WO2018100060A1 - Syntenin inhibitors and uses thereof - Google Patents

Abstract

The present invention relates to peptides and peptide analogues with high affinity for the PDZ domains of syntenin. The peptide or peptide analog interacts with syntenin, blocking the native protein-protein interactions between syntenin and its natural Iigands. The invention furthermore relates to the therapeutic use of these peptides and peptide analogues in prevention and/or treatment of neoplastic disorders, as well as to the use for diagnosing a neoplastic disorder.

Description

Syntenin inhibitors and uses thereof Field of invention The present invention relates to peptides and peptide analogs, which bind to the PDZ domains of syntenin and thereby block syntenin-mediated protein-protein interactions. The invention furthermore relates to therapeutic and diagnostic use of said peptides and peptide analogues.

Background of invention

Protein-protein interactions (PPIs) are vital for most biochemical and cellular processes and are often mediated by scaffold and signal transduction complexes. One of the most abundant classes of human facilitators of PPIs is the family of postsynaptic density protein-95 (PSD-95)/Discs-large/ZO-1 (PDZ) domains. Syntenin is an intracellular scaffolding protein primarily involved in regulation of protein trafficking and cell migration by mediating and facilitating PPIs via its two PDZ domains. Central to syntenin^'s cellular role is its ability to bind and interact with numerous intracellular molecules including various protein partners, as well as membrane phospholipids.

Conventional cancer therapy is based on surgery, chemotherapy or radiation therapy. However, these treatment strategies are associated with severe side effects and there is an urgent need for new cancer treatment strategies. One of the promising therapeutic targets are cancer-enabling PPIs.

A common theme for the intracellular activities of syntenin is the direct and indirect relation to cancer cell invasion and the ability to promote metastasis. Syntenin is overexpressed in several cancers, including melanoma, glioma, breast cancer, urothelial cell carcinoma, small cell lung carcinoma, uveal melanoma. Several studies have identified a direct correlation between overexpression of syntenin and overall patient survival rate. Furthermore, an increase of syntenin expression is directly related to the progression and development of melanoma. Hence, as the benign nevi develop to a radial and vertical growth phase primary melanoma and ultimately to an invasive metastatic melanoma, the expression of syntenin is proportionally increased. Inhibition of syntenin expression has significant effects on cancer cells. Multiple studies demonstrate the importance of syntenin in tumor invasion and progression. Hence, through the use of novel drug design approaches, this protein may provide a worthwhile therapeutic target. As many conventional therapies do not address, or even enhance, tumor invasion, an anti-invasive approach would be a worthwhile addition in cancer therapy. Syntenin is thus a potential target for inhibiting some of the most lethal aspects of cancer progression.

Summary of invention

Targeting syntenin as an explorative and novel pharmacological and integrative cancer treatment approach opens possibilities to modulate and inhibit cancer progression and development of metastasis. The present invention comprises compounds that target and block the PDZ binding sites of syntenin and hence further inhibit downstream signaling events with key binding partners such as c-Src, focal adhesion kinase, Akt, p38MAPK and NF-κΒ, known to promote and induce cell invasion and metastasis, Figure 1 .

The current invention is based on a heptameric peptide, which is originally derived from a phage display library. Surprisingly, the heptameric peptide and subsequently the parent peptide, is not identified in any human proteins. The parent peptide sequence has been further optimized by the inventors, and a number of peptides have been generated, which include both proteinogenic and non-proteinogenic amino acids.

Thus, in a main aspect the present invention concerns a peptide or peptide analog (Pi) comprising at least five amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X₃X₄X₅X₆X₇ (SEQ ID NO: 52) wherein :

a) X₃ is selected from the group consisting of L-tryptophan (W) and ι_-β-(3- benzothienyl)-alanine (BTA),

b) X₄ is selected from the group consisting of L-threonine (T), L-alanine (A), L-valine (V), L-serine (S), L-isoleucine (I), L-tyrosine (Y) and L-f- leucine (TLE),

c) X₅ is selected from the group consisting of L-isoleucine (I), L-Meucine

(TLE) and L-cyclohexylglycine (CHG), d) X₆ is selected from the group consisting of L-aspartic acid (D) and L- glutamic acid (E) and

e) X₇ is selected from the group consisting of L-isoleucine (I), L-valine (V), L-Meucine (TLE) and L-cyclohexylglycine (CHG);

with the proviso that Pi is not WTIDI.

In one aspect, the present invention concerns a peptide or peptide analog as described herein for use as a medicament.

In another aspect, the present invention concerns a method for preventing and/or treating a neoplastic disorder, comprising administering a peptide or peptide analog as described herein, or a composition as described, to a subject in need thereof.

The clinical application of these compounds includes improved diagnostic biomarkers for monitoring and detecting cancer progression and further on the development of novel anti-cancer agents to increase the portfolio of available cancer treatments. Thus, in one aspect, the present invention concerns a method for diagnosing a neoplastic disorder, comprising the steps of:

a) providing a biological sample;

b) contacting the sample of a) with a peptide or peptide analog as described herein, wherein the peptide or peptide analog comprises a detectable moiety;

c) quantitating the level of peptide or peptide analog bound to syntenin in b); d) comparing the quantity of peptide or peptide analog bound to syntenin in c), to a control,

wherein a level of peptide or peptide analog bound to syntenin in the biological sample, higher than the level in a control is indicative of the presence of a neoplastic disorder in the individual from which the biological sample is derived.

Description of Drawings

Figure 1 : Schematic illustration of proposed mode of action. Upon stimulation, the scaffolding protein syntenin forms a tetrameric complex comprising the intracellular tail of integrin, c-Src and FAK, which stimulates downstream cell signalling pathways and further on tumor cell invasion and metastatic spread. By blocking the PDZ binding sites in syntenin with a peptide inhibitor, the connection between FAK and c-Src is blocked, thereby regulating the progression of cancer.

Figure 2: Structure of ligand 40 (SEQ ID NO: 40).

Figure 3: Saturation binding affinities (K_d) for fluorescently labelled ligands 3 (SEQ ID NO: 3) and 40 (SEQ ID NO: 40) towards PDZ12 of syntenin. Data is shown as mean ± SEM, n>3. Figure 4: Competitive binding affinities ( ) for ligands 40 (SEQ ID NO: 40), 41 (SEQ ID NO: 41 ) and 45 to 50 (SEQ ID NO: 45 to 50) towards PDZ12 of syntenin. Data is shown as mean ± SEM, n>3.

Figure 5: In vitro plasma stability of ligands 3 (SEQ ID NO: 3), 40 (SEQ ID NO: 40) and 45 to 50 (SEQ ID NO: 45 to 50). Data is shown as mean ± SEM, n=3.

Figure 6: In vitro hepatic clearance of ligands 3 (SEQ ID NO: 3), 40 (SEQ ID NO: 40) and 45 to 50 (SEQ ID NO: 45 to 50). Data is shown as mean ± SEM, n=3. Figure 7: Intraluminal budding of mCherry-syntenin in MCF-7 cells. Cells were treated for 24 h with different concentrations of compound 45 to 50 (SEQ ID NO: 45 to 50) after which the number of filled syntenin endosomes were quantified. Data is presented as mean ± SD, n=2. Figure 8: Pulldown of syntenin from mouse brain lysate using ligand 66 (SEQ ID NO: 66) as analysed by SDS-PAGE and Western blot.

Figure 9: High throughput screening of binding of ligand 40 (SEQ ID NO: 40) to the human PDZome. Ligand 40 shows strongest binding intensity (Bl) to syntenin PDZ1 out of the 255 tested single PDZ domains of the human PDZome. Tandem syntenin PDZ12 and PSD-95 PDZ12 were used as positive and negative control, respectively.

Figure 10: Inhibition of A2058 (A), MCF-7 (B) and HT-29 (C) cancer cell proliferation by treatment with increasing concentrations of ligands 40 (SEQ ID NO: 40) and 46 to 48 (SEQ ID NO: 46 to 48) for 72 h. The non-malignant lung fibroblasts MRC-5 cell line was used as a control (D). Data is expressed as mean ± SD, n=3.

