Mirror-image technology for the identification of highly stable and selective peptides

Abstract

The present invention relates to methods of identifying cyclic peptide binders comprising D-amino acids to protein targets of interest. The invention further relates to cyclic peptide inhibitors of matrix metalloproteinase 7 (matrilysin), pharmaceutical formulations, and methods of use thereof.

Description

[0001] MIRROR-IMAGE TECHNOLOGY FOR THE IDENTIFICATION OF HIGHLY STABLE AND SELECTIVE PEPTIDES

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the fields of amino acid mutagenesis and mRNA display libraries, and relates to methods of identifying D-amino acid containing cyclic peptides that bind to target polypeptides of interest. The present invention further relates to novel cyclic peptide inhibitors of matrix metalloproteinase 7.

[0004] BACKGROUND OF THE INVENTION

[0005] Random nonstandard Peptides Integrated Discovery (RaPID) system is one of the most powerful methods for the selection of de novo macrocyclic peptide binders for proteins of interest by using a combination of flexible in vitro translation system (FIT) and mRNA display technology (Yamagishi, Y. et al., Chem. & Biol. 2011, 18, 1562-1570; Roberts and Szostak, Proc. Natl. Acad. Sci. USA 1997, 94, 12297-12302; Passioura and Suga, Chem. Commun. 2017, 53, 1931-1940; Passioura, T. et al., Ann. Revi. Biochem. 2014, 83, 727-752). FIT consists of an artificial tRNA aminoacylating ribozyme called flexizyme (Goto, Y. et al., Nature Prot. 2011, 6, 779-790), which is capable of charging tRNAs with various non-canonical amino acids, including P-amino acids (Katoh, T. et al., Nature Chem. 2020, 12, 1081-1088) and D-amino acids (Goto, Y. et al., Nature Prot. 2011, 6, 779-790; Katoh, T. et al., Nature Chem. 2020, 12, 1081-1088; Murakami, H. et al., Nature Methods 2006, 3, 357-359; Fujino, T. et al., J. Am. Chem. Soc. 2013, 135, 1830-1837). P-amino acids have demonstrated the ability to modulate peptide conformations, and P-amino acid-containing peptides have shown better serum stability compared to those consisting of only a-amino acids (Cabrele, C. et al., J. Med. Chem. 2014, 57, 9718-9739; Gopi, H.N. et al., FEBS letters 2003, 535, 175-178; Seebach, D. et al., Helv. Chim. Acta 2001, 84, 3503-3510). Moreover, peptides that comprise D-amino acids are highly resistant against protease digestion, thus having longer half-life and lower immunogenicity compared to their L-counterparts (Werle and Bernkop-Schnurch, Amino Acids 2006, 30, 351- 367). The high diversity of the RaPID library, exceeding 1012members of macrocyclic peptides, led to the discovery of potent molecules that bind to different protein targets through affinity selection (Stefan, E. et al., Elife 2021, 10, e67732; Bashiruddin, N. K. et al. Bioconj. Chem. 2018, 29, 1847-1851; Goto and Suga, Acc. Chem. Res. 2021, 54, 3604-3617; Liu, W. et al., J. Am. Chem. Soc. 2021, 143, 18481-18489; Ford, D.J. et al., J. Med. Chem. 2021, 64, 7853-7876). However, the ribosomal incorporation of consecutive D-amino acids was not possible due to low incorporation efficiencies that are mainly attributed to slow accommodation of D- aminoacyl-tRNAs onto the ribosome and slow or inhibited peptidyl transfer between D-amino acids (Imanishi, S. et al., J. Am. Chem. Soc. 2021, 143, 5680-5684). Suga and coworkers were able to construct a hybrid L / D RaPID library with a maximum of five D-amino acids incorporated into one macrocyclic peptide, albeit not consecutively (ibid.~). Thus, identifying peptide binders to a target of interest composed of mainly and / or consecutively-linked D-amino acids in a high-throughput manner remains a serious challenge.

[0006] Matrix metalloproteinases (MMPs), a family of multidomain zinc-dependent endopeptidases, have been reported to hydrolyze the components of the extra-cellular matrix (ECM) and thereby induce tissue remodelling (Roy, R. et al. J. Clin. Oncol. 2009, 27, 5287- 5297). The MMPs share three common domains, which are the pro-domain, the zinc-dependent catalytic domain and the hemopexin-like C-terminal domain (Tailant, C. et al., Biochim. Biophys. Acta 2010, 1803, 20-28). MMPs are typically expressed as inactive precursors (zymogens) that contain the pro-domain. The zymogens are subsequently activated via the removal of the pro-domain (Gaffney, J. et al., Matrix Biology 2015, 44-46, 191-199). To date, the MMPs activation mechanisms are not fully understood. The activity of the MMPs is tightly regulated by their endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPS). Failure to maintain a balance between the activity of MMPs and their inhibitors has been widely recognized as a contributor to pathological conditions such as cancer cell invasion and metastasis (Kessenbrock, K. et al., Cell 2010, 141, 52-67; Egeblad and Werb, Nature Rev. Cancer 2002, 2, 161-174; Coussens, L.M., et al., Science 2002, 295, 2387-2392). In humans, at least 23 different MMPs have been identified to date. These MMPs are divided into six subclasses based on their substrate specificity, namely, collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and other MMPs (Egeblad and Werb, Nature Rev. Cancer 2002, 2, 161-174; Nagase, H. et al., Cardiovasc. Res. 2006, 69, 562-573; Laronha, H. et al., Cells 2020, 9, 1076).

[0007] MMP7, which is the smallest MMPs, has an important role in various physiological and pathological processes (Yokoyama, Y. et al., Clin. Cancer Res. 2008, 14, 5503-5511). Previous studies have reported that MMP7 hydrolyzes a range of ECM substrates and non-ECM proteins. To date, the identified substrates of MMP7 include laminin, fibronectin and non-fibrillar collagen, gelatin, elastin, entactin / nidogen and tenascin-C (Heinz, A. et al., Biochimie 2011, 93, 187-194; Wang, F.Q. et al., Int. J. Cancer 2005, 114, 19-31), tumor necrosis factor a (TNF- a), Fas ligand, heparin-binding epidermal growth factor precursor (HB-EGF), P-integrin and E-cadherin (li, M. et al., Exper. Bio. and Med. 2006, 231, 20-27). MMP7 is associated with human cancer invasion, apoptosis, growth and angiogenesis (Yokoyama, Y. et al., Clin. Cancer Res. 2008, 14, 5503-5511). MMP7 has been found to be overexpressed in various cancers, including gastric, colorectal, lung, and liver cancers (Yamashita, K. et al., Int. J. Cancer 1998, 79, 187-194; Sasaki, H. et al., J. Surg. Res. 2001, 101, 242-247; Liu, D. et al., Lung Cancer 2007, 58, 384-391; Han, J.C., et al., World J. Surg. Oncol. 2015, 13, 5). The upregulated MMP7 in cancer cells have been found to be related with tumor metastasis and poor survival rates of cancer patients (Yamashita, K. et al., Int. J. Cancer 1998, 79, 187-194; Sasaki, H. et al., J. Surg. Res. 2001, 101, 242-247; Liu, D. et al., Lung Cancer 2007, 58, 384-391; Han, J.C., et al., World J. Surg. Oncol. 2015, 13, 5; Szarvas, T. et al, Cancer Sci. 2010, 101, 1300-1308; Jones, L. E. et al., Clin. Cancer Res. 2004, 10, 2832-2845; Wang, W.S. et al., Carcinogenesis 2006, 27, 1113-1120). In recent years, the serum level of MMP7 has been widely recognized as a predictive biomarker for cancer progression such as pancreatic cancer and gastric cancer (Jones, L. E. et al., Clin. Cancer Res. 2004, 10, 2832-2845; Wang, W.S. et al., Carcinogenesis 2006, 27, 1113-1120; Kuhlmann, K.F. et al., Cancer Epidemiol. Biomarkers Prev. 2007, 16, 886-891). Hence, MMP7 has emerged as a promising therapeutic target in cancer treatment. To date, there are only a few peptide-based or small molecule MMP7 inhibitors that have been developed (Tabuse, H. et al., J. Med. Chem. 2022, 65, 13253-13263; Li, M.-H. et al., RSC advances 2015, 5, 104725-104732). However, these MMP7 inhibitors show moderate selectivity over other MMPs. Moreover, these inhibitors did not show great stability. Therefore, there is a need to develop new selective and stable inhibitors to target MMP7.

[0008] Schumacher et al. (Science. 1996, 271, 1854) reported a phage display technology in which a D-enantiomer of a protein or catalytic site was prepared by chemical synthesis and used to isolate L-amino acid peptide ligands that interact with it from a phage display library. Once a strongly binding L-amino acid peptide ligand was isolated, the D-enantiomer of that ligand was synthesized and then tested for binding to the natural protein or catalytic site. As a proof of concept, the L- and D-enantiomers of the chicken Src homology (SH3) domain were chemically synthesized and used to screen a phage display library. The D-SH3 screening resulted in a novel L-amino acid peptide. The resulting peptide was then synthesized as the D- enantiomer and was shown to bind the L-SH3 domain with micromolar binding affinity. There remains an urgent need to develop high-throughput selection methods for identifying D-amino acid-based cyclic peptide binders.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides a method and system for identifying peptides that bind to a target polypeptide of interest, from a very large and random collection of unnatural peptides in an mRNA display library, using a D-amino acid analog of the target polypeptide (D- polypeptide analog) as a panning moiety. In particular, the method is used to identify cyclic peptides and / or peptides comprising D-amino acids and other unnatural amino acids, such as P-amino acids

[0011] The present invention further provides, according to other embodiments, novel cyclic peptide inhibitors of the matrix metalloproteinase MMP7, the cyclic peptides containing a plurality of D-amino acids. The novel cyclic peptide inhibitors were identified using the novel method disclosed herein. It is shown that peptide binders identified using the method of the present invention are more stable and active than peptides identified using a similar method that does not include the use of the D-polypeptide analog of the target.

[0012] The present invention provides, according to one aspect, a method for identifying a cyclic peptide comprising D-amino acids capable of binding to a target polypeptide, the method comprising:

[0013] (i) synthesizing a D-amino acid analog of the target polypeptide (D-polypeptide analog), or of a binding domain thereof;

[0014] (ii) preparing an mRNA display library encoding random cyclic peptides, wherein each mRNA display library molecule comprises, from 5’ to 3’: a first cyclization codon; a series of at least 6 randomized codons; a second cyclization codon, a spacer encoding 2-10 amino acids; a translation stop element; a linker; and puromycin, and wherein the randomized codons and the spacer codons do not include the first or second cyclization codons;

[0015] (iii) preparing an in vitro translation system comprising aminoacyl-tRNAs in which a tRNA that responds to the first cyclization codon and a tRNA that responds to the second cyclization codon are each aminoacylated with amino acids capable of reacting together to form a cyclic peptide; and wherein the in vitro translation system does not include any other aminoacyl-tRNAs that respond to the first cyclization codon, to the second cyclization codon, or whose amino acid can interfere with the cyclization reaction;

[0016] (iv) translating the mRNA display library molecules obtained in step (ii) in the in vitro translation system of step (iii) and providing conditions for the amino acids incorporated in response to the cyclization codons to cyclize, to produce a library of cyclic peptides wherein each peptide is conjugated to its progenitor mRNA;

[0017] (v) incubating the library obtained in step (iv) with the D-polypeptide analog of step (i) to allow binding of cyclic peptides to the D-polypeptide analog;

[0018] (vi) isolating at least one cyclic peptide binder and identifying its amino acid sequence by the reverse transcribed DNA of its conjugated progenitor mRNA;

[0019] (vii) synthesizing a cyclic peptide having inverse stereochemistry to the at least one isolated cyclic peptide binder and confirming its binding to the target polypeptide.

[0020] According to some embodiments, the D-polypeptide analog is synthesized by solidphase peptide synthesis (SPPS). According to some embodiments, the D-polypeptide analog is immobilized on a solid support, before performing step (v).

[0021] According to some embodiments, the cyclic peptide of step (vii) is synthesized by SPPS.

[0022] According to some embodiments, the target polypeptide is a natural polypeptide.

[0023] According to some embodiments, each mRNA display library molecule comprises between 6 and 15 randomized codons and the cyclic peptide comprises between 8 and 17 amino acid residues.

[0024] According to some embodiments, the first cyclization codon is AUG and the second cyclization codon is UGG, the randomized codons and the spacer codons do not comprise AUG and UGG and the translation system does not comprise L-Met, L-Trp, or their cognate aminoacyl-tRNA synthetases (aaRS).

[0025] According to some embodiments, the randomized codons comprise NNU codons.

[0026] According to some embodiments, the spacer encodes 4-10 amino acids. According to some embodiments, the spacer encodes 5-9 amino acids. According to some embodiments, the spacer encodes 6-8 amino acids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the spacer encodes 6 amino acids. According to some embodiments, the spacer encodes the amino acid sequence Ser-Ser-Asn-Val-Ser-Ala (SEQ ID NO: 3). According to some embodiments, the spacer comprises the nucleic acid sequence AGUUCUAACGUAAGCGCU (SEQ ID NO: 4).

[0027] According to some embodiments, the translation stop element is a stop codon.

[0028] According to some embodiments, the translation system does not include a catalytically active release factor (RF).

[0029] According to some embodiments, the translation stop element is a stop codon and the translation system does not include a catalytically active RF.

[0030] According to some embodiments, the stop codon is UAG.

[0031] According to some embodiments, the linker comprises between 10-40 nucleotides. Each possibility represents a separate embodiment of the invention. According to some embodiments, the linker comprises between 12-35 codons. Each possibility represents a separate embodiment of the invention. According to some embodiments, the linker comprises between 15-30 codons. Each possibility represents a separate embodiment of the invention. According to some embodiments, the linker comprises 15 nucleotides. According to some embodiments, the linker comprises the sequence GACGGGGGGCGGAAA (SEQ ID NO: 5).

[0032] According to some embodiments, the mRNA display library molecule comprises a ribosome binding site upstream of the initiation codon. According to some embodiments, the ribosome binding site is a Shine-Dalgarno sequence comprising the consensus sequence AGGAGG.

[0033] According to some embodiments, the mRNA display library molecule comprises a sequence of 4-13 nucleotides between the Shine-Dalgarno consensus sequence and the first cyclization codon.

[0034] According to some embodiments, the mRNA display library molecule comprises a sequence of 4-13 nucleotides upstream of the Shine-Dalgarno consensus sequence.

[0035] According to some embodiments, the in vitro translation system is a purified E. coli translation system.

[0036] According to some embodiments, the aminoacyl-tRNAs that respond to the first and second cyclization codons are provided to the in vitro translation system as pre-charged aminoacy 1-tRN A s . According to some embodiments, the pre-charged aminoacyl-tRNAs are prepared via a reaction comprising a ribozyme comprising the sequence SEQ ID NO: 6 or SEQ ID NO: 7.

[0037] According to some embodiments, the amino acid encoded by one of the cyclization codons comprise a reactive thiol and the amino acid encoded by the other cyclization codon comprises an N-terminal 2-halogen-acetyl group, wherein the halogen is selected from chloro, bromo, and iodo.

[0038] According to some embodiments, the amino acids capable of reacting together to form a cyclic peptide are D-Cys and A-chloroacctyl-D-tyrosinc (ClAc-D-Tyr).

[0039] According to some embodiments, the aminoacyl-tRNAs that respond to the cyclization codons are Cl Ac-D-Tyr-tRNAI Vkl, and D-Cys-tRNA^^ccA.

[0040] According to some embodiments, the aminoacyl-tRNA that responds to the first cyclization codon is ClAc-D-Tyr-tRNA0^61.

