12 or 15 to 24.

A method of detecting the presence or absence of TCTP in a sample, wherein the method comprises

a. contacting the sample with a peptide of any one of claims 1 to 12, and b. analyzing the sample resulting from a. to identify the presence or absence of the binding product of TCTP and the peptide of any one of claims 1 to 12.

The method of claim 34, wherein the peptide comprises a label.

The method of claim 35, wherein the label is an optically identifiable label emitting an optically detectable signal in case of the present of TCTP.

A microarray comprising a peptide of any one of claims 1 to 12 for detection of TCTP in a sample.