Figure 1 1 : High throughput screening of growth inhibition on 60 cancer cell lines (NCI60 cancer cell library) using ligands 40 or 46 (SEQ ID NO: 40 and SEQ ID NO: 46). Ligand 46 inhibit NCI-H522 (non-small cell lung cancer), KM12 (colon cancer cell line), SK-MEL-5 (melanoma), UO-31 (renal cancer) and T-47D (breast cancer cell line) cell growth. Figure 12: Cell viability of glioblastoma cells treated with ligands 40 or 46 (SEQ ID NO: 40 and SEQ ID NO: 46) at increasing concentrations. A reduced cell viability of glioblastoma cells is observed at increasing concentrations of ligand 46.

Figure 13: Survival rate following treatment with peptides 40 and 46. A significant difference between the two groups was observed, p < 0.05. GBM.

Detailed description of the invention Definitions

The term "Akt", or Protein kinase B, refers to a multifunctional protein kinase, which regulates cell migration, cell proliferation, transcription and metabolism.

The term "albumin binding moiety" refers to a moiety capable of binding to albumin, i.e. having albumin binding affinity.

The term "streptavidin binding moiety" refers to a moiety capable of binding to streptavidin, i.e. having streptavidin binding affinity. One example of a streptavidin binding moiety is biotin.

The term "reactive group", as used herein, refers to a chemical entity, which comprises a reactive functional group, which is capable of reacting with and forming a bond with a second chemical entity. The reactive group may be a nucleophilic group or an electrophilic group. As exemplified in the present invention, the reactive group may be a cysteine, which is capable of reacting with an electrophilic second chemical entity, thereby linking the peptide to said second chemical entity. Proteinogenic "amino acids" are named herein using either its 1 -letter or 3-letter code according to the recommendations from lUPAC, see for example

http://www.chem.qmul.ac.uk/iupac/AminoAcid/. Capital letter abbreviations indicate L- amino acids, whereas lower case letter abbreviations indicate D-amino acids.

The term "amide-bonded", as used herein, refers to chemical entities, exemplified herein as proteinogenic or non-proteinogenic amino acids, which are connected via an amide bond. An amide bond is a chemical bond formed by a reaction between a carboxylic acid and an amine (and concomitant elimination of water). Where the reaction is between two amino acid residues, the bond formed as a result of the reaction is also known as a peptide linkage or a peptide bond.

The term "peptide analog", as used herein, refers to a peptide which is modified or functionalized. Examples of modifications or functionalizations include, but are not limited to, N-methylation of the peptidic backbone, stapling of the peptide, insertion of peptide bond isosteres and attachment of a second moiety, such as for example a CPP or an albumin binding moiety. The term "c-Src", or proto-oncogene tyrosine-protein kinase Src, refers to a multifunctional non-receptor kinase, which controls cell invasion, cell proliferation and angiogenesis.

The term "CPP" (cell penetrating peptide) refers to a peptide characterised by the ability to cross the plasma membrane of mammalian cells, and thereby may give rise to the intracellular delivery of cargo molecules, such as peptides, proteins or

oligonucleotides to which it is linked.

The term "focal adhesion kinase" refers to a multifunctional protein regulator of cell signalling, which controls cell motility, survival and proliferation.

The term "K_d" refers to a dissociation constant and is a measure of the affinity of a molecule for another molecule. The lower the K_d, the higher the affinity of a peptide for its binding site. The term "K," refers to a inhibitory constant of a protein-inhibitor complex. The lower the Ki, the higher the affinity of a peptide for its binding site.

The term "non-proteinogenic amino acids", also referred to as non-coded, non- standard, non-cognate, unnatural or non-natural amino acids, refers to amino acids, which are not encoded by the genetic code. Examples are L-Meucine (TLE), i_- cyclohexylglycine (CHG), L-3-(3-benzothienyl)-alanine (BTA) and L-3-(1 -naphthyl)- alanine (1 NAL). The term "NF-κΒ", or nuclear factor kappa-light-chain-enhancer of activated B cells, refer to a protein complex that controls transcription of DNA, cytokine production and cell survival.

The term "Λ/PEG", is a linker derived from the classical polyethylene glycol (PEG) moiety, but where one or more of the backbone oxygen atoms is replaced with a nitrogen atom.

The term "PDZ" refers to Postsynaptic density protein-95 (PSD-95), Drosophila homologue discs large tumor suppressor (DlgA), Zonula occludens-1 protein (zo-1 ).

The term "retroinverso" refers to peptides that are composed of D-amino acids assembled in the reverse order from that of the parent L-amino acid sequence.

The term "retroinverso-D-Tat sequence" refers to a 9-mer CPP sequence made by reverting the Tat sequence and using D-amino acids (rrrqrrkkr, SEQ ID NO: 60), which facilitates permeability across biological membranes, including the blood-brain barrier, and whose structure increases its stability towards protease enzymes.

The term "syntenin" refers to the protein syntenin/MDA-9, e.g. human syntenin-1 (Uniprot: 000560).

The term "Tat sequence" refers to an 1 1 -mer CPP sequence (YGRKKRRQRRR, SEQ ID NO: 59) derived from the human immunodeficiency virus-type 1 (H IV-1 ) Tat protein, which facilitates permeability across biological membranes, including the blood-brain barrier. The peptide or peptide analog

In a main aspect, the present invention concerns a peptide or peptide analog (Pi) comprising at least five amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X3X4X5 6 7 (SEQ ID NO: 52) wherein:

a) X₃ is selected from the group consisting of L-tryptophan (W) and ι_-β-(3- benzothienyl)-alanine (BTA),

b) X₄ is selected from the group consisting of L-threonine (T), L-alanine (A), L-valine (V), L-serine (S), L-isoleucine (I), L-tyrosine (Y) and L-f- leucine (TLE),

c) X₅ is selected from the group consisting of L-isoleucine (I), L-Meucine (TLE) and L-cyclohexylglycine (CHG),

d) X₆ is selected from the group consisting of L-aspartic acid (D) and L- glutamic acid (E) and

e) X₇ is selected from the group consisting of L-isoleucine (I), L-valine (V), L-Meucine (TLE) and L-cyclohexylglycine (CHG);

with the proviso that Pi is not WTIDI.

In some embodiments, the peptide or peptide analog comprises at least seven amide- bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X₁X₂X₃X₄X₅X₆X₇ (SEQ ID NO: 53), wherein:

a) Xi is selected from the group consisting of L-serine (S), L-alanine (A) and D-serine (s),

b) X₂ is selected from the group consisting of L-histidine (H), L-alanine (A) and D-histidine (h),

c) X₃ is selected from the group consisting of L-tryptophan (W) and ί-β-(3- benzothienyl)-alanine (BTA),

d) X₄ is selected from the group consisting of L-threonine (T), L-alanine (A), L-valine (V), L-serine (S), L-isoleucine (I), L-tyrosine (Y) and L-f- leucine (TLE),

e) X₅ is selected from the group consisting of L-isoleucine (I), L-Meucine (TLE) and L-cyclohexylglycine (CHG),

f) X₆ is selected from the group consisting of L-aspartic acid (D) and L- glutamic acid (E) and g) X₇ is selected from the group consisting of L-isoleucine (I), L-valine (V), L-Meucine (TLE) and L-cyclohexylglycine (CHG).

In some embodiments, the peptide or peptide analog is capable of binding specifically to syntenin, and preferably to human syntenin-1 (Uniprot: 000560).

In some embodiments, the peptide or peptide analog further comprises a conjugated moiety. The conjugated moiety may be selected from the group consisting of a cell penetrating peptide (CPP), an albumin binding moiety, a detectable moiety, a streptavidin binding moiety, a reactive group and a linker (L). In some embodiments, the peptide or peptide analog has the generic structure of Formula I :

Z - L - P, (I) wherein Z is a CPP, an albumin binding moiety, a detectable moiety, a streptavidin binding moiety or a reactive group and L is an optional linker.

In some embodiments, the peptide or peptide analog is conjugated to a CPP. Said CPP may have a polycationic structure, such as comprising at least 4 amino acid residues individually selected from the group consisting of L-lysine (K) and L-arginine (R). In some embodiments, the CPP comprises a retroinverso peptide. In further embodiments, the CPP comprises a Tat peptide, a retroinverso-D-Tat peptide or a polyarginine peptide. Preferably, the CPP is selected from the group consisting of SEQ ID NO: 57 to 65.

In some embodiment, the CPP comprises at least 4 amino acids having cationic or basic side chains that are analogous to L-arginine (R) or L-lysine (K), such as for example 5-hydroxylysine, ornithine, 2-amino-3 (or-4)-guanidinopropionic acid, and homoarginine.