[0041] According to some embodiments, the aminoacyl-tRNA that responds to the second cyclization codon is D-Cys-tRNAProlE2ccA.

[0042] According to some embodiments, the aminoacyl-tRNA whose amino acid can interfere with the cyclization reaction comprises an amino acid comprising a reactive thiol group.

[0043] According to some embodiments, the aminoacyl-tRNA whose amino acid can interfere with the cyclization reaction is Cys-tRNACys.

[0044] According to some embodiments, the in vitro translation system does not comprise L- Cys or L-Cys aminoacyl-tRNA synthetase (aaRS).

[0045] According to some embodiments, the cyclic peptide comprises an at least one additional unnatural amino acid.

[0046] According to some embodiments, the at least one additional unnatural amino acid is a P-amino acid.

[0047] According to some embodiments, the P-amino acid is aminoacylated onto a tRNA that responds to a cysteine codon.

[0048] According to some embodiments, the P-amino acid is aminoacylated onto a tRNA via a reaction comprising a ribozyme comprising the sequence SEQ ID NO: 6 or SEQ ID NO: 7.

[0049] According to some embodiments, the peptide comprises at least one additional P-amino acid, aminoacylated onto a tRNA that responds to an AUU, AUC, CUU, or CUC codons. According to some embodiments, the in vitro translation system does not comprise L- L-Cys, L-Leu, L-Ile, or their cognate aminoacyl-tRNA synthetases (aaRS).

[0050] According to some embodiments, the in vitro translation system does not comprise the L-amino acids Cys, Leu, He, Met, Trp, Gin, Glu, or their cognate aaRS.

[0051] According to some embodiments, the in vitro translation system does not comprise release factor 1 (RF1).

[0052] According to some embodiments, the P-amino acid is selected from (lS,2S)-2- aminocyclohexane carboxylic acid, (lR,2S)-2-aminocyclopentane carboxylic acid, and (lR,2R)-2-aminocyclopentane carboxylic acid.

[0053] According to some embodiments, the in vitro translation system further comprises one or more aminoacyl-tRNAs selected from (lS',2S')-2-ACHC-tRNAProlE2GAu, (lR,2S)-2-ACPC- tRNA^^GAG, and (lR,2R)-2-ACPC-tRNAGluE2GCA.

[0054] According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least 108molecules. According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least IO10molecules. According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least 1012molecules.

[0055] According to some embodiments, the D-polypeptide analog comprises a biotin tag. According to some embodiments, the biotin tag is connected to the N-terminus of the D- polypeptide analog. According to some embodiments, the solid support comprises avidin, e.g., streptavidin. According to some embodiments, the solid support comprises streptavidin beads.

[0056] According to some embodiments, the reverse transcribed DNA is prepared before performing step (v).

[0057] According to some embodiments, the reverse transcribed DNA of step (vi) is used to enrich the cyclic peptide binder. According to some embodiments, the method comprises at least two rounds of enrichment.

[0058] According to some embodiments, the target polypeptide is a matrix metalloproteinase (MMP).

[0059] According to some embodiments, the MMP is a human MMP or a catalytically active variant or fragment thereof. According to some embodiments, the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the sequence of SEQ ID NO: 1, or a catalytically active variant thereof having at least 80% sequence identity.

[0060] According to some embodiments, the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the catalytic domain set forth in SEQ ID NO: 2, or a catalytically active variant thereof having at least 80% sequence identity.

[0061] The present invention also provides, according to another aspect, a cyclic peptide that binds to human matrix metalloproteinase 7 (MMP7, matrilysin-1) and inhibits its activity, particularly a cyclic peptide comprising D-amino acids and at least one 0-amino acid.

[0062] According to some embodiments, the cyclic peptide that binds to human MMP7 and inhibits its activity comprises the amino acid sequence (D-Ser)-((77?,27?)-2- aminocyclohexylcarboxy)-(D-Arg)-(D-Tyr)-(D-Thr)-(D-Val)-(D-Phe)-(D-Val)-(D-Ala)-(D- Val)-(D-Tyr)-(D-Val)-(D-Ser) (SEQ ID NO: 8); or a derivative thereof comprising 1-3 modifications to SEQ ID NO: 8.

[0063] According to some embodiments, the cyclic peptide that comprises SEQ ID NO: 8 further comprises an N-terminal and a C-terminal residues that form a cyclic peptide.

[0064] According to some embodiments, the N-terminal and a C-terminal residue react to form a thioether bond.

[0065] According to some embodiments, the residues before cyclization are N-chloroacetyl-L- tyrosine and L-cysteine.

[0066] According to some embodiments, the cyclic peptide comprises an N-terminal N- chloroacetyl-L-tyrosine and a C-terminal L-cysteine, before cyclization.

[0067] According to some embodiments, the cyclic peptide comprises the structure (D’20):

[0068] According to some embodiments, the cyclic peptide comprises a C-terminus that is, amidated, carboxylated, or substituted with an alcohol moiety or with a moiety the improves at least one property of the peptide.

[0069] According to some embodiments, the moiety the improves at least one property of the peptide is a polymer.

[0070] According to some embodiments, the C-terminus is PEGylated.

[0071] According to some embodiments, the present invention provides a conjugate comprising the cyclic peptide and a moiety that increases solubility, stability and / or permeability.

[0072] According to some embodiments, the moiety is PEG.

[0073] According to some embodiments, the conjugate of the cyclic peptide comprises 4-30 linear PEG units attached to its C-terminus. According to some embodiments, the conjugate comprises a linear PEG of 6 units attached to its C-terminus. According to some embodiments, the conjugate comprises a linear PEG of 27 units attached to its C-terminus.

[0074] According to some embodiments, the conjugate of the cyclic peptide has the structure (D’20-PEG6):

[0075] According to some embodiments, the conjugate of the cyclic peptide has the structure

[0076] 5 (D’20-PEG27):

[0077]

[0078] According to some embodiments, the cyclic peptide or conjugate binds a human MMP7 polypeptide comprising SEQ ID NO: 1 or a catalytically variant thereof with at least 80% sequence identity thereto.

[0079] According to some embodiments, the cyclic peptide binds the catalytic domain of human MMP7 comprising the sequence SEQ ID NO: 2 or a catalytically variant thereof with at least 80% sequence identity thereto.

[0080] According to some embodiments, the cyclic peptide binds the catalytic domain of human MMP7 with an IC50 of 90 nM.

[0081] According to some embodiments, the present invention provides a pharmaceutical composition comprising a cyclic peptide or conjugate and an acceptable salt, diluent or carrier.

[0082] According to some embodiments, the pharmaceutical composition is formulated for enteral or parenteral administration. According to some embodiments, the pharmaceutical composition is formulated for intravenous, oral, intra-articular, intramuscular, subcutaneous, topical, transdermal, intradermal, nasal or intrathecal administration. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. According to some embodiments, the pharmaceutical composition is formulated for oral administration.

[0083] According to some embodiments, the pharmaceutical composition is for use in inhibiting an activity of MMP7.

[0084] According to some embodiments, the pharmaceutical composition is for use in preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7.

[0085] According to some embodiments, the disease or disorder is selected from a cancer or a tumor, a kidney disease, disorder or injury, angiogenesis related disorders, including but not limited to idiopathic pulmonary fibrosis and biliary atresia.

[0086] According to some embodiments, the use involves at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

[0087] According to some embodiments, the present invention provides a method of preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7, comprising administering to a subject in need a pharmaceutical composition as disclosed herein.

[0088] According to some embodiments, the disease or disorder is selected from a cancer or a tumor, a kidney disease, disorder or injury, angiogenesis related disorders, including but not limited to idiopathic pulmonary fibrosis, and biliary atresia.

[0089] According to some embodiments, the cancer is a solid cancer or a metastatic cancer.

[0090] According to some embodiments, the solid cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, gastric cancer, esophageal cancer, gallbladder cancer, and brain cancer.

[0091] According to some embodiments, the cancer is hematologic cancer.

[0092] According to some embodiments, the pharmaceutical composition is administered before, together with, or following, an additional anti-cancer treatment.

[0093] According to some embodiments, the additional anti-cancer treatment is selected from the group consisting of surgery, chemotherapy, radiotherapy, and immunotherapy. According to some embodiments, the method results in at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

[0094] BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0096] Fig. 1 shows the steps of a representative method of the present invention.

[0097] Fig. 2A shows the sequence of the catalytic domain of human MMP7 (SEQ ID NO:2), which consists of 173 amino acids, MMP7(95-267). Since this domain does not carry any cysteine residues, the synthetic approach used was based on Fmoc-solid phase peptide synthesis (Fmoc- SPPS) of four peptide segments that were combined by three native chemical ligation (NCE) reactions at the Eysl31-Alal32, Hisl78-Alal79, and Ala211-Ala212 junctions (shown as underlined and bolded), with temporary substitution of three Ala residue (A132, A179, and A212) with protected Cys in the form of Thz (Z) to facilitate native chemical ligation (NCE) reactions followed by a final desulfurization step.

[0098] Fig. 2B shows the synthetic scheme of the NCL steps used to synthesize the L- and D- enantiomeric polypeptides of the catalytic domain of human MMP7 (L-MMP7catand D- MMP7cat)

[0099] Fig. 2C shows the final HPEC chromatogram and electron spray ionization mass spectrogram of synthesized L-MMP7cat.

[0100] Fig. 2D shows the final HPEC chromatogram and electron spray ionization mass spectrogram of D-MMP7cat.

[0101] Fig. 3A shows an overlay of circular dichroism spectra for L-MMP7catand D-MMP7cat. Fig. 3B shows an overlay of catalytic activity of L-MMP7catcompared with recombinant MMP7 as measured by monitoring the hydrolysis of the general MMP Anorogenic peptide substrate FS-6 at Xext = 320 nm and Xem= 400 nm.

[0102] Fig. 4A shows the codon reassignments for the unnatural amino acids used in the cyclic peptide library.

[0103] Fig. 4B shows the structures of the three P-amino acids, (lS,2S)-2-aminocyclohexane carboxylic acid ((15,25)-2-ACHC), (lR,2S)-2-aminocyclopentane carboxylic acid ((lR,2S)-2- ACPC) and (lR,2R)-2-aminocyclopentane carboxylic acid ((lR,2R)-2-ACPC).

[0104] Fig. 4C shows the design of a representative mRNA display library molecule used.

[0105] Fig. 4D shows the sequences of the successful binders, LIO and L16 for L-MMP7catand D20 and D21 for D-MMP7cat, that were selected for further testing for in vitro activity against wild type MMP7. D20 and D21 were synthesized with inverted stereochemistry (termed D'20 and D'21, as shown in the mirror) before proceeding with further testing.

[0106] Fig. 4E shows the chemical structure of L16. Shown in grey is the thioether linkage as well as the P-amino acid (lR,2S)-2-ACPC.

[0107] Fig. 4F shows the chemical structure of D'20. Shown in grey is the thioether linkage as well as the P-amino acid (lR,2R)-2-ACPC.

[0108] Fig. 5A shows a general scheme of synthesis of cyclic peptides using Fmoc-solid phase peptide synthesis (SPPS) and formation of thioether cyclization.

[0109] Fig. 5B shows the structure of Ud-PEGe. Shown in grey is the thioether linkage and (1 R,2S)- 2-ACPC.

[0110] Fig. 5C shows the structure of L76-PEG27. Shown in grey is the thioether linkage and (1 R,2S)- 2-ACPC.

[0111] Fig. 5D shows the structure of D'20-PEG6. Shown in grey is the thioether linkage and (1R,2R)- 2-ACHC.

[0112] Fig. 5E shows the structure of D'2d-PEG27- Shown in grey is the thioether linkage and (1R,2R)- 2-ACHC.

[0113] Fig. 5F shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified LI 0-PEGe. Fig. 5G shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified L / d-PEGe.

[0114] Fig. 5H shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified D’20-PEG6.

[0115] Fig. 51 shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified D’27-PEG6.

[0116] Fig. 5J shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified L / 6-PEG27.

[0117] Fig. 5K shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified D’20-PEGzi.

[0118] Fig. 6A shows the dose response curve for in vitro inhibition activity of L16 on human MMP7, which was examined using the Anorogenic peptide (FS-6).

[0119] Fig. 6B shows the dose response curve for in vitro inhibition activity of D'20 on human MMP7, which was examined using the Anorogenic peptide (FS-6).

[0120] Fig. 6C shows double reciprocal plot of 1 / V vsl / [S] for L16 used to determine mode of inhibition in Example 6.

[0121] Fig. 6D shows double reciprocal plot of 1 / V v.v 1 / [SJ for D’20 used to determine mode of inhibition in Example 6.

[0122] Fig. 6E shows selectivity of L16lo MMP7 over other MMPs (MMP1, MMP8, MMP9, MMP10 and MMP-14). All data points represent mean ± SD for each condition and n = 3. Curves were fitted to the data using the equation y = 1 + 10^-logVGo)), where, y is remaining activity of the enzyme and x is the inhibitor concentration.

[0123] Fig. 6F shows selectivity of D'20 to MMP7 over other MMPs (MMP1, MMP8, MMP9, MMP10 and MMP-14). All data points represent mean ± SD for each condition and n = 3. Curves were fitted to the data using the equation y = 1 + 10^-logVGo)), where, y is remaining activity of the enzyme and x the inhibitor concentration.

[0124] Fig. 7A shows the results of the degradation assay of D’20 in simulated gastric Auid (SGF).

[0125] Fig. 7B shows the results of the degradation assay of L16 in SGF.

[0126] Fig. 7C shows the results of the degradation assay of D’20 in simulated intestinal Auid (SIF). Fig. 7D shows the results of the degradation assay of LI 6 in SIF.

[0127] Fig. 8A shows representative pictures of transwell migration of CFPAC-1 cells after treatment with different concentrations of D’20.

[0128] Fig. 8B shows representative pictures of transwell migration of CFPAC-1 cells after treatment with different concentrations of L16.

[0129] Fig. 8C shows bar plots of transwell migration comparing D’20 and LI 6, wherein the bar represents the mean ± SE of three replicates. Student’s t-test p-values are reported.

[0130] DETAILED DESCRIPTION OF THE INVENTION

[0131] The present invention provides, according to some embodiments, methods for the identification of novel cyclic peptides capable of binding to a target polypeptide, wherein the cyclic peptides comprise a plurality of D-amino acids. According to other embodiments, the cyclic peptides further comprise other unnatural amino acids, such as P-amino acids. The method of the present invention demonstrates, for the first time, that an mRNA display technology, such as RaPID, can be used to identify novel cyclic D-amino acid peptides. The present invention further provides, according to other embodiments, novel cyclic peptide inhibitors of the matrix metalloproteinase MMP7 (matrilysin) containing a plurality of D-amino acids. The novel cyclic peptide inhibitors of the present invention were identified by utilizing the novel method disclosed herein.

[0132] Cyclic peptides containing a plurality of D-amino acids are attractive due to their resistance to proteolytic degradation, consequently demonstrating improved stability and lower immunogenicity compared to L-amino acids cyclic peptides. Identifying peptides, specifically cyclic peptides, comprising D-amino acids using mRNA display or the improved RaPID would be particularly attractive, given the possibility for large library sizes as well as ease of the technique. However, this has proven elusive, since it is difficult to prepare in vitro translation systems comprising D-aminoacyl-tRNAs. More importantly, it is well-established in the art that D-amino acids are poorly tolerated by the ribosome and other components of the translational machinery, specifically in incorporating multiple and / or consecutive D-amino acids.