In some embodiments, the CPP has an amphipathic structure and comprises an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids, such as penetratin (SEQ ID NO: 54), retroinverso-penetratin (SEQ ID NO: 55) or amphipathic model peptide (SEQ ID NO: 56). The peptide or peptide analog may also be conjugated to a detectable moiety that is a fluorophore. Said fluorophore is preferably 5,6-carboxyltetramethylrhodamine ( TAMRA) or indodicarbocyanine (Cy5). The peptide or peptide analog may also be conjugated to a detectable moiety that comprises or consists of a radioisotope. Said radioisotope is preferably selected from the group consisting of ¹²⁵l, ^99mTc, ¹¹¹ In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,⁸⁹Zr, ¹²³l, ¹⁸F and ²⁰¹TI.

In some embodiments, peptide or peptide analog is conjugated to a linker (L), which comprises or consists of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, Λ/PEG or any combination thereof.

Said peptide or peptide analog may comprise between 5 and 25 amino acid residues., such as at least 5 amino acid residues, such as at least 6 amino acid residues, such as at least 7 amino acid residues, such as at least 8 amino acid residues, such as at least 9 amino acid residues, such as at least 10 amino acid residues, such as at least 1 1 amino acid residues, such as at least 12 amino acid residues, such as at least 13 amino acid residues, such as at least 14 amino acid residues, such as at least 15 amino acid residues, such as at least 16 amino acid residues, such as at least 17 amino acid residues, such as at least 18 amino acid residues, such as at least 19 amino acid residues, such as at least 20 amino acid residues, such as at least 21 amino acid residues, such as at least 22 amino acid residues, such as at least 23 amino acid residues, such as at least 24 amino acid residues, such as 25 amino acid residues.

In one embodiment, said peptide or peptide analog comprises no more than 25 amino acid residues, such as no more than 24 amino acid residues, such as no more than 23 amino acid residues, such as no more than 22 amino acid residues, such as no more than 21 amino acid residues, such as no more than 20 amino acid residues, such as no more than 19 amino acid residues, such as no more than 18 amino acid residues, such as no more than 17 amino acid residues, such as no more than 16 amino acid residues, such as no more than 15 amino acid residues, such as no more than 14 amino acid residues, such as no more than 13 amino acid residues, such as no more than 12 amino acid residues, such as no more than 1 1 amino acid residues, such as no more than 10 amino acid residues, such as no more than 9 amino acid residues, such as no more than 8 amino acid residues, such as no more than 7 amino acid residues, such as no more than 6 amino acid residues, such as no more than 5 amino acid residues. In one embodiment, the peptide or peptide analog is SHW(TLE)(CHG)DI (SEQ ID NO: 40).

In one embodiment, compound of Formula I is YGRKKRRQRRR-SHW(TLE)(CHG)DI (SEQ ID NO: 45). In another embodiment, compound of Formula I is rRrGrKkRr- SHW(TLE)(CHG)DI (SEQ ID NO: 46). In yet another embodiment, compound of Formula I is cyclo[KrRrGrKkRrE]-SHW(TLE)(CHG)DI (SEQ ID NO: 47). In yet another embodiment, compound of Formula I is KrRrGrKkRrE-SHW(TLE)(CHG)DI (SEQ ID NO: 48). In yet another embodiment, compound of Formula I is RRRRRRRRR- SHW(TLE)(CHG)DI (SEQ ID NO: 49). In a further embodiment, compound of Formula I is KRRRRRRRRRE-SHW(TLE)(CHG)DI (SEQ ID NO: 50). In an even further embodiment, compound of Formula I is cyclo[KRRRRRRRRRE]-SHW(TLE)(CHG)DI (SEQ ID NO: 51 ).

In one embodiment, the peptide or peptide analog inhibits the syntenin/c-Src, and/or syntenin/focal adhesion kinase, and/or syntenin/Akt, and/or syntenin/p38MAPK, and/or syntenin/ NF-κΒ interactions.

In one embodiment, the peptide or peptide analog has a K_d for syntenin below 1 μΜ, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

In another embodiment, the peptide or peptide analog has a K, for syntenin below 1 μΜ, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

The peptide or peptide analog may be part of a composition. In one embodiment, said composition is a pharmaceutical composition. In one aspect, the present invention concerns a peptide or peptide analog as described herein for use as a medicament.

Method for treating neoplasms

In one aspect, the present invention concerns a method for preventing and/or treating a neoplastic disorder, comprising administering a peptide or peptide analog as described herein, or a composition as described, to a subject in need thereof. In some embodiments, the neoplastic disorder is a solid tumor, or is associated with the formation of solid tumors. The solid tumor is selected from the group consisting of prostate cancer, breast cancer, lung cancer, small cell lung carcinoma, non-small cell lung carcinoma, colorectal cancer, skin cancer, melanomas, uveal melanoma, bladder cancer, brain/CNS cancer, glioma, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, urothelial cell carcinoma, liver cancer, lymphomas, ovarian cancer, testicle cancer, pancreatic cancer, thyroid cancer, renal cancer and sarcomas. In a preferred embodiment, the solid tumour is a melanoma. In another preferred embodiment, the solid tumour is a breast cancer. In yet another preferred embodiment, the solid tumour is a glioma.

In some embodiments, the neoplastic disorder is a neoplastic hematologic disorder. Said neoplastic hematologic disorder may be selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

Method for diagnosing a neoplastic disorder

Syntenin is upregulated in numerous cancers and several studies have identified a direct correlation between overexpression of syntenin and overall patient survival rate. Furthermore, an increase of syntenin expression is directly related to the progression and development of melanoma. Thus, in one aspect the present invention concerns a method for diagnosing a neoplastic disorder, comprising the steps of:

a) providing a biological sample; b) contacting the sample of a) with a peptide or peptide analog as described herein, wherein the peptide or peptide analog comprises a detectable moiety;

c) quantitating the level of peptide or peptide analog bound to syntenin in b); d) comparing the quantity of peptide or peptide analog bound to syntenin in c), to a control,

wherein a level of peptide or peptide analog bound to syntenin in the biological sample, higher than the level in a control is indicative of the presence of a neoplastic disorder in the individual from which the biological sample is derived. In one embodiment, the sample is a body fluid and/or a tissue sample. In another embodiment, step c) of the method comprises a fluorescence assay.

Method of isolation In one embodiment, a method for isolating syntenin from whole brain lysates by using the peptide or peptide analog according to the present invention is provided, wherein said peptide or peptide analogue is immobilized on a solid support.

When using cell lysate, full length syntenin is pulled down. Hence, both PDZ1 and PDZ2 are isolated. For differentiation between PDZ1 and PDZ2, single PDZ domains are used. Thus, in one embodiment, the incvention concerns a method for isolating syntenin PDZ1 , but not syntenin PDZ2 by using the peptide or peptide analog according to the present invention is provided, wherein said peptide or peptide analogue comprises biotin at the N-terminus.

Examples

Example 1 : Peptide synthesis

Ligands 1 to 50 (SEQ ID NO: 1 to 50), exemplified by ligand 40 (SEQ ID NO: 40) in Figure 2, were manually synthesized using Fmoc-based solid phase peptide synthesis on 2-chlorotrityl chloride resin. The resin was first swelled in dichloromethane (DCM) for 1 h at room temperature after which 2 eq. (relative to the resin loading) of the first amino acid was dissolved in 8 eq. diisopropylethylamine (DIPEA) in DCM and added to the resin. The reaction was allowed to proceed for 1 h under agitation and repeated. The resin was washed in DCM and capped by treatment of 5 ml DCM/methanol (MeOH)/diisopropylethylamine (17:2:1 ) for 5 min. The capping procedure was repeated twice followed by extensive washing with DCM and dimethylformamide (DMF). The resin was swelled in DMF for 30 min before Fmoc-deprotection was carried out by treating the resin with 20% piperidine in DMF for 2 x 2 min. The resin was washed extensively with DMF before the next Fmoc-protected amino acid was coupled to the resin by reaction with 4 eq. of amino acid dissolved in 4 eq. Λ/,Λ/,Λ/',Λ/'-Tetramethyl-O- (1 H-benzotriazol-1 -yl)uronium hexafluorophosphate (HBTU), 8 eq DIPEA in DMF or in 4 eq. 1 -[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-ib]pyridinium 3-oxid hexafluorophosphate (HATU), 8 eq. DIPEA in DMF. The coupling reaction was allowed to proceed for 30 min - 16 h at room temperature. The Fmoc-deprotection and coupling of amino acids was allowed to proceed until the desired peptide sequence was obtained. Finally, the Fmoc-group was removed from the N-terminus on the last amino acid. For ligands 1 and 2 (SEQ ID NO: 1 and 2), the fluorescent tag 5,6- carboxyltetramethylrhodamine ( TAMRA) (1 .5 eq.), was coupled to the N-terminus by reaction with 1 .5 eq. (benzotriazol-l -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 3 eq. DIPEA in A/-methyl-2-pyrrolidone (NMP) for 16 h at room temperature. For ligands 45 to 50 (SEQ ID NO: 45 to 50), the N-terminus was capped on resin by treatment with 870 μΙ acetic anhydride, 470 μΙ DIPEA, 15 ml DMF for 2 x 5 min followed by washing with DMF. The cyclic ligand 47 (SEQ ID NO: 47) was treated with 20 eq PhSiH₃ and 0.2 eq Pd(PPh₃)₄ in DCM under nitrogen for 2 x 15 min to remove the allyl and alloc protecting groups on Lys1 and Glu1 1 side chains, respectivly. The resin was washed with DCM followed by DMF. The cyclisation reaction between the side chains of Glu and Lys was performed by treatment with 2 eq. HATU and 2 eq. DIPEA in DMF for 16 h at room temperature followed by DMF wash. The resin was washed extensively with DCM and dried. The peptide was cleaved from the solid support by treatment with trifluoroacetic acid (TFA)/triisopropylsilane (TIPS)/water (95:2.5:2.5) for 2 h at room temperature followed by evaporation and precipitation with ice-cold diethyl ether. The crude peptide was collected by centrifugation and purified by RP-HPLC followed by lyophilisation. The final peptide ligands were characterised by LC-MS and UPLC (214 nm).

In conclusion, example 1 demonstrates that the ligands can be synthesized, purified and obtained in pure form. Example 2: Methods for determining affinity to PDZ12 of syntenin The binding affinity to PDZ12 of syntenin was measured with fluorescent polarisation (FP) by first obtaining saturation binding curves for the TAMRA labelled ligands 1 (SEQ ID NO: 1 ) and 2 (SEQ ID NO: 2) by using increasing concentrations of PDZ12 of syntenin in the presence of 100 nM fluorescent labelled ligand. The FP of the sample was measured at excitation/emission wavelength of 530/585 nm and the generated FP values were fitted to a one site binding model using the GraphPad Prism software. The K_d value was determined at 50% of the maximum response, Table 1 and Figure 3. The affinity between the non-fluorescent labelled ligands and PDZ12 syntenin was determined by a competitive FP based inhibition assay, where 100 nM of ligand 1 (SEQ ID NO: 1 ) or 2 (SEQ ID NO: 2) and 2500 nM or 100 nM PDZ12 of syntenin, respectively, was titrated with increasing concentrations of non-fluorescent labelled ligand. The FP values were fitted to a one-site competition with variable slope model in GraphPad Prism. The generated IC₅₀ value in GraphPad Prism was converted to the competition inhibition constant (K,), as previously described (Nikolovska-Coleska et al, Anal Biochem, 2004, 332, 261 -273).

Table 1 . Affinity of fluorescent labelled peptides to PDZ12 of syntenin.

Sequence Compound FP

K_d (μΜ)

7 UWR/4-NNG-SHWTIDI 1 0.84 ± 0.80

(n>3) In conclusion, example 2 describes how to determine the affinity of ligands binding to PDZ12 of syntenin.

Example 3: Determining affinity to PDZ12 of syntenin

The affinity of synthesized ligands towards PDZ12 of syntenin was determined using FP as described in example 2. Ligands 3 to 50 (SEQ ID NO: 3 to 50) were diluted in 25 mM HEPES, 0.15 M NaCI, 1 mM β-mercaptoethanol, pH 7.4 and incubated with 100 nM of ligand 1 (SEQ ID NO: 1 ) or 2 (SEQ ID NO: 2) and 2500 nM or 100 nM PDZ12 of syntenin, respectively, at increasing concentrations at room temperature. The FP values were determined and converted to K.-values based on the K_d values of ligands 1 (SEQ ID NO: 1 ) and/or 2 (SEQ ID NO: 2), see tables 2 to 7. The affinity of parent ligand 3 (SEQ ID NO: 3) to PDZ12 of syntenin was determined to 2 μΜ. A subset of ligands 4 to 39 (SEQ ID NO: 4 to 39) show significantly lower affinities towards PDZ12 of syntenin compared to the parent ligand 3 (SEQ ID NO: 3). Introduction of two mutations (TLE and CHG) at position X₄ and X₅, respectively, generating ligand 40 (SEQ ID NO: 40), resulted in a synergistic increase in affinity. The affinity of ligand 40 (SEQ ID NO: 40) was determined to 40 nM, corresponding to a 50-fold improvement of the affinity compared to the parent peptide 3. As syntenin is an intracellular target, various CPPs were introduced at the N-terminus of ligand 40 (SEQ ID NO: 40), generating ligand 45 to 50 (SEQ ID NO: 45 to 50). The affinities of ligands 45 to 50 (SEQ ID NO: 45 to 50) were determined to be in the range of 270-470 nM, Figure 4 and Table 7.

Table 2. Binding affinities (Kj), presented as mean ± SEM and n=3, of N-terminally truncated peptides.

Sequence Compound K, (μΜ)

SHWTI DI 3 2.1 1 ± 0.32

SHWTI DI- NH₂ 4 > 250

HWTI DI 5 3.93 ± 1 .59

WTI DI 6 5.93 ± 1 .39

TI DI 7 >103.5

Table 3. Binding affinities (Kj), presented as mean ± SEM and n=3, for Ala scan.

Sequence Compound Ki (μΜ)

AHWTI DI 8 1.51 ± 0.29

SAWTIDI 9 4.82 ± 0.93

SHATIDI 10 >103.5

SHWAIDI 11 9.23 ± 2.53

SHWTADI 12 71 .59 ± 18.08

SHWTIAI 13 36.07 ± 5.88

SHWTI DA 14 >103.5

Table 4. Binding affinities (Kj), presented as mean ± SEM and n=3, for D-amino acid scan.

Sequence Compound Ki (μΜ)

sHWTIDI 15 3.53 ± 0.26

ShWTIDI 16 2.30 ± 0.29

SHwTIDI 17 58.24 ± 15.33

SHWtl DI 18 >500

SHWTiDI 19 >500

SHWTIdl 20 >500

SHWTI Di 21 >500

Table 5.

Sequence Compound Ki (μΜ)

SHFTIDI 22 47.06 ± 5.48

SHYTIDI 23 124.29 ± 35.03

SH(1 NAL)TIDI 24 18.53 ± 0.19

SH(BTA)TIDI 25 7.03 ± 0.30 SHWVIDI 26 1.95 ± 0.05

SHWSIDI 27 6.12 ± 0.19

SHW(TLE)IDI 28 0.67 ± 0.01

SHWII DI 29 1.45 ± 0.05

SHWYIDI 30 2.68 ± 0.20

SHWTTDI 31 >500

SHWTVDI 32 15.29 ± 1 .72

SHWT(TLE)DI 33 5.63 ± 0.14

SHWT(CHG)DI 34 0.82 ± 0.019

SHWTIEI 35 5.49 ± 0.52

SHWTI DT 36 280.0 ± 33.20

SHWTI DV 37 4.89 ± 0.33

SHWTI D(TLE) 38 3.64 ± 0.15

SHWTI D(CHG) 39 2.20 ± 0.053

Table 6. Binding affinities (K,), presented as mean ± SEM and n=3, using 2.

Sequence Compound Ki (μΜ)

SHW(TLE)(CHG)DI 40 0.040 ± 0.003

D(CHG)(TLE)HIWS 41 >500

HW(TLE)(CHG)DI 42 0.216 ± 0.015

W(TLE)(CHG)DI 43 0.222 ± 0.01 1

Biotin-PEG2-SHW(TLE)(CHG)DI 44 0.186 ± 0.010

Table 7. In vitro characterisation of lead peptide with cell penetrating peptides.