[0133] In the approach of the present invention, mRNA display is used to perform affinity selection against the mirror-image form of the target protein (consisting of D-amino acids and achiral glycine) using cyclic peptides that are synthesized by the ribosome and are comprised of mostly L-amino acid residues. The identified cyclic peptide binders are subsequently synthesized with their inverted stereochemistry, i.e., mostly D-amino acid residues. These cyclic peptides are used to target the corresponding L-protein (Fig. 1). This remarkable strategy allows, for the first time, for the selection of highly specific and stable binders from a large library (1012) using mRNA display. In addition, the use of evolved ribozymes to charge tRNAs with unnatural amino acids along with a purified translation system allows for the development of a cyclic peptide library that includes additional unnatural amino acids, such as P-amino acids. This strategy holds tremendous potential for the development of highly stable cyclic therapeutic peptides.

[0134] According to one embodiment, the present invention provides a method for identifying a cyclic peptide comprising D-amino acids capable of binding to a target polypeptide, the method comprising preparing an mRNA display library encoding random cyclic peptides, wherein each mRNA display library molecule comprises, from 5’ to 3’: a first cyclization codon; a series of at least 6 randomized codons; a second cyclization codon, a spacer encoding 2-10 amino acids; a translation stop element; a linker; and puromycin, and wherein the randomized codons and the spacer codons do not include the first or second cyclization codons.

[0135] As is known in the art, mRNA display, and the improved RaPID, use an in vitro translation system to translate a modified mRNA encoding a peptide sequence, usually with a randomized portion, ligated at its 3’ end with puromycin.

[0136] Translation is process of synthesizing proteins using an RNA template, known as messenger RNA (mRNA). The mRNA dictates the order of the amino acids in the protein chain by a three-nucleotide sequence known as a codon, that results in the addition of one specific amino acid to the protein being generated. The matching of a three-nucleotide sequence to a specific amino acid is called the genetic code.

[0137] The process of translation occurs in all living organisms, however, performing translation in an in vitro system that provides the necessary components is well-established. Translation is mediated by the ribosome, a large ribonucleoprotein complex with two subunits; a smaller subunit that binds the mRNA template and a large subunit that is responsible for catalyzing peptide bond formation between sequential amino acids. Translation comprises three steps: initiation, elongation, and termination. These steps are further facilitated by protein factors. In bacteria, for example, the three steps are facilitated by initiation factors 1, 2, 3 (IF1, IF2, and IF3), elongation factors Tu (EF-Tu) and G (EF-G), and release factors 1, 2, 3 (RF1, RF2, and RF3), respectively.

[0138] In bacteria, initiation occurs when the ribosome encounters a ribosome binding site (RBS) sequence on the mRNA. Downstream of the RBS, the ribosome assembles around on the start codon, generally AUG. In cellular translation, the first tRNA is generally tRNA^Hhat is aminoacylated with the formylated methionine. However, non-canonical initiation of translation with non- AUG codons is also known.

[0139] The ribosome facilitates decoding of the mRNA into amino acids by inducing the binding of complementary transfer RNA (tRNA) anticodon sequences to mRNA codons. A tRNA is a bifunctional adaptor molecule with a molecular weight of approximately 25 KDa. At one end, the tRNA has a three-nucleotide sequence, known as an anticodon, which binds a complementary codon encoded by the mRNA. In addition, 3 ’-CCA end of tRNAs can have an amino acid covalently linked by an acyl linkage to the 3’ ribose OH group. Such tRNAs are known as aminoacyl-tRNAs (aa-tRNAs).

[0140] Both subunits of the ribosome comprise three binding sites: the aminoacyl-tRNA site (A site), the peptidyl-tRNA site (P site) and the tRNA exit site (E site). Elongation occurs when an incoming aa-tRNA is ferried to the ribosome by EF-Tu. The A site of the small subunit of the ribosome displays the next mRNA codon to be decoded. When the anticodon of the incoming aa-tRNA binds the displayed mRNA codon, the aa-tRNA is the accommodated into the A site of the large subunit of the ribosome. Peptide bond formation then occurs in the large subunit between the A-site bound aa-tRNA and the peptidyl-tRNA in the P site, which connects the growing peptide chain attached to a tRNA. As a result of peptide bond formation, the entire chain is transferred to the A site tRNA and the P site tRNA is deacylated. The A site tRNA is then moved to the P site along with concomitant movement of the mRNA by one codon. The deacylated tRNA enters the E site and dissociates. The A site is then ready to accept another aa-tRNA.

[0141] As used herein, the term “responds to a codon” refers to the binding event between mRNA codon in the A site and the anticodon of the tRNA that leads to the abovementioned accommodation. It should be noted that the binding event is independent from the nature of the amino acid at the 3’ end of the tRNA. Thus, according to the principles of the present invention, a tRNA may be aminoacylated with an amino acid that is not cognate or is unnatural and may be accommodated into the ribosome and become incorporated into the growing peptide chain following peptide bond formation. Furthermore, codons are reassigned. The term “codon reassignment” or variations thereof refers to the utilization of a codon that encodes for an amino acid or a stop codon according to the rules of the genetic code, to encode for a different amino acid.

[0142] In cells, termination occurs with an mRNA stop codon in the A site. Generally, release factors recognize the stop codons and catalyze the release of the peptide from the P-site bound tRNA.

[0143] As used herein, the term "mRNA display library molecule" refers to the modified mRNA and refers a randomized codon sequence that may be translated using in an in vitro translation system to generate a cyclic peptide. In addition, the mRNA display library molecule can form a linkage with its own translated peptide, via puromycin that is covalently linked to its 3’ end. Puromycin is an antibiotic derived from Streptomyces alboniger that mimics the 3’ end of aminoacylated tRNA. Puromycin enters the A site of the large subunit and forms a peptide bond with the P-site bound peptidyl tRNA, leading to the formation of a puromycylated nascent chain and premature chain release. Once translation is complete, rather the peptide being released from the peptidyl-tRNA bound in the ribosome peptidyl-tRNA site (P site), the 3’ puromycin that is covalently linked to the 3’ end of the mRNA display library molecule binds the ribosome aminoacyl-tRNA site (A site) and reacts with the P-site bound peptide chain. This reaction covalently links the peptide with its progenitor mRNA to form a conjugated mRNA- cyclic peptide molecule to be used in a binding assay to a target polypeptide of interest. According to the principles of the present invention, the target polypeptide of interest is synthesized as its mirror-image, i.e., as the D-amino acid enantiomer (D-polypeptide analog) and used for screening mRNA-cyclic peptide library.

[0144] When binders collected (e.g. after incubating with a D-polypeptide analog target, washing and eluting), the mRNA that is linked to the eluted cyclic peptide binder serves as an identification tag; by reverse transcribing the conjugated mRNA into cDNA followed by PCR amplification and sequencing, the peptide amino acid sequence may be easily determined. The PCR amplified product may then be transcribed to mRNA and ligated with puromycin to form the mRNA library display molecules for iterative rounds of translation followed by binding to the D-polypeptide analog of the target polypeptide. These steps are repeated until enrichment of successful binders is achieved. According to some embodiments, the mRNA display library molecule comprises a ribosome binding site upstream of the initiation codon. According to some embodiments, the ribosome binding site is an internal ribosome entry site. According to some embodiments, the ribosome binding site is a Shine-Dalgarno sequence comprising the consensus sequence AGGAGG.

[0145] According to some embodiments, the mRNA display library molecule comprises a sequence upstream of the Shine-Dalgarno consensus sequence. According to some embodiments, the sequence upstream of the Shine-Dalgarno consensus sequence comprises 4- 13 nucleotides. According to some embodiments, the sequence upstream of the Shine-Dalgarno consensus sequence comprises 11 nucleotides. According to some embodiments, the sequence upstream of the Shine-Dalgarno consensus sequence comprises the sequence GGGUUAACUUU (SEQ ID NO: 30).

[0146] According to some embodiments, the mRNA display library molecule comprises a first cyclization codon. According to some embodiments, the first cyclization codon is AUG. According to some embodiments, the mRNA display library molecule comprises an initiation codon. According to some embodiments, the initiation codon is AUG. According to some embodiments, the first cyclization codon is the initiation codon. According to some embodiments, the first cyclization codon is not the initiation codon. According to some embodiments, the first cyclization codon is the initiation codon and is AUG. Translation initiation on non-AUG codons, also known as non-canonical translation initiation, is also within the scope of this invention.

[0147] According to some embodiments, the mRNA display library molecule comprises a sequence of 4-13 nucleotides between the Shine-Dalgarno consensus sequence and the initiation codon. According to some embodiments, the mRNA display library molecule comprises a sequence of 4-13 nucleotides between the Shine-Dalgarno consensus sequence and the first cyclization codon. According to some embodiments, the mRNA display library molecule comprises a sequence of 11 nucleotides between the Shine-Dalgarno consensus sequence and the initiation codon. According to some embodiments, the mRNA display library molecule comprises a sequence of 11 nucleotides between the Shine-Dalgarno consensus sequence and the first cyclization codon. According to some embodiments, the sequence of 11 nucleotides comprises the sequence GAGAUAUACAU (SEQ ID NO: 29). According to some embodiments, the mRNA display library molecule comprises a series of at least 4 randomized codons. According to some embodiments, the mRNA display library molecule comprises a series of at least 6 randomized codons. According to some embodiments, the mRNA display library molecule comprises a series of at least 10 randomized codons. According to some embodiments, each mRNA display library molecule comprises between 6 and 38 randomized codons. According to some embodiments, each mRNA display library molecule comprises between 6 and 28 randomized codons. According to some embodiments, each mRNA display library molecule comprises between 6 and 18 randomized codons. According to some embodiments, each mRNA display library molecule comprises between 6 and 15 randomized codons. According to some embodiments, the randomized codons are NNU codons.

[0148] According to some embodiments, the mRNA display library molecule comprises a second cyclization codon. According to some embodiments, the second cyclization codon is UGG. According to some embodiments, the first and second cyclization codons are each independently selected from AUG and UGG. According to some embodiments, the randomized codons do not comprise AUG and UGG. According to further embodiments, the translation system does not comprise L-Met, L-Trp, or their cognate aminoacyl-tRNA synthetases (aaRS).

[0149] According to some embodiments, the mRNA display library molecule comprises a spacer. According to some embodiments, the mRNA display library molecule comprises a spacer encoding 2-10 amino acids. According to some embodiments, the mRNA display library molecule comprises a spacer encoding 4-10 amino acids. According to some embodiments, the mRNA display library molecule comprises a spacer encoding 5-9 amino acids. According to some embodiments, the mRNA display library molecule comprises a spacer encoding 6-8 amino acids. According to some embodiments, the spacer encodes 6 amino acids. According to some embodiments, the spacer does not comprise AUG and UGG codons. According to some embodiments, the spacer encodes the amino acid sequence Ser-Ser-Asn-Val-Ser-Ala (SEQ ID NO: 3). According to some embodiments, the spacer comprises the nucleic acid sequence AGUUCUAACGUAAGCGCU (SEQ ID NO: 4).

[0150] According to some embodiments, the mRNA display library molecule comprises a translation stop element. According to the principles of the present invention, translation by the ribosome is stopped with a peptidyl site (P site)-bound peptidyl-tRNA comprising the translated peptide and an empty aminoacyl site (A site). This configuration is required in order to allow puromycin that is linked to the mRNA to bind the A site and react to produce a peptide-mRNA conjugate. As used herein, “translation stop element” refers to any element that causes the ribosome to stop translating but without causing defects to the catalytic functions of the ribosome (e.g., peptidyl transfer). According to some embodiments, the translation pausing element is an mRNA-DNA junction wherein, for example, a DNA oligomer is ligated to the 3’ end of the mRNA. An RNA-DNA junction on an mRNA display library molecule is formed, for example, if the linker that follows the translation stop element is comprised of DNA. According to some embodiments, the translation stop element is a stop codon. According to some embodiments, the translation stop element is a stop codon selected from UAG, UGA, and UAA. According to some embodiments, the in vitro translation system is devoid of catalytically active release factors. According to some embodiments, the translation stop element is a stop codon and wherein the in vitro translation system is devoid of catalytically active release factors. It is to be understood that “catalytically active” includes recognition of stop codons and / or binding the ribosome in response to the stop codon and / or catalyzing peptide release. According to some embodiments the stop codon is UAG. According to some embodiments, the stop codon is UAG and the in vitro translation system is devoid of catalytically active bacterial or eukaryotic release factor 1 (RF1). According to some embodiments, the stop codon is UAG and the in vitro translation system is devoid of catalytically active bacterial RF1. According to some embodiments, the stop codon is UAG, the in vitro translation system is derived from E. coli and is devoid of catalytically active E. coli RF1.

[0151] According to some embodiments, the mRNA display library molecule comprises a linker. According to some embodiments, the linker comprises 10-40 nucleotides, including each value within the specified range. Each possibility represents a separate embodiment of the invention. According to some embodiments, the linker comprises 12-35 nucleotides. According to some embodiments, the linker comprises 15-30 nucleotides. According to some embodiments, the linker comprises 15 nucleotides. According to some embodiments, the linker comprises the sequence GACGGGGGGCGGAAA (SEQ ID NO: 5). According to some embodiments, the linker comprises RNA nucleotides. According to some embodiments, the linker comprises DNA nucleotides. According to some embodiments, the linker comprises polyethyleneglycol (PEG). According to other embodiments, the linker comprises PEG-CA.

[0152] According to some embodiments, puromycin is covalently linked to the 3’ end of the mRNA display molecule. According to some embodiments, puromycin is covalently linked to the 3’ end of the linker. Variations of puromycin, such as, for example, as C-puromycin and CC-puromycin, which more fully mimic the aminoacylated 3 ’-CCA end of tRNA are also contemplated.

[0153] According to some embodiments, the mRNA display library molecule comprises the sequence of SEQ ID NO: 27. According to some embodiments, the mRNA display library molecule comprises the sequence of SEQ ID NO: 27 with puromycin linked to the 3’ end.

[0154] According to some embodiments, the present invention provides a method for identifying a cyclic peptide comprising D-amino acids capable of binding to a target polypeptide, the method comprising preparing an in vitro translation system comprising aminoacyl-tRNAs in which a tRNA that responds to the first cyclization codon and a tRNA that responds to the second cyclization codon are each aminoacylated with amino acids capable of reacting together to form a cyclic peptide; and wherein the in vitro translation system does not include any other aminoacyl-tRNAs that respond to the first cyclization codon, to the second cyclization codon, or whose amino acid can interfere with the cyclization reaction.

[0155] According to some embodiments, the method comprises preparing an in vitro translation system comprising aminoacyl-tRNAs in which a tRNA that responds to the first cyclization codon and a tRNA that is responds to the second cyclization codon are each aminoacylated with amino acids capable of reacting together to form a cyclic peptide. According to some embodiments, the in vitro translation system does not include any other aminoacyl-tRNAs that respond to the first cyclization codon, to the second cyclization codon, or whose amino acid can interfere with the cyclization reaction.

[0156] As used herein, the terms “aminoacylate” “aminoacylated”, “aminoacylation”, “charging”, “charge”, “charged” and the like, describes the process of covalently linking an amino acid to a tRNA. The term “amino acid activation” may also be used. In cells, this process is mediated by aminoacyl-tRNA synthetases (aaRSs), enzymes that recognize the anticodon of specific tRNAs that encode for given amino acid and aminoacylate the correct amino acid onto its cognate tRNA, i.e., aminoacylating the correct tRNA with the correct amino acid, thereby ensuring translation fidelity. Separate, dedicated aaRSs are found for each amino acid. Thus, the term “cognate tRNA” or “cognate aa-tRNA” used herein, refers to an aminoacyl-tRNA charged with a naturally-occurring amino acid and with an anticodon that binds a codon that encodes for the same amino acid, as dictated by the genetic code.