Sequence Compound Tl/2 Tl/2

(μΜ) plasma clearance

(h) (min)

YGRKKRRQRRR-SHW(TLE)(CHG)DI 45 0.25 ± 0.039 1 .0 >60

rRrGrKkRr-SHW(TLE)(CHG)DI 46 0.30 ± 0.028 >24 >60 c[KrRrGrKkRrE]-SHW(TLE)(CHG)DI 47 0.27 ± 0.031 >24 >60

KrRrGrKkRrE-SHW(TLE)(CHG)DI 48 0.47 ± 0.030 >24 >60

RRRRRRRRR-SHW(TLE)(CHG)DI 49 0.45 ± 0.073 1 .8 >60

KRRRRRRRRRE-SHW(TLE)(CHG)DI 50 0.39 ± 0.030 4.2 >60

Ki values are based on the K_d value of probe 2 and presented as mean ± SEM, n=3.

D-amino acids are denoted with small letters.

c indicates side chain to side chain cyclisation between K and E.

In conclusion, example 3 lists the affinities of the developed peptides. Most importantly, by introducing two specific mutations at position X₄ and X₅ in the parent peptide 3, generating ligand 40 (SEQ ID NO: 40), the affinity for PDZ12 of syntenin significantly increased to the low nM range. By introducing CPPs at the N-terminus of ligand 40 (SEQ ID NO: 40), the affinity decreased around 10-fold.

Example 4: In vitro plasma stability of ligand 3 (SEQ ID: 3), 40 (SEQ ID: 40) and 45 to 50 (SEQ ID: 45 to 50)

The in vitro stability of ligand 3 (SEQ ID NO: 3), 40 (SEQ ID NO: 40) and 45 to 50 (SEQ ID NO: 45 to 50) in human plasma was determined by incubating 200 μΜ ligand in undiluted plasma at 37 °C for 0-60 minutes or for 0-24 h, depending on stability, see table 7. The ligands were extracted from 45 μΙ plasma by pretreatment with 50 μΙ 6 M urea or 26 mg guanidium hydrochloride for 10 min followed by addition of 50 μΙ 20% trichloroacetic acid (TCA) in water or 90 μΙ 10 % TCA in acetone. The samples were finally centrifuged at 13400 rpm for 15 minutes. The supernatant was filtered and analysed by UPLC to determine the amount of ligand remaining, Figure 5 and table 7.

In conclusion, example 4 demonstrates a method of assessing the stability in human plasma in vitro. Ligands comprising D-amino acids have increased stability and half-life compared to only L-amino acid comprising peptides.

Example 5: In vitro hepatic clearance of ligands 3 (SEQ ID: 3), 40 (SEQ ID: 40) and 45 to 50 (SEQ ID: 45 to 50)

The in vitro stability of ligand 3 (SEQ ID NO: 3), 40 (SEQ ID NO: 40) and 45 to 50 (SEQ ID NO: 45 to 50) in mouse microsomes (BALB-C) was determined by incubating 0.5 mg/ml mouse microsomes with 3 mM MgCI₂, 1 mM NADPH and 25 μΜ ligand at 37 °C for 0, 5, 15, 30, 45 and 60 minutes, see table 7. The ligands were extracted from the microsomes by pretreatment with 50 μΙ 6 M urea or 26 mg guanidium hydrochloride for 15 min followed by addition of 100 μΙ acetonitrile or 10% trichloroacetic acid (TCA) in acetone. The samples were finally centrifuged at 13400 rpm for 15 minutes. The supernatant was filtered and analysed by UPLC and the amount of ligand remaining after each time point was determined, Figure 6 and table 7.

In conclusion, example 5 demonstrates a method of assessing the stability and hepatic clearance in mouse liver microsomes in vitro. Ligands comprising a CPP tag and/or D- amino acids have increased stability and half-life compared to peptides comprised exclusively of L-amino acids and without a CPP tag.

Example 6: Inhibition of intracellular budding in cancer cells by treatment of ligands 45-50 (SEQ ID NO: 45 to 50)

MCF-7 cells were co-transfected with mCherry-syntenin and cerulean-Rab5Q79L. Cells were treated for 24 h with increasing concentrations of ligands 45 to 50 (SEQ ID NO: 45 to 50) or with vehicle (0.01 % DMSO in sterilized water). The number of cerulean- Rab5Q79L endosomes filled with mCherry-syntenin was determined by confocal microscopy, Figure 7. In conclusion, example 6 demonstrates a method of assessing the inhibition of syntenin filled endosomes by treatment of ligands 45 to 50 (SEQ ID NO: 45 to 50). Ligands 46 to 47 and 49 to 50 (SEQ ID NO: 45 to 47 and 49 to 50), show a dose-dependent inhibition of mCherry-syntenin filled endosomes and hence are drug leads for targeting the intracellular protein syntenin.

Example 7: Pulldown of syntenin

After cervical dislocation, whole brains of C57BL/6 mice, were removed from the scull and rapidly homogenized in 1 ml_ lysate buffer (20 mM Hepes, 100 mM KCH₃COOH, 40 mM KCI, 5 mM EGTA, 5 mM MgCI₂, 5 mM DTT, 1 mM PMSF, 1 % Triton X, protease inhibitor Roche complete, pH 7.2) per 200 mg tissue using a pistol homogenizer (8 strokes at 900 rpm). The homogenate was centrifuged at 10.000 x g for 15 min. Subsequently, the supernatant was flash-frozen in liquid nitrogen and stored at -80 'Ό. Frozen lysates were thawed and centrifuged at 1000 x g. The total protein concentration of the supernatant was determined using a BCA Kit. Supernatants and pellets were frozen at -80 'Ό. Before further analysis, the samples were centrifuged at 10.000 x g after thawing.

Peptides 66 (SEQ ID NO: 66; peptide 40 having an N-terminal cysteine reactive group for immobilization on resin) and 67 (SEQ ID NO: 67; peptide 41 having an N-terminal cysteine reactive group for immobilization on resin) were attached to magnetic Dynabeads M-270 epoxy resin beads by first dissolving the peptide in 10 % DMSO, 2 M ammonium sulfate in PBS, 10 mM TCEP, pH 7.6 and incubated at 50 °C for 15 min. Next, 165 μΙ resin slurry was transferred into eppendorf tubes, the storage solvent was removed and the resin was washed with 4 x 1 ml PBS. Finally, the solution of peptide 66 (SEQ ID NO: 66) or 67 (SEQ ID NO: 67) was transferred to the resin and incubated at 37 ^<Ό for 48 h. Incorporation efficiency was analysed by LC-MS of the peptide solutions before and after the incubation. The peptide solution was removed and the resin was washed with 3 x 1 ml PBS and 1 x 1 ml PBST (0.5% Triton X-100). The beads were incubated with 450 μΙ of diluted brain lysate (final protein concentration was 6.3 μg/μL; diluted with PBS) and incubated at 37 °C for 15 min (1000 rpm). The flow- through was removed and the resin washed with 2 x 1 ml PBS. The beads were transferred into new tubes and washed again with 1 ml PBS. Proteins were eluted from the resin using 10 μΙ 4xNuPage sample buffer containing 10 mM TCEP, followed by centrifugation for 15 s, vortex for 10 s and separation of resin from supernatant using a magnetic rack for 2 min. The eluates were collected and the elution procedure was repeated twice. The eluates were pooled and analysed by SDS-PAGE and Western Blot, Figure 8. In conclusion, example 7 demonstrates a method for isolating syntenin from whole brain lysates by using ligand 66 (SEQ ID NO: 66). Ligand 66 binds syntenin with high affinity in whole brain lysate of C57BL/6 mice. Peptide 66, but not peptide 67, can be used to isolate syntenin from highly complex mixtures, such as brain lysate. Example 8: High throughput screening of the human PDZome using peptide 44 (SEQ ID NO: 44)

To determine the PDZ binding profile of peptide 40 (SEQ ID NO: 40), an N-terminally biotinylated peptide, peptide 44 (SEQ ID NO: 44), was designed and synthesised. Peptide 44 was used to screen the human PDZome, as previously described (Vincentelli R, et al. Nat. Methods, 2015, 12(8):787-793). Specifically, automated holdup assays were performed on a Tecan Evo200 robot with a 96-tip pipetting head and an eight-needle pipetting arm using MZHVNOW 384 well plates (Millipore).