[0157] According to the principles of the present invention, any tRNA may be used. According to some embodiments, a tRNA is transcribed into RNA from a DNA transcript. According to some embodiments, tRNAs are purified from cells, i.e., a tRNA pool. tRNAs that respond to specific codons may be purified from the tRNA pool using methods that are known in the art. According to some embodiments, the tRNA does not comprise post-transcriptional modifications. According to some exemplified embodiments, the tRNA is an engineered tRNA. Engineered tRNAs include, for example, tRNAs optimized for the incorporation of an unnatural amino acid into the growing peptide chain of the P-site -bound peptidyl-tRNA. According to some exemplified embodiments, the tRNA is an engineered tRNA that is transcribed into RNA from a DNA transcript.

[0158] According to some embodiments, the aminoacyl-tRNAs that that responds to the first and second cyclization codons are charged within the in vitro translation system. According to some embodiments, the in vitro translation system comprises orthogonal tRNA / aminoacyl- tRNA synthetase pairs that charge the orthogonal tRNAs that respond to the first and second cyclization codons with amino acids that are capable of reacting together to form a cyclic peptide. As used herein, orthogonal tRNA / aminoacyl-tRNA synthetase pairs are not recognized by the rest of the translational machinery of a given in vitro system and may be co-evolved for the orthogonal aaRS to charge the paired orthogonal tRNA with an unnatural amino acid of interest.

[0159] According to some embodiments, the in vitro translation system is a cell-free translation system. According to some embodiments, lysates of E. coli, rabbit reticulocytes, or wheat germ may be used. According to some exemplary embodiments, the in vitro translation system is a purified E. coli translation system.

[0160] According to some embodiments, the aminoacyl-tRNAs that respond to the first and second cyclization codons are provided to the in vitro translation system as pre-charged aminoacyl-tRNAs. According to some embodiments, pre-charged aminoacyl-tRNAs are prepared by a method selected from ribozyme, orthogonal tRNA / aminoacyl-tRNA synthetase pairs, and chemically synthesized aminoacyl-tRNAs. According to some embodiments, precharged aminoacyl-tRNAs are prepared by the ribozyme method. The ribozyme, also known as flexizyme, are engineered RNA catalysts that recognizes the 3 ’-CCA end of tRNA and an amino acid derivatized with either cyanomethyl ester or dintirobenzyl ester and catalyze tRNA charging. According to some embodiments, the pre-charged aminoacyl-tRNAs are prepared via a reaction comprising a ribozyme comprising the sequence SEQ ID NO: 6 or SEQ ID NO: 7. According to some embodiments, the tRNA that responds to the first cyclization codon and a tRNA that is responds to the second cyclization codon are each aminoacylated with amino acids capable of reacting together to form a cyclic peptide. Any cyclization reaction that may be effected by two reactive moieties of amino acids, whether natural or unnatural, acylated onto tRNAs are within the scope of the present invention. Non-limiting examples include alkyneazide cyclizations, disulfide linkages, diselenide linkages, lactam linkages between Lys and Glu side chains, and Diels-Alder type cyclizations. According to some embodiments, the amino acid encoded by one of the cyclization codons comprises a reactive thiol and the amino acid encoded by the other cyclization codon comprises an N-terminal 2-halogen-acetyl group, wherein the halogen is selected from chloro, bromo, and iodo. Each possibility represents a separate embodiment of the invention. According to some embodiments, the halogen is chloro. According to some embodiments, the cyclic peptide is cyclized by a thioether linkage. According to some embodiments, the thioether linkage is of the form -S-CH2-C(O)-.

[0161] It should be understood that the first cyclization codon and the second cyclization codon do not have to be the first and last codons that encode the cyclic peptide. Thus, according to some embodiments, the cyclic peptide can include at least one tail of non-cyclized amino acids.

[0162] According to some embodiments, the amino acids capable of reacting together to form a cyclic peptide are D-Cys and N-chloroacetyl-D-tyrosine (ClAc-D-Tyr). According to some embodiments, the aminoacyl-tRNAs that respond to the cyclization codons are ClAc-D-Tyr- tRNAIMcl, and D-Cys-tRNAProlE2ccA. According to some embodiments, the aminoacyl-tRNA that responds to the first cyclization codon is CIAc-D-Tyr-tRNAI Vkl. According to some embodiments, the aminoacyl-tRNA that responds to the second cyclization codon is D-Cys- tRNA^^ccA. It is to be understood that the amino acids capable of reacting together may be aminoacylated onto a tRNA that responds to the first cyclization codon or to the second cyclization codon. Thus, for example, the aminoacyl-tRNA that responds to the first cyclization codon can be D-Cys-Tyr-tRNA0^61and the aminoacyl-tRNA that responds to the second cyclization codon can be ClAc-D-Tyr-tRNA^^ccA.

[0163] According to some embodiments, the aminoacyl-tRNA whose amino acid can interfere with the cyclization reaction comprises an amino acid comprising a reactive thiol group. According to some embodiments, the aminoacyl-tRNA whose amino acid can interfere with the cyclization reaction is Cys-tRNACys. According to some embodiments, the in vitro translation system does not comprise L- Cys or L-Cys aminoacyl-tRNA synthetase (aaRS). According to some embodiments, the in vitro translation system does not comprise cysteine, Cys aminoacyl-tRNA synthetase, methionine, Met-aaRS, tryptophan, Trp-aaRS, glutamine, Gln-aaRS, lysine, Lys-aaRS, glutamic acid, and Glu-aaRS. According to some embodiments, the in vitro translation system does not comprise cysteine, Cys aminoacyl-tRNA synthetase, methionine, Met-aaRS, tryptophan, Trp-aaRS, glutamine, Gln-aaRS, lysine, Lys-aaRS, glutamic acid, Glu-aaRS and RF1.

[0164] According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least 108molecules. According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least IO10molecules. According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least 1012molecules. According to some embodiments, the library of cyclic peptides each conjugated to its progenitor mRNA comprises between 108and 1013molecules. Each possibility represents a separate embodiment of the invention.

[0165] According to some embodiments, the D-polypeptide analog is immobilized on a solid support. According to some embodiments, the D-polypeptide analog comprises a tag that allows for it to be immobilized on a solid support. Any tag known in the art, i.e., biotin, FLAG, His6, may be used. According to some embodiments, the tag may be linked to any residue of the D- polypeptide analog that does not affect binding of a cyclic peptide to the D-polypeptide analog. According to some embodiments, the tag is connected to the N-terminus or the C-terminus of the D-polypeptide analog. Each possibility represents a separate embodiment of the invention. According to some embodiments, the tag is connected to the N-terminus of the D-polypeptide analog. According to some embodiments, the D-polypeptide analog comprises a biotin tag. According to some embodiments, the biotin tag is connected to the N-terminus of the D- polypeptide analog. According to some embodiments, the solid support comprises avidin, e.g., streptavidin. According to some embodiments, the solid support comprises beads. According to some embodiments, the solid support comprises magnetic beads. According to some embodiments, the solid support comprises streptavidin beads. According to some embodiments, the solid support comprises magnetic streptavidin beads.

[0166] According to some embodiments, the reverse transcribed DNA is prepared before or after performing step (v). Each possibility represents a separate embodiment of the invention. According to some embodiments, the reverse transcribed DNA is prepared before performing step (v). According to some embodiments, the reverse transcribed DNA of step (vi) is used to enrich the cyclic peptide binder. According to some embodiments, the method comprises at least two rounds of enrichment. According to some embodiments, the method comprises at least three rounds of enrichment. According to some embodiments, the method comprises at least four rounds of enrichment. According to the principles of the present invention, the reserve transcribed DNA that encodes the isolated cyclic peptide binder is PCR amplified followed by transcription to RNA, for example, using T7 RNA polymerase. Puromycin is then covalently linked to the RNA to form the mRNA display library molecule. The mRNA display library molecules are then added to the in vitro translation system as described, for example, in step (iv) herein to form the cyclic peptide-mRNA conjugates followed by a binding assay to the D- polypeptide analog (step (v) herein). The repetition of these steps enriches the pool of cyclic peptides with binders to the D-polypeptide analog. As used herein, the terms “enrichment” and “rounds of enrichment” refer to the iterative rounds of using cDNA to form mRNA display library molecules to be translated to the cyclic peptide-mRNA conjugates followed by binding to the D-polypeptide analog. An indication of enrichment as used herein is a value of positivity recovery percentage vs. negative recovery percentage (P / N ratio). According to some embodiments, the cyclic peptides with the 30 highest P / N ratios are selected. According to some embodiments, the cyclic peptides with the 20 highest P / N ratios are selected. According to some embodiments, the cyclic peptides with the 10 highest P / N ratios are selected. According to some embodiments, the cyclic peptides with the 5 highest P / N ratios are selected.

[0167] According to some embodiments, the present invention provides a method for identifying a cyclic peptide comprising D-amino acids capable of binding to a target polypeptide, the method comprising translating the mRNA display library molecules obtained in step (ii) in the in vitro translation system of step (iii) and providing conditions for the amino acids incorporated in response to the cyclization codons to cyclize, to produce a library of cyclic peptides wherein each peptide is conjugated to its progenitor mRNA.

[0168] According to some embodiments, the cyclic peptide comprises at least 6 amino acid residues. According to some embodiments, the cyclic peptide comprises at least 8 amino acid residues. According to some embodiments, the cyclic peptide comprises between 8-40 amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cyclic peptide comprises between 8-30 amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cyclic peptide comprises between 8-20 amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cyclic peptide comprises between 8 and 17 amino acid residues. Each possibility represents a separate embodiment of the invention.

[0169] According to some embodiments, the cyclic peptide comprises D-amino acids. According to some embodiments, the cyclic peptide comprises at least two D-amino acid residues. According to some embodiments, the cyclic peptide comprises at least three, at least four, or at least five D-amino acid residues. According to some embodiments, the cyclic peptide comprises between 10-38 D-amino acid residues. According to some embodiments, the cyclic peptide comprises between 6-28 D-amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cyclic peptide comprises between 6-18 D-amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cyclic peptide comprises between 6 and 15 D-amino acid residues. Each possibility represents a separate embodiment of the invention. According to some embodiments, at least half of the residues are D-amino acid residues.

[0170] According to the principles of the present invention, the method provides cyclic peptides that comprise D-amino acid residues. However, linear peptides comprising D-amino acid residues are also within the scope of the present invention.

[0171] According to some embodiments, the cyclic peptide further comprises an additional unnatural amino acid residue. According to some embodiments, the term "unnatural amino acid" refers to an amino acid which is not a DNA-encoded amino acid (i.e., an amino acid which is one of the known 20 amino acids which are encoded for by nucleic acid sequences and are utilized by a ribosome in the translation process of protein synthesis) and is not a D-amino amino acid of the 19 chiral amino acids which are encoded for by nucleic acid sequences and are utilized by a ribosome in the translation process of protein synthesis. Unnatural amino acids may refer to amino acids that do not occur in nature or naturally-occurring amino acids that are not normally utilized by a ribosome in the translation process of protein synthesis (e.g., norvaline, ornithine, homocysteine, etc.). According to some embodiments, the unnatural amino acid is a P-amino acid. According to some embodiments, the unnatural amino acid is a cyclic P-amino acid. According to some embodiments, the P-amino acid is selected from (lS,2S)-2-aminocyclohexane carboxylic acid, (lR,2S)-2-aminocyclopentane carboxylic acid, and (lR,2R)-2-aminocyclopentane carboxylic acid. According to some embodiments, the method of the present invention comprises synthesizing a cyclic peptide having inverse stereochemistry to the at least one isolated cyclic peptide binder. It is to be understood that “inverse stereochemistry” means that all L-amino acid residues of the isolated cyclic peptide binder will be D-amino acid residues in the synthesized cyclic peptide. Likewise, all D-amino acid residues of the isolated cyclic peptide binder will be L-amino acid residues in the synthesized cyclic peptide. In addition, for example, a P-amino acid with the stereochemistry (1S,2S) in the isolated cyclic peptide binder will have the stereochemistry (1R,2R) synthesized cyclic peptide.

[0172] According to one embodiment, the present invention provides a method for identifying a cyclic peptide comprising a plurality of D-amino acids capable of binding to a target polypeptide, the method comprising synthesizing a D-polypeptide analog of the target polypeptide, or a binding domain thereof.

[0173] The term "target polypeptide " refers to any polypeptide that can function on its own (i.e., is stable without other domains) for which is it desired to identify a specific and potent cyclic peptide binder that comprises a plurality of D-amino acids. The polypeptide may be found in nature or a variant thereof. The polypeptide may also be designed. A “binding domain” of a target polypeptide refers to any domain or portion of the polypeptide to which a peptide can bind. Any protein domain or part thereof wherein a peptide can bind included within the scope of the invention. According to some embodiments, the binding domain is a catalytic domain. The target polypeptide or binding domain thereof may be a eukaryotic or prokaryotic- derived polypeptide. According to some embodiments, the target polypeptide or binding domain thereof is a eukaryotic-derived polypeptide. According to some embodiments, the target polypeptide or binding domain thereof is a human-derived polypeptide.

[0174] As used herein, the term "D-amino acid analog of a target polypeptide” or “D- polypeptide analog” refers to a polypeptide with the identical amino acid sequence as the target polypeptide, but wherein the amino acids (except for achiral glycine) possess inverted stereochemistry at the a-carbon of the amino acid. Thus, wherein the target polypeptide is made up of a specific sequence of amino acids of L-chirality and achiral glycine, the D-polypeptide analog is made up of the same specific sequence of amino acids, only of D-chirality and achiral glycine. According to some embodiments, the D-polypeptide analog is the enantiomer of the target polypeptide or binding domain thereof.

[0175] Any method known in the art may be used to synthesize the D-polypeptide analog. According to some embodiments, the D-polypeptide analog is synthesized by solid phase peptide synthesis (SPPS). SPPS is described in further detail hereinbelow. A limitation of SPPS is the size of polypeptides that may be synthesized by SPPS is between about 10-70 amino acids, thereby limiting the size of polypeptides that may be synthesized. Thus, a strategy for synthesizing a larger D-polypeptide analog by SPPS comprises synthesizing segments of the polypeptide followed by sequentially ligating the segments to obtain the full polypeptide. Any method known in the art may be used to ligate the segments. One of the most effective methods for synthesizing larger polypeptides is native chemical ligation (NCL), in which an ionized thiol group of an N-terminal Cys residue of an unprotected peptide reacts with a C-terminal thioester of a second unprotected peptide to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full-length ligation product having a native peptide bond at the ligation site.

[0176] According to some embodiments, the D- polypeptide analog is synthesized by solidphase peptide synthesis (SPPS). According to some embodiments, the D-polypeptide analog by fmoc-SPPS. According to some embodiments, the D-polypeptide analog is synthesized by a strategy of synthesizing segments of the D-polypeptide analog by SPPS, followed by a chemical ligation strategy that provides the full D-polypeptide analog. According to some embodiments, the D-polypeptide analog is synthesized by a strategy of synthesizing segments of the D- polypeptide analog by SPPS, in which the segments are capable of being combined sequentially by NCL to obtain the full D-polypeptide analog. According to some embodiments, the D- polypeptide analog is synthesized by a strategy of synthesizing segments of the D-polypeptide analog by SPPS, in which the segments are subsequently combined sequentially by NCL to obtain the full D-polypeptide analog.

[0177] According to some embodiments, the target polypeptide or binding domain thereof is a human matrix metalloproteinase. According to some embodiments, the target polypeptide is a human matrix metalloproteinase or a catalytically active variant thereof. According to some embodiments, the human matrix metalloproteinase is human matrix metalloproteinase 7 (MMP7). According to some embodiments, the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the sequence of SEQ ID NO: 1, or a catalytically active variant thereof having at least 80% sequence identity. According to some embodiments, the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the catalytic domain set forth in SEQ ID NO: 2, or a catalytically active variant thereof having at least 80% sequence identity. According to some embodiments, the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the catalytic domain set forth in SEQ ID NO: 2, or a catalytically active variant thereof having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity. Each possibility represents a separate embodiment of the invention. According to some embodiments, the target polypeptide is human MMP7 consisting of the catalytic domain set forth in SEQ ID NO: 2.

[0178] As used herein, the terms “matrix metalloproteinase 7”, “MMP7”, “matrilysin” and “matrilysin-1” are used interchangeably.

[0179] As used herein, the terms "mirror image", "enantiomer", "inverse stereochemistry" all relate to the chirality, or handedness, of an amino acid or a target polypeptide of interest.

[0180] The term “variant” as used herein refers to a polypeptide sequence that possesses some modified structural property of the wild type or parent protein. For example, the variant may be truncated at either the amino or carboxy terminus or both termini or may have one or more amino acids deleted, inserted and / or substituted. Each possibility represents a separate embodiment.

[0181] The term “amino acids” used in the invention are those that are natural, those that are available commercially or are available by routine synthetic methods. Natural coded amino acids and their derivatives are represented by either the one-letter code or three-letter codes according to IUPAC conventions. Typically, variants will include conservative substitutions of amino acids as known to those skilled in the art. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g., aliphatic, aromatic, positively charged, and negatively charged. These substitutions may enhance activity, stability and the like of the MMP7 native protein. One of skill in the art will recognize that individual substitutions, deletions or additions to the protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence are “conservatively modified variants” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0182] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T);

[0183] 2) Aspartic acid (D), Glutamic acid (E);

[0184] 3) Asparagine (N), Glutamine (Q);

[0185] 4) Arginine (R), Lysine (K), Histidine (H);

[0186] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0187] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0188] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0189] Solid Phase Peptide Synthesis

[0190] Solid phase peptide synthesis (SPPS) procedures are well known in the art and further described in "Solid-Phase Synthesis: A Practical Guide", Ed. Steven A. Kates and Fernando Albericio, CRC Press; 1st Edition (2000). A skilled artesian may synthesize any of the peptides of the present invention manually or by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. The methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis.

[0191] Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3- oxazolidinyl) phosphinic chloride (BOP-CI), benzotriazolyl-N-oxytrisdimethyl- aminophosphonium hexafluoro phosphate (BOP), 1 -oxo- 1 -chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.

[0192] The use of additional coupling reagents including, but not limited to: coupling reagents such as PyBOP (Benzo triazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP (Bromo-tris-pyrrolidino-phosphonium hexafluoro-phosphate), HBTU (2-(lH- B enzo triazole- 1-yl)- 1,1, 3, 3- tetramethyluronium hexafluoro-phosphate), TBTU (2-(lH- B enzo triazole- 1-yl)- 1,1, 3, 3 -tetramethyluronium tetrafluoroborate), may be also utilized for synthesizing the peptide compounds of the present invention.

[0193] Additional coupling chemistries may be used, such as pre-formed urethane-protected N-carboxy anhydrides (UNCA'S), pre-formed acyl halides most preferably acyl chlorides.

[0194] Such coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.

[0195] Synthetic peptides can be purified by preparative high performance liquid chromatography (HPLC) and the composition of which can be confirmed via amino acid sequencing, mass spectrometry (MS) or other techniques known in the art.

[0196] The present invention also provides, according to another aspect, a cyclic peptide that binds to human matrix metalloproteinase 7 (MMP7, matrilysin-1) and inhibits its activity. According to further embodiments, the cyclic peptide comprises D-amino acids and at least one P-amino acid.

[0197] According to some embodiments, the cyclic peptide that binds to human MMP7 and inhibits its activity comprises the amino acid sequence (D-Ser)-((77?,27?)-2- aminocyclohexylcarboxy)-(D-Arg)-(D-Tyr)-(D-Thr)-(D-Val)-(D-Phe)-(D-Val)-(D-Ala)-(D- Val)-(D-Tyr)-(D-Val)-(D-Ser) (SEQ ID NO: 8); or a derivative thereof comprising 1-3 modifications to SEQ ID NO: 8.

[0198] According to some embodiments, a derivative according to the present invention comprises substitutions, deletions or additions of 1 to 3 amino acids. In some embodiments, a derivative has at least about 80% identity to the sequence of the peptide of the invention, for example at least about 85%, at least about 90%, at least about 93%, at least about 94%, at least about 95% identity to the sequence of the cyclic peptide of the invention. According to some embodiments, the substitution may be conservative or non-conservative, as long as the substituted cyclic peptide has the same or improved binding to the target polypeptide as the cyclic peptide of the present invention.

[0199] According to some embodiments, the cyclic peptide that comprises SEQ ID NO: 8 further comprises an N-terminal and a C-terminal residues that form a cyclic peptide. According to some embodiments, the N-terminal and a C-terminal residue react to form a thioether bond.

[0200] According to some embodiments, the residues before cyclization are N-chloroacetyl-L- tyrosine and L-cysteine. According to some embodiments, the cyclic peptide comprises an N-terminal N- chloroacetyl-L-tyrosine and a C-terminal L-cysteine, before cyclization.

[0201] According to some embodiments, the cyclic peptide comprises the structure:

[0202] According to some embodiments, the cyclic peptide comprises SEQ ID NO: 9. According to some embodiments, the cyclic peptide consists of SEQ ID NO: 9.

[0203] According to some embodiments, the cyclic peptide comprises a C-terminus that is PEGylated, amidated, carboxylated, or substituted with an alcohol moiety. According to some embodiments, the cyclic peptide comprises a C-terminus that is PEGylated.

[0204] According to some embodiments, the present invention provides a conjugate comprising the cyclic peptide and a moiety that improves a property of the cyclic peptide, including but not limited to solubility, stability and / or permeability. A conjugate according to the present invention comprises any cyclic peptide of the present invention, conjugated to a carrier peptide, protein or another moiety which improves the peptide’s solubility, stability or permeability. According to some embodiments, a cyclic peptide identified by the method of the present invention can be provided as a conjugate comprising the cyclic peptide and a moiety that increases solubility, stability and / or permeability.

[0205] Water soluble, synthetic polymers, particularly polyethyleneglycols, are widely used to conjugate therapeutically active molecules such as peptides. These therapeutic conjugates have been shown to alter pharmacokinetics favorably by prolonging circulation time and decreasing clearance rates, decreasing systemic toxicity, and in several cases, displaying increased clinical efficacy. The process of covalently conjugating polyethylene glycol, PEG, to proteins is commonly known as "PEGylation". Where appropriate, the abbreviation PEG is used in combination with a numeric suffix, which indicates the number of repeating ethylene glycol units. Other moieties, such as hydrophilic amino acid and sequences may alternatively be used.

[0206] According to some embodiments, the cyclic peptide or conjugate comprises a solubility tag. According to some embodiments, the solubility tag may be any tag that improves solubility without adversely affecting binding of the cyclic peptide or conjugate thereof to the target. According to some embodiments, the solubility tag is selected from a PEG tag and a polyionic tag. According to some embodiments, the tag is a poly-Arg tag. According to some embodiments, the solubility tag is a PEG tag.

[0207] As used herein "PEG" refers to any compound including at least one polyethylene glycol moiety. PEGs exist in linear forms and branched forms comprising a multi-arm and / or grafted polyethylene glycols. A PEG may be modified by alkylation of the terminal hydroxy group. A PEG may further comprise a functional group. A PEG may be mono-, di-, or multifunctional polyethylene glycols.

[0208] According to some embodiments, the conjugate comprises a cyclic peptide of the present invention attached to a PEG moiety. According to some embodiments, the moiety is PEG. According to some embodiments, the conjugate of the cyclic peptide comprises a PEG moiety attached to its C-terminus.

[0209] According to some embodiments, the conjugate of the cyclic peptide comprises 1-40 linear PEG units attached to its C-terminus. Each possibility represents a separate embodiment of the invention. According to some embodiments, the conjugate of the cyclic peptide comprises 2-40 linear PEG units attached to its C-terminus. According to some embodiments, the conjugate of the cyclic peptide comprises 2-35 linear PEG units attached to its C-terminus. According to some embodiments, the conjugate of the cyclic peptide comprises 2-33 linear PEG units attached to its C-terminus. According to some embodiments, the conjugate of the cyclic peptide comprises 4-30 linear PEG units attached to its C-terminus. According to some embodiments, the conjugate comprises a linear PEG of 6 units attached to its C-terminus. According to some embodiments, the conjugate comprises a linear PEG of 27 units attached to its C-terminus.

[0210] According to some embodiments, the conjugate of the cyclic peptide has the structure:

[0211] According to some embodiments, the conjugate of the cyclic peptide has the structure:

[0212] (D’20-PEG27).

[0213] According to some embodiments, the cyclic peptide or conjugate binds human MMP7 comprising SEQ ID NO: 1 or a catalytically active variant or fragment thereof with at least 80% sequence identity thereto.

[0214] According to some embodiments, the cyclic peptide binds the catalytic domain of human MMP7 comprising the sequence SEQ ID NO: 2 or a catalytically variant thereof with at least 80% sequence identity thereto.

[0215] Pharmaceutical compositions According to some embodiments, the present invention provides a pharmaceutical composition comprising a cyclic peptide or conjugate and an acceptable salt, diluent or carrier.

[0216] The compounds of the present invention can be formulated into various pharmaceutical forms for purposes of administration. The pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant, and a mixture thereof, as detailed for example in "Handbook of Pharmaceutical Excipients"; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA. For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier. Such a carrier can depend on the route of administration, such as oral, rectal, percutaneous, or parenteral injection. A "carrier" as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. For example, in preparing the compositions in oral dosage form, media such as water, glycols, oils, and alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions. Alternatively, solid carriers such as starches, sugars, kaolin, lubricants, binders, and disintegrating agents can be used, for example, in powders, pills, capsules, or tablets.

[0217] The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art, e.g., diluents such as lactose (Pharmatose DCL 21), starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agent; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants / antiadherants; chelating agents; vehicles; bulking agents; stabilizers; preservatives; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; chelating agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof. The disintegrants useful in the present invention include but not limited to starch or its derivatives, partially pregelatinized maize starch (Starch 1500®), croscarmellose sodium, sodium starch glycollate, clays, celluloses, alginates, pregelatinized corn starch, crospovidone, gums and the like used either alone or in combination thereof. The lubricants useful in the present invention include but not limited to talc, magnesium stearate, calcium stearate, sodium stearate, stearic acid, hydrogenated vegetable oil, glyceryl behenate, glyceryl behapate, waxes, Stearowet, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate and the like used either alone or in combination thereof. The anti-adherents or glidants useful in the present invention are selected from but not limited to a group comprising talc, corn starch, sodium lauryl sulfate, and magnesium, calcium, and sodium stearates, and the like or mixtures thereof. In another embodiment of the present invention, the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol. In an embodiment of the present invention, the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, and / or tocopherol. In another embodiment, the dosage form of the present invention additionally comprises at least one wetting agent(s) such as a surfactant selected from a group comprising anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, or mixtures thereof. The wetting agents are selected from but not limited to a group comprising oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate and the like, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises at least one complexing agent such as cyclodextrin selected from a group comprising but not limited to alpha-cyclodextrin, betacyclodextrin, betahydroxy-cyclodextrin, gammacyclodextrin, and hydroxypropyl betacyclodextrin, or the like. In yet another embodiment, the dosage form of the present invention additionally comprises of lipid(s) selected from, but not limited to, glyceryl behenate such as Compritol® ATO888, Compritol® ATO 5, and the like; hydrogenated vegetable oil such as hydrogenated castor oil e.g. Lubritab®; glyceryl palmito stearate such as Precirol® ATO 5 and the like, or mixtures thereof used either alone or in combination thereof. It will be appreciated that any given excipient may serve more than one function in the compositions according to the present invention.

[0218] For parenteral compositions, the carrier can comprise sterile water. Other ingredients may be included to aid in solubility. Injectable solutions can be prepared where the carrier includes a saline solution, glucose solution, or a mixture of both. Injectable suspensions can also be prepared. In addition, solid preparations that are converted to liquid form shortly before use can be made. For percutaneous administration, the carrier can include a penetrationenhancing agent or a wetting agent.

[0219] It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier. Apart from other considerations, the fact that the novel active ingredients of the invention are peptides, peptide analogs, or peptidomimetics, dictates that the formulation be suitable for delivery of these types of compounds.

[0220] According to some embodiments, the pharmaceutical composition is formulated for enteral or parenteral administration. According to some embodiments, the pharmaceutical composition is formulated for intravenous, oral, intra-articular, intramuscular, subcutaneous, topical, transdermal, intradermal, nasal or intrathecal administration. Each possibility represents a separate embodiment of the invention. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. According to some embodiments, the pharmaceutical composition is formulated for oral administration.

[0221] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a cyclic peptide or conjugate described herein, and optionally, a pharmaceutically acceptable carrier, excipient or diluent.

[0222] The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the cyclic peptide or conjugate according to the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0223] The amount of the cyclic peptide or conjugate which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disease or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0224] It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the cyclic peptide or conjugate molecules according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a “therapeutically effective amount” refers to the amount of a molecule required to alleviate one or more symptoms associated with a disease or condition being treated over a period of time.

[0225] Although an appropriate dosage of a molecule of the invention varies depending on the administration route, type of molecule, age, body weight, sex, or conditions of the patient, it will be determined by the physician in the end. The dosage can be administered, for example, in daily, weekly, biweekly, monthly or bimonthly regimens. Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

[0226] Toxicity and therapeutic efficacy of the peptidomimetic and prodrugs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0227] According to some embodiments, the cyclic peptide or conjugate binds the catalytic domain of human MMP7 with an IC50 of at least 1 nM. According to some embodiments, the cyclic peptide or conjugate binds the catalytic domain of human MMP7 with an IC50 of at least 10 nM. According to some embodiments, the cyclic peptide or conjugate binds the catalytic domain of human MMP7 with an ICsoof at least 100 nM. According to some embodiments, the cyclic peptide binds the catalytic domain of human MMP7 with an ICsoof 100-200 nM.

[0228] The preferred doses for administration of such pharmaceutical compositions range from about 0.1 p g / kg to about 20 mg / kg body weight, of the active ingredient. Preferably, the amount of the active ingredient is in the range of from about 10 to 5000 pg / kg.

[0229] In some embodiments, the cyclic peptide or conjugate of the invention are administered at a dose ranging from about 20 ng / kg to about 100 ng / kg of the subject weight. In other embodiments, the peptide of the invention is administered at a dose ranging from about 0.1 mg / kg to about 10 mg / kg of the subject weight. In yet other embodiments, the peptide of the invention is administered at a dose ranging from 0.1, 1, 10, 20, 30, 50, 100, 200, 400, 500, 700, 900 or 1000 ng / kg of the subject weight, to about 100, 200, 400, 500, 700, 900, 1000, 1200, 1400, 1700, or 2000 ng / kg of the subject weight. Each possibility represents a separate embodiment of the invention. In yet other embodiments, the peptide of the invention is administered at a dose ranging from about 0.01, 0.05, 0.1, 0.5, 0.7, 1, or 2 mg / kg of the subject weight, to about 0.05, 0.1, 0.5, 0.7, 1, 2, 5, 10, 15, 20, 50, 100, 250, or 500 mg / kg of the subject weight. Each possibility represents a separate embodiment of the invention.

[0230] Methods of Treatment

[0231] According to some embodiments, the pharmaceutical composition is for use in inhibiting an activity of MMP7.

[0232] According to some embodiments, the pharmaceutical composition is for use in preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7.