1 .5 ml_ of streptavidin sepharose high-performance beads (GE Healthcare) were equilibrated in 45 ml_ buffer A (50 mM Tris, pH 8.0, 300 mM NaCI, 10 mM imidazole, 5 mM DTT). The beads were re-suspended by pipetting vigorously up-and-down before being transferred by the 96-needle robotic pipetting arm into the MZHVNOW filter plate placed on the T-Vac vacuum system (Tecan). 75 μΙ_ of suspension (representing 2.5 μΙ_ of beads/well) were transferred into the wells of the filter plate. The liquid phase was discarded by vacuum filtration. Beads were equilibrated by adding 30 volumes of buffer A per volume of beads, followed by immediate removal of the buffer by vacuum filtration. Eight volumes per bead volume of a 42 μΜ stock solution of biotinylated peptide were transferred into 288 wells of a filter plate. Eight volumes per bead volume of a 42 μΜ stock solution of biotin were transferred into the remaining wells of the filter plate serving as negative control. The filter plate containing the biotinylated peptide- resin and biotin-resin mixtures was transferred onto a multiwell plate shaker and incubated under vigorous agitation at 1 ,200 r.p.m. for 15 min. After saturation of the beads with biotinylated peptide or biotin, the liquid phase was removed by vacuum filtration. Remaining free streptavidin sites on the beads were blocked by incubating a 1 mM biotin solution in every well under agitation at 1 ,200 r.p.m. for 15 min. Liquid phases were extracted by vacuum filtration.

In this set up, each of the 4 ^χ 96 His-MBP-PDZ domains were transferred into three wells containing beads saturated with biotinylated peptides and one well containing beads saturated with biotin (negative control). The PDZ bank was made up of three plates (Plate A, B and C) of soluble protein extract adjusted to 4 μΜ with buffer A. Plate A and B contained 90 PDZ domains and six control proteins (PDZ1 -2 of syntenin and PDZ1 -2 of PSD-95 at 1 , 4 and 8 μΜ, respectively) and Plate C contained 86 PDZ domains (a total of 266 PDZ) and 10 empty wells used as controls. Analytes (His-MBP- PDZ) used in this study were cleared soluble E. coli overexpression extracts adjusted to 4 μΜ with buffer A. The bacterial overexpression extracts all contained 4 μΜ lysozyme provided by the lysis buffer for internal calibration of the binding. Two volumes per resin volume of analytes were transferred into the filter plate. The binding of peptide 44 was assessed in triplicates against the whole PDZome. At the end of the holdup assay, the binding intensities (Bl) were calculated by analysing the three holdup plates on a calliper GX II. The results were analyzed as previously described (Vincentelli R, et al. Nat. Methods, 2015, 12(8)787-793), Figure 9. In conclusion, example 8 demonstrates a method for isolating syntenin PDZ1 , but not syntenin PDZ2, by using ligand 44 (SEQ ID NO: 44). Peptide 44 has the strongest binding intensity (Bl) to syntenin PDZ1 out of the 255 tested single PDZ domains of the human PDZome. Example 9: Inhibition of cancer proliferation by treatment of ligands 40 (SEQ ID NO: 40) and 46-48 (SEQ ID NO: 46 to 48)

The effect of ligands 40 (SEQ ID NO: 40) and 46 to 48 (SEQ ID NO: 46 to 48) were tested on three human cancer cell lines, namely A2058 (melanoma), MCF-7 (breast carcinoma), HT29 (colon carcinoma) and as a control, the non-malignant lung fibroblasts MRC-5 cell line. The cancer cells were treated with increasing concentrations of ligands after which the cell viability was determined after 72 h by a colorimetric assay, as previously described in Hanssen et al Anticancer Res. 2102, 32, 4287-4297. Ligands 46 to 48 (SEQ ID NO: 46 to 48) showed a dose-dependent inhibition of A2058 cells in the low μΜ range, whereas the control ligand 40 (SEQ ID NO: 40) without a CPP showed no inhibitory effect. In addition, ligand 46 (SEQ ID NO: 46) showed an inhibitory effect on MCF-7 cells at 30 μΜ, Figure 10.

In conclusion, example 9 demonstrates a method of assessing the inhibition of cancer cell proliferation by treatment of ligands 46 to 48 (SEQ ID NO: 46 to 48). Ligands 46 to 48 (SEQ ID NO: 46 to 48), but not ligand 40 (SEQ ID NO: 40), show a dose-dependent inhibition of melanoma A2058 cell proliferation and hence are drug leads for treatment of cancer by targeting the intracellular protein syntenin.

Example 10: High throughput screening of the NCI60 cancer cell line library using peptide 46 (SEQ ID NO: 46)

The human tumor cell lines of the NCI60 cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96-well microtiter plates in 100 μΙ_ medium at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5 % C0₂, 95 % air and 100 % relative humidity for 24 h prior to addition of peptide 46 (SEQ ID NO: 46). After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of peptide addition (Tz). Peptide 46 (SEQ ID NO: 46) was dissolved in DMSO at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of peptide addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration (10 μΜ) with complete medium containing 50 μg/mL gentamicin. Aliquots of 100 μΙ_ of drug were added to the appropriate microtiter wells containing 100 μΙ_ of cell culture. Following addition of peptide 46 (SEQ ID NO: 46), the plates were incubated for an additional 48 h at 37 °C, 5 % C0₂, 95 % air, and 100 % relative humidity.

For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μΙ_ of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4^<Ό. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) staining solution (100 μΙ_) at 0.4 % (w/v) in 1 % acetic acid was added to each well, and the plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1 % acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was recorded on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μΙ of 80 % TCA (final concentration, 16 % TCA), Figure 1 1 .

In conclusion, example 10 demonstrates that peptide 46 (SEQ ID NO: 46; peptide 40 with an N-terminally linked CPP), but not ligand 40 (SEQ ID NO: 40), inhibits NCI-H522 (non-small cell lung cancer), KM12 (colon cancer cell line), SK-MEL-5 (melanoma), UO-31 (renal cancer) and T-47D (breast cancer cell line) cell growth.

Example 11 : Inhibition of glioblastoma (GBM) cell viability by treatment with peptide 46 (SEQ ID NO: 46)

GBM001 -002 cells were maintained through subcutaneous xenografting in the flanks of BALB/c (nu/nu) mice. Tumors were dissected out and dissociated using papain dissociation system (Worthington Biochemical). Acutely (max culture time 24 h post dissection from mice) dissociated cells were cultured in Neurobasal A media supplemented with B27 supplement minus vitamin A (Invitrogen), epidermal growth factor and basic fibroblast growth factor (10 ng/mL, Invitrogen). Cells were cultured at 37 °C in an atmosphere of 5% C0₂. Single cells were plated into a 96-well plate at 3000 cells/well in triplicates. Next day, peptide 40 (SEQ ID NO: 40) or 46 (SEQ ID NO: 46) were added and cell viability was measured after 72 hours using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Results were calculated as relative fold change in ATP with each group internally normalized to the respective vehicle control. The experiments were performed three times in triplicate and the data is presented as mean ± SD, Figure 12.

In conclusion, example 1 1 demonstrates a method of assessing the inhibiton of glioblastoma cell viability by treatment with ligand 46 (SEQ ID NO: 46; peptide 40 with an N-terminally linked CPP). Ligand 46 (SEQ ID NO: 46), but not ligand 40 (SEQ ID NO: 40), show a dose-dependent inhibition of glioblastoma cell viability and hence is a drug lead for treatment of glioblastoma. Example 12: In vivo study of treatment of glioblastoma using peptide 40 or 46.

Patient derived glioblastoma multiforme (GBM) cells, T10, were preincubated with 50 μΜ peptide 40 (SEQ ID NO: 40) or peptide 46 (SEQ ID NO: 46) for 24 hrs to allow for cellular uptake of the peptides. Subsequntly, 10,000 viable cells (counting was performed using Countess automated cell counter, Life Technologies; dead cells were excluded prior to counting using the trypan blue method) were stereotactically implanted into the right frontal lobes of NMRInu-F mice (female, 8 weeks). Mice were monitored daily for neurological impairment and weight loss, at which point they were sacrificed. Figure 13 show the survival rate following treatment with peptide 40 and peptide 46 respectively. A significant difference between the two groups was observed, p < 0.05. GBM animal study described was approved by the Danish Regulations for Animal Welfare (Protocol Number 2012-15-2934-00636).

In conclusion, example 12 demonstrates that peptide 46 (SEQ ID NO: 46,

corresponding to peptide 40 having a CPP attached in the N-terminal end) provides better treatment of glioblastoma than peptide 40 comprising no CPP.

Example 13: Diagnostic method

A correlation between the expression of syntenin and the advancing tumor grade of higher invasive melanoma, glioma, urothelial cell carcinoma and breast cancer has been determined previously (Kegelman et al Expert Opin. Ther. Targets, 2015, 19(1 ):97-1 12). Accordingly, a ligand capable of binding to syntenin can be used for detection of tumor progression and/or diagnosis of proliferative disorders e.g. cancer. Ligand 1 , 2 or 40 (SEQ ID NO: 1 , 2 or 40) labelled with ²⁵l-indodicarbocyanine, indodicarbocyanine, ²⁵l-indotricarbocyanine or indotricarbocyanine are used to monitor syntenin expression and tumor progression, as described in Becker et al Nature Biotech., 2001 , 19(4):327-331 . The ligands of the invention are injected intravenously in tumor bearing mice. The fluorescence and radioactivity signals are monitored over time. The quantification and biodistribution parameters are determined. Cryo-preserved tumor tissue is used for histopathology analysis of tumor progression, by staining the cancer sections with labelled ligand 1 or 2 (SEQ ID NO: 1 or 2). First, the sections are washed in PBS, followed by incubation with labelled ligand 1 or 2 (SEQ ID NO: 1 or 2) e.g. in PBS overnight at 4°C and additional washing with PBS is conducted. The slides are analysed using fluorescent microscopy. „

o

The results from this study will demonstrate if a certain ligand is useful for detecting tumors and/or for the diagnosis of cancer progression.

Example 14: List of compounds

38 SHWTI D(TLE)

39 SHWTI D(CHG)

40 SHW(TLE)(CHG)DI LHK040

41 D(CHG)(TLE)HIWS

42 HW(TLE)(CHG)DI

43 W(TLE)(CHG)DI

44 Biotin-PEG2-SHW(TLE)(CHG)DI

45 YGRKKRRQRRR- SEQ I D NO: 40 + 59

SHW(TLE)(CHG)DI

46 rRrGrKkRr-SHW(TLE)(CHG)DI SEQ I D NO: 40 + 61

47 cyclo[KrRrGrKkRrE]- SEQ I D NO: 40 + 62

SHW(TLE)(CHG)DI

48 KrRrGrKkRrE-SHW(TLE)(CHG)DI SEQ I D NO: 40 + 63

49 RRRRRRRRR-SHW(TLE)(CHG)DI SEQ I D NO: 40 + 57

50 KRRRRRRRRRE- SEQ I D NO: 40 + 64

SHW(TLE)(CHG)DI

51 cyclo[KRRRRRRRRRE]- SEQ I D NO: 40 + 65

SHW(TLE)(CHG)DI

52 X3X4X5X6X7 X₃ is selected from the group consisting of

W and BTA; X₄ is selected from the group consisting of T, A, V, S, I, Y and TLE; X₅ is selected from the group consisting of I, TLE and CHG; X₆ is selected from the group consisting of D and E; and X₇ is selected from the group consisting of I, V, TLE and CHG.

53 Xi X2X3X4X5X6X7 Xi is selected from the group consisting of

S, A and s; X₂ is selected from the group consisting of H, A and h; X₃ is selected from the group consisting of W and BTA; X₄ is selected from the group consisting of T, A, V, S, I, Y and TLE; X₅ is selected from the group consisting of I, TLE and CHG; X₆ is selected from the group consisting of D and E; and X₇ is selected from the group consisting of I, V, TLE and CHG.

54 RQIKIWFQNRRMKWKK Penetratin

55 kkwkmrrnqfwikiqr retroinverso-penetratin

56 KLALKLALKLAKAALKA amphipathic model peptide

57 RRRRRRRRR

58 RGGRLSYSRRRFSTSTGRA SynB1

59 YGRKKRRQRRR Tat peptide

60 rrrqrrkkrgy Retroinverso-D-Tat peptide

61 rRrGrKkRr

62 cyclo[KrRrGrKkRrE] 63 KrRrGrKkRrE

64 KRRRRRRRRRE

65 Cyclo[KRRRRRRRRRE]

66 CSHW(TLE)(CHG)DI Cys-SEQ I D NO:40

67 CD(CHG)(TLE)HIWS Cys-SEQ I D NO:41

References

Kegelman et al. (2015) Targeting tumor invasion: the roles of MDA-9/Syntenin. Expert Opinion Therapeutic Targets 19(1 ):97-1 12.

Becker et al. (2001 ) Receptor-targeted optical imaging of tumors woth near- infrared fluorescent ligands. Nature Biotechnology 19(4):327-331 .

Vincentelli R, et al. (2015) Quantifying domain-ligand affinities and specificities by high-throughput holdup assay Nat. Methods 12(8)787-793.

Claims

Claims

A peptide or peptide analog (Pi) comprising at least five amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence

X3X₄X5X6X7 (SEQ ID NO: 52) wherein:

a. X₃ is selected from the group consisting of L-tryptophan (W) and ί-β-(3- benzothienyl)-alanine (BTA),

b. X₄ is selected from the group consisting of L-threonine (T), L-alanine (A), L- valine (V), L-serine (S), L-isoleucine (I), L-tyrosine (Y) and L-Meucine (TLE), c. X₅ is selected from the group consisting of L-isoleucine (I), L-Meucine (TLE) and L-cyclohexylglycine (CHG),

d. X₆ is selected from the group consisting of L-aspartic acid (D) and L- glutamic acid (E) and

e. X₇ is selected from the group consisting of L-isoleucine (I), L-valine (V), i-t- leucine (TLE) and L-cyclohexylglycine (CHG);

with the proviso that Pi is not WTIDI.

The peptide or peptide analog according to claim 1 , wherein Pi comprises at least seven amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X ₁X₂X3X₄X5X6X7 (SEQ ID NO: 53) wherein:

a. Xi is selected from the group consisting of L-serine (S), L-alanine (A) and D- serine (s),

b. X₂ is selected from the group consisting of L-histidine (H), L-alanine (A) and D-histidine (h),

c. X₃ is selected from the group consisting of L-tryptophan (W) and ί-β-(3- benzothienyl)-alanine (BTA),

d. X₄ is selected from the group consisting of L-threonine (T), L-alanine (A), L- valine (V), L-serine (S), L-isoleucine (I), L-tyrosine (Y) and L-Meucine (TLE), e. X₅ is selected from the group consisting of L-isoleucine (I), L-Meucine (TLE) and L-cyclohexylglycine (CHG),

f. X₆ is selected from the group consisting of L-aspartic acid (D) and L-glutamic acid (E) and

g. X₇ is selected from the group consisting of L-isoleucine (I), L-valine (V), L-t- leucine (TLE) and L-cyclohexylglycine (CHG). The peptide or peptide analog according to any one of the preceding claims wherein Pi is capable of binding specifically to syntenin.

The peptide or peptide analog according to any one of the preceding claims wherein Pi is capable of binding specifically to human syntenin-1 (Uniprot: 000560).

The peptide or peptide analog according to any one of the preceding claims, further comprising one or more conjugated moieties.

The peptide or peptide analog according to any one of the preceding claims, wherein the conjugated moiety is selected from the group consisting of a Cell Penetrating Peptide (CPP), an albumin binding moiety, a detectable moiety and/or a linker (L).

The peptide or peptide analog according to any one of the preceding claims, wherein said peptide or peptide analog has the generic structure of Formula I

Z - L - P! wherein Z is a CPP, an albumin binding moiety, a detectable moiety, a streptavidin binding moiety or a reactive group and L is an optional linker.

The peptide or peptide analog according to any one of the preceding claims, wherein the conjugated moiety is a CPP.

The peptide or peptide analog according to any one of the preceding claims, wherein the CPP has a polycationic structure.

10. The peptide or peptide analog according to any one of the preceding claims, wherein the CPP comprises at least 4 amino acid residues individually selected from the group consisting of lysine (K) and arginine (R). 1 1 . The peptide or peptide analog according to any one of the preceding claims, wherein the CPP comprises a retroinverso peptide.

12. The peptide or peptide analog according to any one of the preceding claims, wherein the CPP comprises a Tat peptide, a Retroinverso-D-Tat peptide or a polyarginine peptide.

13. The peptide or peptide analog according to any one of the preceding claims, wherein the CPP is selected from the group consisting of SEQ ID NO: 57 to 65.

14. The peptide or peptide analog according to any one of the preceding claims, wherein the CPP comprises at least 4 amino acids having cationic or basic side chains that are analogous to arginine (R) or lysine (K), such as for example 5- hydroxylysine, ornithine, 2-amino-3 (or-4)-guanidinopropionic acid, and homoarginine.