[0233] According to some embodiments, the disease or disorder is selected from a cancer or a tumor a kidney disease, disorder or injury, angiogenesis related disorders, including but not limited to idiopathic pulmonary fibrosis and biliary atresia.

[0234] According to some embodiments, the use involves at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

[0235] According to some embodiments, the present invention provides a method of preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7, comprising administering to a subject in need a pharmaceutical composition as disclosed herein.

[0236] According to some embodiments, the disease or disorder is selected from a cancer or a tumor a kidney disease, disorder or injury, angiogenesis related disorders, including but not limited to idiopathic pulmonary fibrosis and biliary atresia.

[0237] According to some embodiments, the cancer is a solid cancer or a metastatic cancer. According to some embodiments, the cancer is a solid cancer. According to some embodiments, the cancer is a metastatic cancer. According to some embodiments, the solid cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, gastric cancer, esophageal cancer, gallbladder cancer, and brain cancer.

[0238] According to some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

[0239] According to some embodiments, the cancer is hematologic cancer.

[0240] According to some embodiments, the pharmaceutical composition is administered before, together with, or following, an additional anti-cancer treatment.

[0241] According to some embodiments, the additional anti-cancer treatment is selected from the group consisting of surgery, chemotherapy, radiotherapy, and immunotherapy.

[0242] According to some embodiments, the method results in at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

[0243] The terms “comprise”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0244] The term “consisting of’ means “including and limited to”.

[0245] The term “consisting essentially of’ means that the composition, method, or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0246] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0247] The term “about” as used herein refers to a numeric value ± 10%, or according to some embodiments, ± 5%.

[0248] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and / or" and not the exclusive sense of "either / or." Throughout this application, various embodiments of this invention may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0249] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0250] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

[0251] EXAMPLES

[0252] Example 1: Total chemical synthesis of L- and £)-enantiomers of the catalytic domains of MMP7

[0253] Materials and Methods

[0254] All Fmoc-L-amino acids, and Boc-L-Thz-OH (Boc-L-thiazoline-4-carboxylic acid) were obtained either from CS Bio Co. (Menlo Park, CA) or Matrix innovation (Quebec City, Canada), while Fmoc-D-amino acids and Boc-D-Thz-OH were obtained from Chem-Impex, all of which with the following side chain protecting groups: Arg(Pbf), Asp(OtBu), Glu(OtBu), Ser(tBu), Thr(tBu), Cys(Trt), Lys(Boc), Tyr(tBu), Asn(Trt). (Pbf = 2, 2, 4, 6, 7- pentamethy 1-2,3 - dihydrobenzofuran-5-sulfonyl). TentaGel® R RAM resin (loading 0.18 mmol / g), and chlorotrityl resin (loading 0.5 mmol / g) were purchased from Rapp Polymer GmbH (Germany), GL Biochemical (China) or Chem-Impex (USA). All P-amino acids, Fmoc-(lS,2S)-2-aminocyclopentane carboxylic acid, Fmoc-(lS,25')-2- aminocyclohexane carboxylic acid, Fmoc-cA-(lR,25')-2-aminocyclopentane carboxylic acid, and Fmoc-(lR,2R)-2-aminocyclohexane carboxylic acid were obtained from Chem-Impex. Recombinant human catalytic domain of MMP7 was obtained from Giotto Biotech, Italy. Recombinant MMP1, MMP8, MMP9, MMP10 and MMP14 were obtained from the Shifman lab, The Hebrew University of Jerusalem.

[0255] Buffer for ligation reactions was prepared using MilliQ water (Millipore, Merck). Ultrapure guanidinium chloride (GmHCl, MP Biomedicals, LLC, France) was used in all ligation buffer. NaiHPO-r I2H2O, tris(2-carboxyethyl)phosphine hydrochloride (TCEP • HC1), 4-mercaptophenylacetic acid (MPAA), methyl 3 -mercaptopropionate (MMP), triisopreopylsilane (TIPS), tert-butyl mercaptane, acetylacetone (Acac), Fmoc-Gly- Thr(psiMe,Mepro)-OH, D-biotin, MMP7 substrate FS-6, human serum, and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (Merck, Israel). AA,A’,A’-Tetramethyl-O- (6-chloro-lH-benzotriazol-l-yl)uronium hexafluorophosphate (HCTU), 1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoropho sphate (H ATU) , benzo triazol- 1 -y loxy tripyrrolidinopho sphonium hexafluorophosphate (PyBOP) and ethylcyano(hydroxyimino)acetate (OxymaPure) were purchased from Luxembourg Biotechnologies Ltd. (Rehovot, Israel). Fmoc-NH-PEGe- CH2CH2COOH, Fmoc-NH-PEG27-CH2CH2COOH and A-Succinimidyl 2-chloroacetate were obtained from Holland Moran. All solvents: AA-dimethylformamide (DMF), N-methyl-2- pyrollidone (NMP), dichloromethane (DCM), acetonitrile (ACN), AA-diisopropylethyl amine (DIEA), piperidine (Pip), diethyl ether (Et2O) and trifluoroacetic acid (TFA) were purchased from Bio-Lab (Jerusalem, Israel) and were peptide synthesis, HPLC or UPLC-grade.

[0256] High Performance Liquid Chromatography (HPLC)- The analytical reverse phase- HPLC (RP-HPLC) and semi-preparative RP-HPLC were performed on a reverse-phase Waters Alliance HPLC with a UV detector (detection was set at 220 nm and 280 nm) using an X-Bridge C4 column (300 A, 3.5 pm, 4.6 xl50 mm) and C18 column (3.5 pm, 130 A, 4.6 x 150 mm), respectively. Preparative RP-HPLC was performed on a Waters LCQ150 system (XBridge C4 column, 5 pm, 19 x 250 mm). Linear gradients of MeCN with 0.1% TFA (buffer B) and water with 0.1% TFA (buffer A) were used for all systems to elute peptides. The flow rates were 1 mL / min (analytical), 3.34 mL / min (semi-preparative) and 10 mL / min (C4 preparative).

[0257] Mass spectrometry (MS)- Electron Spray Ionization MS (ESLMS) was performed on a

[0258] Thermo Scientific-LCQ Fleet Ion-Trap mass spectrometer. Peptide masses were calculated from the experimental mass to charge (m / z) ratios from the observed multiply-charged species of a peptide using MagTran vl.03 software.

[0259] General procedure for Fmoc-SPPS- Peptides were prepared by using an automated peptide synthesizer (CS136XT, CS Bio Inc., CA, USA), typically on 0.25 mmol scale for L- MMP7 and 0.125 mmol scale for D-MMP7. Fmoc-amino acids (1 mmol in 5 mL DMF, 4 equiv.) were activated with HCTU or HATU (1 mmol in 5 mL DMF, 4 equiv.) and DIEA (2 mmol in 5 mL of DMF, 8 equiv.) for 5 min and coupled for 25 min, with constant shaking. The Fmoc deprotection step was carried out with a solution of 0.1 M Oxyma Pure (Ethyl cyano(hydroxyimino)acetate, Novabiochem) and 20% piperidine in DMF for 2 x 10 min, and 0.4 M LiCl in DMF was used for washing the resin. The resulting resins were washed with DMF (3x), DCM (3x) and dried.

[0260] Deprotection and cleavage from the resin- Peptides were cleaved off resin using a TFA:triisopropylsilane (TIPS):H2O (95:2.5:2.5) cocktail for 2.5 h at room temperature. The cleavage mixture was filtered, and the resin was washed with TFA. The combined solutions were concentrated by N2 bubbling, to which cold ether was added. The precipitated crude peptides were centrifuged, the ether was removed, and the crude peptides were dissolved in a solution of MeCN: water (1: 1) containing 0.1% TFA and lyophilized.

[0261] Loading of 2 -chlorotrityl chloride resin- 0.25 mmol of 2-chlorotrityl chloride resin was washed with DMF and DCM, then swelled with 10 mL DMF for 1 h. Next, 1 mmol of amino acid and 1 mmol of DIEA in 5 mL DMF was added to the resin and coupling was allowed to proceed for 1 h. The coupling step was then performed one more time. After this, the unreacted sites on the resin were capped by using 5 mL of 10% MeOH in DMF for 10 min (this step was performed 2x). Finally, the resin was washed well with DMF (3x) followed by DCM (3x).

[0262] Synthesis of Cl-Trt-NHNFh (hydrazide-functionized) resin- 2-chlorotrityl chloride resin (0.5 mmol / g) was swollen in DMF for 1 h and treated twice with freshly prepared 10% hydrazine in DMF for 30 min, then drained. The resin was washed well with DMF and then treated twice with 10% MeOH in DMF for 15 min.

[0263] The selection method of the present invention utilizes the novel mirror-image system disclosed herein to perform affinity selection against the mirror-image form of a target protein (wherein the target protein consists of D-amino acids and achiral glycine). Following the requisite rounds of selection, the mirror-image forms of the identified cyclic peptide binders are subsequently synthesized and used to target the corresponding L-protein. The steps of a representative method of the present invention, and those used in the Examples herein, are depicted in Fig. 1.

[0264] As a proof of principle to demonstrate that a highly selective, potent, and stable binder could be selected using the present invention, human matrix metalloproteinase 7 (MMP7) was selected as the target protein. The catalytic domain of MMP7 was chemically synthesized as the biotinylated L-enantiomer (L-MMP7cat) and D-enantiomer (D-MMP7cat). A cyclic peptide library containing P-amino acids was then screened against L-MMP7catand D-MMP7cat(as described in Example 3 hereinbelow).

[0265] The primary sequence of the catalytic domain of human MMP7 (SEQ ID NO: 1, Uniprot accession code P09237) consists of 173 amino acids, MMP7(95-267) (MMP7cat, SEQ ID NO: 2)). Since this domain does not carry any cysteine residues, the synthetic approach was based on Fmoc-solid phase peptide synthesis (Fmoc-SPPS) of four peptide segments that were combined by three native chemical ligation (NCL) reactions at the Lysl31-Alal32, Hisl78- Alal79, and Ala211-Ala212 junctions (shown as underlined and bolded in Fig. 2A, SEQ ID NO: 2), with temporary substitution of three Ala residue (A132, A179, and A212) with protected Cys in the form of Thz (Z) to facilitate native chemical ligation (NCL) reactions followed by a final desulfurization step.

[0266] The corresponding C-terminal segment MMP7(212-267)(A212C) was synthesized by standard Fmoc-SPPS. MMP7(179-211)(A179Z)-carboxy thioester (COSR), MMP7(132- 178)(A132Z)-COSR, and the N-terminally biotinylated (biotin is required for immobilization) MMP7(95-131)-COSR were prepared by Fmoc-SPPS as C-terminal thioester surrogates. All Met residues were replaced with norleucine (Nle) to avoid oxidation. The chemical synthesis approach is depicted in Fig. 2B.

[0267] It is to be understood that the same approach was taken for the synthesis of L-MMP7catand D-MMP7cat, the only difference being the scale of Fmoc-SPPS, which was typically on a 0.25 mmol scale for L-MMP7catand on a 0.125 mmol scale for D-MMP7cat, as described in the Materials and Methods. Therefore, the synthesis described hereinbelow applies equally for both enantiomers and only the yields of each enantiomer are described separately where relevant.

[0268] Synthesis of MMP7(212-267)

[0269] The synthesis of MMP7(212-267)(A212C) was carried out on 2-chlorotrityl chloride resin (0.5 mmol / g) on an automated peptide synthesizer, as described in the Material and Methods. The first amino acid Lys267 was loaded as described in the Material and Methods hereinabove and subsequent steps were completed with standard Fmoc-SPPS. After peptide chain assembly, the peptide was cleaved according to the general procedure described in the Material and Methods and lyophilized. The corresponding peptide MMP7(212-267)(A212C) was obtained in 65% yield (251 mg for L-MMP7 (212 -267) and 50 mg for D-MMP7 (212 -267)) by preparative RP-HPLC using a gradient of 5% B over 1 min followed by 25-50% B over 40 min. The products were characterized by ESI-MS.

[0270] Synthesis of MMP7( 179-211 )(A179Thz)-COSR

[0271] The hydrazide-functionalized chlorotrityl resin was used for standard Fmoc-SPPS (described in the Material and Methods), where the coupling of the amino acids was carried out on an automatic synthesizer. Gly203 was manually coupled as (Dmb)Gly (1.5 equiv. Fmoc- (Dmb)Gly-OH, 1.5 equiv. HATU and 3 equiv. DIEA) and Asp202 was doubly coupled to prevent aspartimide formation. Moreover, 5 equiv. of Boc-Thz-OH was activated (using 5 equiv. of 0.4 M HCTU and 10 equiv. of 0.8 M DIEA in DMF) and coupling allowed to proceed for 2 h. The cleavage was performed as described in the Material and Methods hereinabove to afford 300 mg of L-MMP7(179-211)(A179Thz)-CONHNH2and 150 mg of D-MMP7(179- 21 l)(A179Thz)-CONHNH2.

[0272] The conversion to thioester was performed by dissolving the peptide in phosphate buffer (200 mM, 6 M Gn • HC1, pH -2.5) and treating with 10 equiv. of acetylacetone (acac) and 50 mM of MPAA for 2h at room temperature. Subsequent purification by RP-HPLC (C4 column) was performed using a linear gradient of 30-60% ACN with 0.1% TFA over 40 min to obtain 180 mg of L-MMP7(179-211)(A179Thz)-COSR and 30 mg of D-MMP7(179-211)(A179Thz)- COSR (60% and 20% yield, respectively). The products were characterized by ESI-MS.

[0273] Synthesis ofMMP7(132-l 78)(A132Thz)-COSR

[0274] The synthesis MMP7(132-178)(A132Thz)-COSR, was carried out on chlorotrityl resin, using the hydrazide-functionalized chlorotrityl resin and standard Fmoc-SPPS, wherein the coupling of the amino acids was carried out on an automatic synthesizer. All Met residues were replaced with Nle. Glyl71 was manually coupled as (Dmb)Gly (1.5 equiv. Fmoc-(Dmb)Gly- OH, 1.5 equiv. HATU and 3 equiv. DIEA) and Asp 171 was doubly coupled to prevent aspartimide formation. Glyl50 and Thrl51 were coupled as pseudoproline dipeptides, (1.5 equiv. of Fmoc-Gly-Thr(psiMe, Mepro)-OH, 1.5 equiv. HATU and 3 equiv. DIEA) and coupled for 2 hours. Alal31 was replaced by Boc-Thz-OH (5 equiv.) which was activated using 5 equiv. of 0.4 M HCTU and 10 equiv. of 0.8 M DIEA in DMF and coupling was allowed to proceed for for 2 h. The cleavage was performed as described in the Material and Methods section hereinabove to afford 290 mg of L-MMP7(132-178)(A132Thz)-CONHNH2and 120 mg of D- MMP7(132-178)(A132Thz)-CONHNH2.

[0275] The conversion to the thioester was performed by separately dissolving the peptides in phosphate buffer (200 mM, 6 M Gn • HC1, pH ~2.5) and treating with 10 equiv. of acetylacetone (acac) and 50 mM of MPAA for 2h at room temperature. Subsequent purification by RP-HPLC (C4 column) was performed using a linear gradient of 30-60% ACN with 0.1% TFA over 40 min to obtain 18 mg of L-MMP7(132-178)(A132Thz)-COSR and 10 mg of D- MMP7(132-178)(A132Thz)-COSR (6% and 9% yield, respectively). The products were characterized by ESI-MS.