15. The peptide or peptide analog according to any one of the preceding claims, wherein the CPP has an amphipathic structure and comprises an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.

16. The peptide or peptide analog according to claim 15, wherein the CPP is selected from the group consisting of penetratin (SEQ ID NO: 54), retroinverso-penetratin (SEQ ID NO: 55) and amphipathic model peptide (SEQ ID NO: 56).

17. The peptide or peptide analog according to any one of the preceding claims, wherein the detectable moiety is a fluorophore. 18. The peptide or peptide analog according to any one of the preceding claims, wherein the detectable moiety is 5,6-carboxyltetramethylrhodamine (TAMRA) or indodicarbocyanine (Cy5).

19. The peptide or peptide analog according to any one of the preceding claims, wherein the detectable moiety comprises or consists of a radioisotope.

20. The peptide or peptide analog according to any one of the preceding claims, wherein the radioisotope is selected from the group consisting of ²⁵l, ^99mTc, In, 6⁷Ga, ⁶⁸Ga, ⁷²As,⁸⁹Zr, ²³l, ⁸F and ²⁰ TI.

21 . The peptide or peptide analog according to any one of the preceding claims, wherein the linker (L) comprises or consists of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, Λ/PEG or any combination thereof.

The peptide or peptide analog according to any one of the preceding claims, wherein P^ comprises between 5 and 25 amino acid residues.

The peptide or peptide analog according to any one of the preceding claims, wherein P^ comprises at least 5 amino acid residues, such as at least 6 amino acid residues, such as at least 7 amino acid residues, such as at least 8 amino acid residues, such as at least 9 amino acid residues, such as at least 1 0 amino acid residues, such as at least 1 1 amino acid residues, such as at least 12 amino acid residues, such as at least 13 amino acid residues, such as at least 14 amino acid residues, such as at least 15 amino acid residues, such as at least 16 amino acid residues, such as at least 17 amino acid residues, such as at least 18 amino acid residues, such as at least 19 amino acid residues, such as at least 20 amino acid residues, such as at least 21 amino acid residues, such as at least 22 amino acid residues, such as at least 23 amino acid residues, such as at least 24 amino acid residues, such as 25 i amino acid residues.

The peptide or peptide analog according to any one of the preceding claims, wherein Pi comprises no more than 25 amino acid residues, such as no more than 24 amino acid residues, such as no more than 23 amino acid residues, such as no more than 22 amino acid residues, such as no more than 21 amino acid residues, such as no more than 20 amino acid residues, such as no more than 19 amino acid residues, such as no more than 1 8 amino acid residues, such as no more than 17 amino acid residues, such as no more than 1 6 amino acid residues, such as no more than 1 5 amino acid residues, such as no more than 14 amino acid residues, such as no more than 13 amino acid residues, such as no more than 12 amino acid residues, such as no more than 1 1 amino acid residues, such as no more than 10 amino acid residues, such as no more than 9 amino acid residues, such as no more than 8 amino acid residues, such as no more than 7 amino acid residues, such as no more than 6 amino acid residues, such as no more than 5 amino acid residues.

The peptide or peptide analog according to any one of the preceding claims, wherein P, is SHW(TLE)(CHG)DI (SEQ ID NO: 40).

26. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is YGRKKRRQRRR-SHW(TLE)(CHG)DI (SEQ ID NO: 45).

27. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is rRrGrKkRr-SHW(TLE)(CHG)DI (SEQ ID NO: 46).

28. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is cyclo[KrRrGrKkRrE]-SHW(TLE)(CHG)DI (SEQ ID NO: 47). 29. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is KrRrGrKkRrE-SHW(TLE)(CHG)DI (SEQ ID NO: 48).

30. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is RRRRRRRRR-SHW(TLE)(CHG)DI (SEQ ID NO: 49).

31 . The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is KRRRRRRRRRE-SHW(TLE)(CHG)DI (SEQ ID NO: 50).

32. The peptide or peptide analog according to any one of the preceding claims, wherein Formula I is cyclo[KRRRRRRRRRE]- SHW(TLE)(CHG)DI (SEQ ID NO:

51 ).

33. The peptide or peptide analog according to any one of the preceding claims, wherein said peptide or peptide analog inhibits the syntenin/c-Src, and/or syntenin/focal adhesion kinase, and/or syntenin/Akt, and/or syntenin/p38MAPK, and/or syntenin/ NF-κΒ interactions.

34. The peptide or peptide analog according to any of the preceding claims, wherein said peptide or peptide analog has a K_d for syntenin below 1 μΜ, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

35. The peptide or peptide analog according to any of the preceding claims, wherein said peptide or peptide analog has a K, for syntenin below 1 μΜ, such as below

900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

36. The peptide or peptide analog according to any of the preceding claims, wherein the peptide has been immobilized on a solid support.

37. A microchip comprising an array of peptides or peptide analogues according to any one of the preceding claims.

38. A composition comprising the peptide or peptide analog according to any of

claims 1 to 35.

39. The composition according to claim 0, wherein the composition is a

pharmaceutical composition.

40. A peptide or peptide analog according to any of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use as a medicament.

41 . A peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use in the prevention and/or treatment of a neoplastic disorder.

42. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 41 , wherein the neoplastic disorder is a solid tumor, or is associated with the formation of solid tumours.

43. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 42 wherein the solid tumor is selected from the group consisting of prostate cancer, breast cancer, lung cancer, small cell lung carcinoma, non-small cell lung carcinoma, colorectal cancer, skin cancer, melanomas, uveal melanoma, bladder cancer, brain/CNS cancer, glioma, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, urothelial cell carcinoma, liver cancer, lymphomas, ovarian cancer, testicle cancer, pancreatic cancer, thyroid cancer, renal cancer and sarcomas.

44. The peptide or peptide analog according to any one of claims 1 to 35, or the composition according to any one of claims 0 to 39, for use according to claim 42, wherein the solid tumour is a melanoma. 45. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 42, wherein the solid tumour is a breast cancer.

46. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 42, wherein the solid tumour is a glioma.

47. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 41 wherein the neoplastic disorder is a neoplastic hematologic disorder.

48. The peptide or peptide analog according to any one of claims 1 to 35, or the

composition according to any one of claims 0 to 39, for use according to claim 47 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

49. Use of the peptide or peptide analog according to any one of claims 1 to 35, or the composition according to any one of claims 0 to 39, for the prevention and/or treatment of a neoplastic disorder as defined in any one of claims 42 to 48.

50. A method for prevention and/or treatment of a neoplastic disorder as defined in any one of claims 42 to 48, comprising administering a peptide or peptide analog according to any one of claims 1 to 35, or a composition according to any one of claims 0 to 39, to a subject in need thereof.

51 . A method for diagnosing a neoplastic disorder, the method comprising:

a. providing a biological sample;

b. contacting the sample of a) with a peptide or peptide analog according to any one of claims 1 to 34, wherein the peptide or peptide analog comprises a detectable moiety; c. quantifying the level of peptide or peptide analog bound to syntenin in b); d. comparing the quantity of peptide or peptide analog bound to syntenin in c), to a control,

wherein a level of peptide or peptide analog bound to syntenin in the biological sample, higher than the level in a control is indicative of the presence of a neoplastic disorder in the individual from which the biological sample is derived.

52. The method of claim 51 , wherein the sample is a body fluid and/or a tissue

sample.

53. The method of any one of claims 51 and 52, wherein step c) comprises a

fluorescence assay.

54. The method of any one of claims 51 to 53, wherein the neoplastic disorder is as defined in any one of claims 42 to 48.

A method for isolating syntenin, the method comprising:

a) providing whole brain lysate,

b) contacting the the whole brain lysate of a) with a peptide or peptide analog according to any one of claims 1 to 36, or the microchip according to claim 37, thus obtaining a peptide:syntenin complex,

c) purifying the syntenin protein bound to the peptide, thus obtaining pure syntenin.

A method for isolating syntenin PDZ1 , essentially free from syntenin PDZ2, the method comprising:

a) contacting a composition comprising syntenin PDZ1 , with an N-terminally biotinylated peptide or peptide analog, wherein the peptiode or peptide analog is as defined in any one of claims 1 to 36, or wherein the peptide has been immobilized on a microchip as defined in claim 37;

b) harvesting the biotin-peptide-PDZ1 cmplex.