[0276] Synthesis of biotin labeled L- and D-MMP7(95-131 )-COSR

[0277] Biotin MMP7(95-131)-Nbz was synthesized on Fmoc-Dbz-resin. 0.25 mmol of TentaGel Ram resin (0.19 mmol / gr) was used for the synthesis. Mono-Fmoc-3,4-diaminobenzoic acid (Fmoc-Dbz-OH, 3 equiv.) was activated with HCTU (3 equiv.) and DIEA (6 equiv.) in DMF and coupled manually to the free amine of the resin for 2 h. The synthesis was completed on an automatic peptide synthesizer. Biotin (5 equiv.) was coupled manually using PyBop (5 equiv) / DIEA (10 equiv) in DMF.

[0278] On-resin Nbz formation was performed by treating the peptide-Dbz-resin with a solution of p-nitrophenyl chloroformate (5 equiv.) in DCM and shaken for 1 h at room temperature. Following this, the resin was washed and a solution of 0.5 M DIEA in DMF and shaken for an additional 1 h to complete Nbz formation (repeated twice). Finally, the peptide-resin was washed using DCM and dried under vacuum. Cleavage was performed as described in the Material and Methods section hereinabove to afford 400 mg of L-MMP7(95-131)-Nbz and 180 mg of D-MMP7(95-131)-Nbz.

[0279] The conversion to thioester was performed by dissolving the peptide in phosphate buffer (200 mM, 6 M Gn • HC1, pH -7) to a final concentration of 2mM and treating with 5 % (v / v) methyl 3 -mercaptopropionate (MMP) for 2 h at room temperature. The peptide was then purified by RP-HPLC (C4 column) using a linear gradient of 25-50% ACN with 0.1% TFA over 40 min to obtain 40 mg of L-MMP7(95-131)-COSR and 12 mg of D-MMP7(95-131)- COSR (10% and 7% yield, respectively). The product was characterized by ESI-MS.

[0280] First native chemical ligation reaction between MMP7(179-211)(A179Thz)-COSR and

[0281] MMP7(212-267) MMP7(212-267)(A212C) (30 mg, 4.8 pmol, 1.1 mM) was dissolved in 5 mL phosphate buffer (200 mM, 6 M Gm HC1, 50 mM TCEP, pH 7) and the reaction mixtures were added to MMP7(179-211)-COSR (16.5 mg, 4.4 pmol, 1 mM). The reaction progress was monitored by HPLC with a gradient of 5% B over 1 min followed by 5-70% B over 20 min and the reaction reached completion after 2 h. Thz opening was performed by addition of 0.5 mL of 2 M McONHi to a final concentration of 0.2 M and reacted overnight at room temperature. The product was then isolated in 35% yield (18 mg) by preparative HPLC (C4 column) and characterized by ESLMS.

[0282] Second native chemical ligation reaction between MMP7(132-178)(A132Thz)-COSR and MMP7(179-267)(Al 79C,A212C)

[0283] MMP7(179-267)(A179C, A212C) (18 mg, 1.85 pmol, 1.1 mM) was dissolved in 2 mL phosphate buffer (200 mM, 6 M Gm HC1, 50 mM TCEP, pH 6.5) and the reaction mixtures were added to MMP7(132-178)(A132Thz)-COSR (10 mg, 1.8 pmol, 1 mM). The reaction progress was monitored by HPLC with a gradient of 5% B over 1 min then 25-55% B over 20 min; the reaction reached completion after 8 h. Thz opening was performed by addition of 0.4 mL of 2 M McONHi to a final concentration of 0.2 M and reacted overnight at room temperature. Then the product was isolated in 36 % yield (10 mg) by semi-preperative HPLC (C4 column) and characterized by ESLMS.

[0284] Third native chemical ligation reaction between biotin-MMP7(95-131)-COSR and MMP7(132-267)(A212C,A179C,A132C)

[0285] MMP7(132-267)(A212C, A179C, A132C) (10 mg, 0.67 pmol, 1.1 mM) was dissolved in 0.7 mL phosphate buffer (200 mM, 6 M Gm HC1, 50 mM TCEP, pH 7) and the reaction mixtures were added to biotin-MMP7(95-131)-COSR (3 mg, 1.8 pmol, 1 mM). The reaction progress was monitored by HPLC with a gradient of 5% B over 1 min then 25-55% B over 20 min; the reaction reached completion after 24 h. The product then was isolated in 39% yield (5 mg) by semi preparative HPLC (C4 column) and characterized by ESLMS.

[0286] Desulfurization of Biotin MMP7(95-267)(A212C,A179C,A132C) and chemical characterization of biotin-L-MMP7cat and biotin-D-MMP7cat

[0287] Biotin-MMP7(95-267)(A212C,A179C,A132C) (5 mg, 1 mM) was dissolved in 500 pL phosphate buffer (200 mM, pH 5) with 600 equiv. TCEP, 60 equiv. of VA-044 (2,2'-Azobis[2- (2-imidazolin-2-yl)propane] Dihydrochloride) and 10 pL of tert-butyl mercaptane. The reaction was performed under argon and allowed to proceed overnight at 37°C. Reaction progress was monitored by HPLC with a gradient of 30-60% B over 20 min; the reaction reached completion after 24 hours.

[0288] The final products, L-MMP7catand D-MMP7catwere isolated in 60% yield for L- (3 mg) and 30% yield (1.5 mg), respectively, by semi-preparative HPLC (C4 column) characterized by ESLMS (HPLC chromatograms and ESLMS spectra of L-MMP7catand D-MMP7catare shown in Figs. 2C-2D, respectively) (henceforth referred to as L-MMP7catand D-MMP7cat). As shown in the chromatograms of Figs. 2C-2D, L-MMP7catand D-MMP7cathad the same retention time, further demonstrating that the two polypeptides are enantiomers. For the ESLMS of L- MMP7cat, the calculated mass was 19284.7 Da and the found mass was 19280.0+2.8 Da; for the ESLMS of D-MMP7cat, the calculated mass was 19284.7 Da and the found mass was 19282.1+5.1 Da. The ESLMS spectra confirmed the identity of both products. Thus, L- MMP7catand D-MMP7catwere successfully synthesized with high yield and purity.

[0289] Example 2: Biochemical characterization of £-MMP7catand D-MMP7cat

[0290] To further characterize L-MMP7catand D-MMP7cat, both enantiomers were folded in 50 mM TRIS-HC1 buffer (pH 7), 10 mM CaCh, 0.1 mM Zn acetate and 5% v / v glycerol at 4 °C for 4 h.

[0291] Subsequently, the structure of both proteins was analyzed by circular dichroism (CD). Far ultraviolet spectra were obtained from 195-260 nm on a J- 180 spectropolarimeter (Jasco) using 20 pM of L-MMP7catand 20 pM of D-MMP7cat. Each spectrum represents the average of 3 scans, and the mean residue ellipticity (OMRE) was calculated according to the following equation:

[0292] The D-MMP7catexhibited an inverted spectrum of L-MMP7cat, demonstrating that the two polypeptides are enantiomers (Fig. 3A).

[0293] The catalytic activity of L-MMP7catwas then determined and compared with the catalytic activity of recombinant MMP7 (Giotto Biotech, Italy) by monitoring the hydrolysis of the general MMP Anorogenic peptide substrate FS-6 (SEQ ID NO: 28) at Xext = 320 nm Xem= 400 nm, following the established protocol (Neumann, U. et al., Anal. Biochem. 2004, 328, 166). As shown in Fig. 3B, L-MMP7catexhibited a similar activity profile compared with recombinant MMP7.

[0294] Example 3: Assessment of cyclic peptide library design by translation of model peptides in the FIT system

[0295] The cyclic peptide library used in the system of the present invention to screen biotinylated L- and D-MMP7catwas designed to incorporate a cyclic peptide library containing one of three P-amino acids, namely (lS,2S)-2-aminocyclohexane carboxylic acid ((15,25)-2- ACHC), (lR,2S)-2-aminocyclopentane carboxylic acid ((lR,2S)-2-ACPC) and (lR,2R)-2- aminocyclopentane carboxylic acid ((lR,2R)-2-ACPC), and two D-amino acids, namely, 2- chloroacetyl-D-Tyr (clAc-D-Tyr) at the N-terminus and D-Cys at the C-terminus. However, prior to generating the full-scale peptide library, the incorporation efficiency of the non- proteinogenic aminoacyl tRNAs in the FIT system was assessed. Three model peptides were designed, containing oneclAcD-Tyr at the N-terminus, one D-Cys at the C-terminus and one P- amino acid in the middle of the sequence. The addition of a P-amino acid was aimed at generating a turn structure to the peptide.

[0296] As the FIT system is a fully purified in vitro translation system in which all components are added, including tRNAs and aminoacyl tRNA synthetases, tRNA codons may be reassigned. The FIT system used herein is described in J. Morimoto, Y. Hayashi, H. Suga, Angew. Chem. Int. Ed. 2012, 124, 3479-3483. The codons assigned for (15,25)-2-ACHC, (lR,2S)-2-ACPC and (lR,2R)-2-ACPC were AUC, CUC and UGC, respectively. The reason for this assignment was that these codons are less likely to encounter misincorporation in the FIT system. The codon assigned for D-Cys was UGG due to the fact that this codon offers high efficiency for the incorporation of D-Cys in the FIT system. SinceclAcD-Tyr is the initial residue of all the peptides that were translated, the codon chosen for this amino acid was AUG. Codon reassignments are shown in Fig. 4A. Structures of the three P-amino acids are shown in Fig. 4B.

[0297] Another advantage of the FIT system is that tRNAs can be charged with virtually any unnatural amino acid using flexizyme, a ribozyme that recognizes the 3’ CCA end of tRNA and an amino acid derivatized with either cyanomethyl ester or dintirobenzyl ester. The run-off transcribed tRNAs used, tRNA^ cAu, tRNAProlE2ccA, tRNAProlE2GAu, tRNAProlE2GAG and tRNAGluE2GCA, were prepared by in vitro transcription of the corresponding DNA with T7 RNA polymerase. Briefly, DNA oligomers (Eurofins) were assembled by PCR. tRNA^ cAu was assembled using oligomers of SEQ ID NOs.: 13-17; tRNA^^VcA was assembled using oligomers of SEQ ID NOs.: 13 and 18-20; IRNA^^GAU was assembled using oligomers of SEQ ID NOs.: 13, 18, 20, and 21; IRNA^^GAG was assembled using oligomers of SEQ ID NOs.: 13, 18, 20, and 22; and 1RNAG1UE2GCA was assembled using oligomers of SEQ ID NOs.: 13, and 23-26. The assembled DNA products were purified using Phenol / Chloroform / Isoamyl alcohol extraction. Once the pure DNA products were obtained, in vitro transcription was performed to generate the corresponding tRNAs. The newly generated tRNAs were purified using denaturing PAGE. The pure tRNA products were harvested by ethanol precipitation and stored at -20°C until use. The DNA oligomers used are listed in Table 3.

[0298] The derivatized amino acids, namely,clAcD-Tyr-cy anomethyl ester (CME), D-Cys- dinitrobenzyl ester (DBE), (lS,2S)-2-ACHC-DBE, (17?,2S)-2-ACPC-DBE and (17?,27?)-2- ACPC-DBE were synthesized as described in Goto, Y. et al., ACS Chem. Biol. 2008, 3, 120- 129.

[0299] Charging reactions were performed with the eFx flexizyme (SEQ ID NO: 7) forclAcD- Tyr-cy anomethyl ester, while DBE-derivatized amino acids were charged with the dFx flexizyme (SEQ ID NO: 6).

[0300] Briefly, initiator tRNA^ cAu was charged withclAcDTyr(CME) using eFx flexizyme (SEQ ID NO: 7). 50 mM HEPES-KOH pH 7.5, 25 pM eFx, 25 pM tRNA^cAu, 0.15 mM MgCh and 5 mMclAcD-Tyr-CME were combined and incubated at 0°C for two hours. tRNA^^ccA was charged with D-Cys-DBE using dFx flexizyme (SEQ ID NO: 6). 50 mM HEPES-KOH pH 7.5, 25 pM dFx, 25 pM tRNA^^ccA, 0.15 mM MgCl2and 5 mM D- Cys-DBE were combined and incubated at 0°C for six hours. tRNAProlE2GAu was charged with (15,2S)-2-ACHC-DBE using dFx (SEQ ID NO: 6). 50 mM Bicine-KOH pH 8.7, 25 pM dFx, 25 pM tRNA^^GAU, 0.15 mM MgCl2and 5 mM 1S,2S- 2-ACHC-DBE were combined and incubated at 0°C for 16 hours. Similarly, tRNA^^GAG and tRNAGluE2GCA were charged with (17?,2S)-2-ACPC-DBE and (17?,27?)-2-ACPC-DBE, respectively, using the same conditions.

[0301] Following the flexizyme charging reactions, all aminoacylated tRNAs were harvested by ethanol precipitation, resuspended in 10 mM potassium acetate pH 5, stored at -80 °C, and were used without further purification. The incorporation efficiency of the non-proteinogenic aminoacyl tRNAs described hereinabove to the ribosome in the FIT system was then assessed by adding them into the FIT system to generate a model peptide. The mass of each model peptide was measured by MALDI- TOF (Autoflex II, Burker). The two model peptides containing the different 2-ACPC P-amino acids were translated without truncation, whereas the model peptide with 2-ACHC showed a large truncated peak, indicating that the incorporation efficiency of 2-ACHC was low. To solve this problem, the translation conditions were optimized by increasing the concentration of the pre-charged 2-ACHC-tRNA (from 25 pM to 40 pM), as well as the concentration of EF-Tu (from 20 pM to 70 pM). EF-Tu is an elongation factor which mediates the accommodation of charged tRNA into the ribosome A site. Using the optimized conditions, the model peptide with 2-ACHC was successfully translated with no truncation. These results indicated that the correct cyclic peptides could be achieved in this system.

[0302] Example 4: Screening of the mRNA display library molecules to identify binders to£- and £>-MMP7eat

[0303] Once the optimized conditions for translation of the peptides containing the P-amino acids were determined, the large-scale peptide library (1012) was prepared using the mRNA display library molecule design shown in Fig. 4C. Specifically, mRNAs with 6-15 repeated NNU sequences for the random region of amino acid sequences were produced by in vitro transcription using corresponding DNAs that were prepared by PCR with the DNA oligomers (SEQ ID NOs.: 10-12 as listed in Table 3) (Eurofins). The in vitro transcribed mRNAs were purified using denature PAGE. The mRNA library was then prepared by mixing the mRNAs with the different number of NNU repeats equally. The final concentration of the mRNA library was 10 pM. Subsequently, the mRNA library was ligated with puromycin according to the protocol described in Hayashi, Y. et al., ACS Chem. Biol. 2012, 7, 607-613.

[0304] The mRNA library ligated with puromycin was added to the FIT system, in which Cys, Gin, Glu, He, Leu, Lys, Met, Trp and their corresponding tRNA synthetases were omitted and ( lS,2S)-2-ACHC-tRNAProlE2GAu, ( lR,2S)-2-ACPC-tRNAProlE2GAG, ( lR,2R)-2- ACPCtRNAGluE2GCA, D-Cys-tRNA^VcA, andclAcD-Tyr-tRNAfMetCAU were included to generate the mRNA-peptide conjugate library. In addition, RF1 was omitted. After that, reverse transcription was performed to generate the mRNA-peptide-cDNA conjugate library as described in J. Morimoto, Y. Hayashi, H. Suga, Angew. Chem. Int. Ed. 2012, 124, 3479-3483. Following a standard protocol (T. Katoh, Y. Goto, H. Suga, Peptide Macrocycles: Methods and Protocols 2022, 247-259), M-280 magnetic Streptavidin beads (Thermo Fisher) were treated with either biotin, L-MMP7cat, or D-MMP7cat. The beads with biotin were used as a negative control. The mRNA-peptide-cDNA library was first mixed with the negative beads in cold PBST buffer and rotated at 4°C for 20 mins to remove nonspecific binders. After that, the recovered library was split and each half was mixed with the L-MMP7cat-saturated beads or the biotin-D-MMP7cat-saturated beads in cold PBST buffer and rotated at 4°C for 30 mins. The supernatants were discarded and the beads were washed three times with PBST buffer and resuspended in PCR buffer (10 mM Tris-HCl pH 8.3, 2.5 mM MgCh, 250 pM dNTPs). The bound cDNAs were recovered by heating the beads at 95°C for 5 mins. The recovered cDNAs were quantified by a real-time qPCR (LightCycler 96, Roche) and then amplified by PCR. The amplified DNA products were used as templates to generate the mRNA display library molecules for the next selection round. The selection rounds were repeated until the cDNA enrichment was observed for each target.

[0305] Six rounds of selection were performed for L-MMP7catand D-MMP7cat. The enrichment of cDNA was observed at round five for D-MMP7cat(0.02% of the input DNA) and at round six for L-MMP7cat(0.01% of the input DNA). The selection was halted at round six and next generation sequencing (NGS) was performed to identify the clone sequences within the enriched cDNA. Overall, the random regions of the top 100 enriched cDNA sequences targeting either L-MMP7cator D-MMP7catwere not highly conserved, but exhibited preference towards hydrophobic amino acids (e.g., Vai and Ala).

[0306] After the selection, the sequences of the obtained cDNAs of round 6 were cloned and sequenced using Miseq (Illumina) and the promising clones were selected for single-clone display. The processes and conditions of the single-clone display were the same with those used for affinity selections described herein. As shown in Table 1, peptide L10 and L16, isolated following screening for affinity to L-MMP7cat, showed good P / N (positivity recovery percentage vs. negative recovery percentage) ratio, and these two peptides appeared to be in the highest frequency when targeting L-MMP7. Regarding the D-MMP7 binders, peptide D6, D20 and D21 showed high positivity recovery percentage (Table 2). The highest P / N ratio was observed in peptide D20, followed by D21 and L16. Based on these results, L10, L16, D20 and D21 were selected for synthesis and further analysis. Table 1: Peptide binders isolated following screening for affinity to L-MMP7cat. All peptides are cyclic and cyclized by a thioether bond. 01 = (15,25)-2-ACHC, 02= (17?,2S)-2- ACPC.

[0307] ACDY= Acetyl-D-Tyr;DC= D-Cys Table 2: Peptide binders isolated following screening for affinity to D-MMP7cat. All peptides are cyclic and cyclized by a thioether bond. 01 = (15,25)-2-ACHC, 02= (17?,2S)-2- ACPC, 03= (l / ?,2 / ?)-2-ACPC.

[0308] ACDY= Acetyl-D-Tyr;DC= D-Cys

[0309] Example 5: Synthesis and characterization of cyclic peptide binders Based on the above data, the four top enriched peptides LIO, LI 6, and the mirror-image forms of D20 and D27 referred here as D’20 and D’21 were synthesized (Figs. 4D). Fig. 4E shows the chemical structure of L16. It is worth noting that the synthetic D’20 (Fig. 4F) and D’21 peptides contain the mirror-image of 01 and 03, i.e. (lR,2Ryi- ACHC and (lS,2S)-2- ACPC, respectively. All peptides were synthesized by Fmoc-SPPS using rink amide, with a C-terminal solubility enhancer, polyethylene glycol PEGe or PEG27, to enhance the solubility of these hydrophobic sequences (Fig. 5A). The synthesized peptides were chloroacetylated at the N- terminus with chloroacetylated-hydroxysuccinimide (Cl-Ac-NHS). The peptides were then cleaved and the linear peptides were treated with triethylamine (TEA) in DMSO to obtain a thioether bond between the N-terminal chloroacetamide group and the sulfhydryl group of the cysteine (Fig. 5A). Figs. 5B-5C shows the structures of LI 6 with PEGe (LI 6- PEGe) and PEG27, (LJ6-PEG27) respectively. Figs. 5D-5E shows the structures of D'20 with PEGe (D’20-PEG6) and PEG27 (£>’20-PEG27), respectively.

[0310] Specifically, the selected cyclic peptides were chemically synthesized by Fmoc-SPPS as described herein and D-amino acids were used to prepare D'20 and D'21. Fmoc-NH-PEG(6)- COOH or Fmoc-(NHPEG27)-COOH (1.1 equiv.) were coupled for 1 h on 0.125 mmol TentaGel R RAM resin (loading 0.18 mmol / g) in DMF solution containing 1.1 equiv. PyBOP and 2.2 equiv. DIEA. P-amino acids were manually coupled using 1.2 equiv. of the corresponding P-amino acid in DMF solution containing 1.2 equiv. HATU and 2.2 equiv. DIEA for 1.5 h. The N-terminal chloroacetyl chloride was coupled using 0.2 M chloroacetyl N- hydroxy succinimide (NHS) ester in NMP for 1 h, and the resin was washed with DMF (3 x 5 mL). The peptide was then cleaved from the resin using TFA / H2O / TIPS (95:2.5:2.5) for 2 h followed by precipitation by cold ether and lyophilization. To form the thioether bond between the N-terminal chloroacetamide group and the sulfhydryl group of the C-terminal Cys, the peptides were dissolved in DMSO and the pH was adjusted to 8 by adding triethylamine (TEA) for 30 min, followed by quenching the cyclization reaction by adding 0.1% TFA in water. Subsequently, the peptides were purified by preparative HPLC using a C4 column and a gradient of 30%-60% B.

[0311] The respective peptides were isolated in milligram quantities and characterized by RP- HPLC and ESI-MS. HPLC chromatograms of L70-PEG6, L76-PEG6, D’20-PEG6and D’21- PEGe are shown in Figs. 5F-5I, respectively. Fig. 5J shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified L / 6-PEG27 Fig. 5K shows the final HPLC chromatogram and electron spray ionization mass spectrogram of purified D’20-PEG27.

[0312] Example 6: In vitro inhibition of MMP7 by cyclic peptide binders

[0313] The activity of the cyclic peptide binders L10, L16, D’20 and D’21 against MMP7 was assessed in competitive inhibition assays. Specifically, In vitro inhibition activity of the cyclic peptides against MMP7, MMP1, MMP8, MMP9, MMP10 and MMP14 was followed by the cleavage of Anorogenic peptide substrate (FS-6) Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- NH2 (SEQ ID NO: 28). The Mca chromophore was monitored using Auorescence (excitation at 320 nm and emission at 400 nm). For the assay, 10 nM of MMP7 and 2 nM of other MMPs were incubated for 30 min at 37°C with different concentration range of each cyclic peptide (0- 225 pM) using 50 mM Tris-HCl buffer (pH 7), 100 mM NaCl, 5 mM CaCl2and 0.05% Brij 35. The reaction was initiated upon addition of 10 pM of the Anorogenic substrate FS-6 and the reaction was followed for 1.5 h. The IC50 was calculated using GraphPad Prism 9.

[0314] All four cyclic peptides inhibited the activity of MMP7. As demonstrated in the data of Figs. 6A-6B, D’20 was the most potent MMP7 inhibitor (IC50 = 90 ± 13 nM) (Fig. 6B), followed by L16 (IC50 = 180 nM) (Fig. 6A). L10 had an IC50 of 2.4 ± 0.87 pM, while D’21 showed weak activity against MMP7 (IC50 = 34.4 ± 16.8 pM). Based on these results, D’20 and L16 were selected for further evaluation.

[0315] Example 6: Mode of inhibition of MMP7 by L16 and D'20

[0316] To determine the mode of inhibition of MMP7 by LI 6 and D'20. the double reciprocal curves were obtained in the presence of various concentration of the substrate FS-6 with or without 1 pM of L16 and 800 nM of D’20. Specifically, 800 nM of D'20 and 1 pM of L16 were incubated with MMP7 in the presence of increasing concentration of FS-6 substrate (0-10 pM). Data were arranged in double reciprocal curves (1 / V vs 1 / [S]) and fitted to linear equation using KaleidaGraph.

[0317] In the case of L16, the curves were intersected at x-axis indicating a non-competitive inhibition (Fig. 6C), while D’20 exhibited the characteristics of competitive inhibition, showing un-parallel lines with different slope and no change in the T-intercept (Fig. 6D).

[0318] Example 7: Selectivity of LI 6 and D'20 to MMP7 over other MMPs

[0319] Next, in vitro inhibition activity against other MMPs was assessed. To determine the selectivity of D’20 and LI 6, these two cyclic peptide binders were screened against other MMPs with similar substrate specificity, namely MMP1, MMP8, MMP9, MMP10 and MMP14. D’20 was found to be highly selective over other MMPs, as shown in Figs. 6E-6F. The inhibitory activity of D’20 against other MMPs was at least 60-fold weaker compared to MMP7 (Fig. 6F). However, screening L16 against other MMPs revealed that L16 also inhibited MMP8 and MMP10 to some extent (Fig. 6E). Example 8: Stereospecificity of the cyclic peptide D'20

[0320] To assess the stereospecificity of D’20 towards the inhibition of MMP7, D20 was prepared, which has the same sequence as D’20 but is mainly composed of L-amino acids and pi (Table 2), and after chemical characterization, the inhibitory activity of D20 was tested against MMP7.

[0321] PEG27-D20 (same sequence as D’20 but composed of L- amino acids instead of D-amino acids) was prepared by solid phase peptide synthesis, as described hereinabove. Following cyclization and purification, the in vitro inhibition activity of D20 against MMP7 was evaluated as described in Example 6. The results revealed that the catalytic activity of MMP7 remained unaffected by D20 (at a concentration as high as 28 pM), suggesting that the inhibition of MMP7 is stereospecific, and is not simply through unspecific hydrophobic interactions.

[0322] Example 9: In vitro stability studies of LI 6 wtAD'20

[0323] The stability of L16 and D’20 was evaluated by monitoring their degradation in human serum, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) at 37 °C. SGF contains pepsin at pH 1.3, while SIF contains pancreatin at pH 6.8.

[0324] The stock solutions of the peptides were prepared by dissolving 2 mg of each cyclic peptide in 1 mL water. For the SGF degradation experiment, the stock solution of pepsin enzyme was prepared by dissolving 5 mg of pepsin in 5 mL of 10 mM HC1 (1 mg / mL), which was diluted to a 0.2 mg / mL with 10 mM HC1. In the degradation process, 34 pL of pepsin (0.2 mg / mL) was added to 100 pL of peptide (2 mg / mL) at pH 1.3 and the reaction was incubated at 37 oC. To monitor the degradation, 10 pL of the reaction aliquots were taken and quenched with 10 pL of 10 mM NaOH every 30 min for several hours and analyzed by HPLC using XSelect C18 column (3.5 pm, 130 A, 4.6 x 150 mm) with a gradient of 5-70% B (0.1% TFA in MeCN) over 20 min at 220 nm.

[0325] For SIF degradation experiment, pancreatin stock solution was prepared by using 68 mg KH2PO4 in 500 pL H2O, 800 pL of 0.2 M NaOH and 10 mg porcine pancreatin. The volume was adjusted to 5 mL with water and the pH was adjusted to 6.8 to obtain 2 mg / mL of porcine pancreatin. For this degradation assay, 3.5 pL of pancreatin (2 mg / mL) was added to 100 pL of peptide (2 mg / mL) at pH 6.8 and the reaction was incubated at 37°C. To monitor degradation progress, 10 pL of the reaction aliquots were removed and quenched with 10 pL of 10 mM HC1 every 30 min for several hours and were analyzed by HPLC using the same conditions as in pepsin degradation assay. The results are shows in Figs. 7A-7D. As shown in Fig. 7A and Fig. 7C, D’20 was stable in both SGF and SIF for at least 12 hours, while L16 was completely degraded in SGF (within 30 minutes, Fig. 7B) and in SIF (within 10 minutes, Fig. 7D).

[0326] In addition, the stability L16 and D'20 in human serum was assessed. The stock solutions of the peptides were prepared by dissolving 2 mg of each peptide separately in 1 mL PBS at pH 7, then 200 pL of each peptide was incubated in human serum (10%) at 37°C for 24 h. At each time of point (0, 6, 24 hours), 17 pL of the reaction was quenched with 20 pL of water containing 0.1% TFA. The mixed solution was centrifuged for 3 min and the supernatant was analyzed by HPLC using XSelect C18 column (3.5 pm, 130 A, 4.6 x 150 mm) with a gradient of 5-70% B (0.1% TFA in MeCN) over 20 min at 220 nm. The results showed that both D’20 and LI 6 were stable in human serum for at least 24 hours.

[0327] Example 10: Impact of MMP7 inhibitory peptides on cell function

[0328] Cyclic peptides L16 and D’20 were used to study the impact of MMP7 inhibition on cell function. The pancreatic cell line CFPAC-1 expresses zymogenic MMP7 as well as activated enzyme and are fully characterized for their MMP7 activity.

[0329] CFPAC-1 cell line was cultured and maintained in DMEM media (Gibco) at 37 °C and 5% CO2. Media was supplemented with 10% dialyzed FBS, ImM sodium pyruvate, ImM penicillin- streptomycin and 2 mM glutamine (Biological industries). Cell line was verified to be mycoplasma-free.

[0330] To test the effect of the cyclic peptides on inhibition of pancreatic cancer cell proliferation and viability, 70% confluent cultures were treated with L16 and D’20. CFPAC-1 cells were seeded in triplicates at a density of 5000 cells / well in 96-well plates and cultured for 24 hours in DMEM with 10% FBS. 70% confluent cultures were washed with PBS and treated with either L16 or D’20 peptides at 0 pM, 0.3 pM, 1 pM and 10 pM concentrations. For proliferation assay, cells were fixed with 0.5% glutaraldehyde every day for three days after drug treatment and for viability assay, cells were fixed after 72 h of drug treatment. The cells were stained with 1% methylene blue solution for 1 h and destained with water until the color ran clear. The plates were dried overnight on the bench and the color was extracted with 0.1 M HC1. Optical density (OD) was measured at 620 nm. Assay was performed in biological duplicates and significance was determined using Student’s t-test. Neither L16 nor D’20 showed any significant inhibition of proliferation and reduction in viability of CFPAC-1 cells. Next, a cell migration assay was performed. To study the impact on cell migration, CFPAC-1 cells were plated in transwell inserts at indicated drug dilutions with fetal bovine serum in the bottom chambers to act as a chemoattractant. L16 and D’20 peptides were dissolved at a concentration of 1 mg / mL in serum free DMEM containing 0.5% DMSO. The peptide solution was sterile filtered with 0.22 pm filter (Merck). CFPAC-1 cells were cultured in serum-free DMEM for 12 h. Then 1.25x105 cells were added to the top chamber of transwell inserts (8 pm pore size, BD Biosciences) in serum-free DMEM, 0.5% DMSO at the following drug concentrations; 0 pM, 0.3 pM, 1 pM and 10 pM. Fetal bovine serum (FBS) was added to the bottom chambers to a final concentration of 10% to act as a chemoattractant and incubated at 37 °C with 5% CO2. After 18 h, cells were fixed with 2.5% glutaraldehyde and stained with 0.05% crystal violet for 1 h. The non-migrating cells on top were gently removed with a Q-tip and migrating cells at the bottom of the trans well were photographed. Total count of migrating cells per image was performed using ImageJ. Assay was performed in biological triplicates and significance was determined using Student’s t-test.

[0331] Figs. 8A-8B shows representative pictures of transwell migration after treatment with different concentrations of D’20 or LI 6, respectively. While L16 did not show significant migration inhibition, D’20 significantly inhibited cell migration (Fig. 8C). The data was used to calculated an IC50 of about 1 pM. This IC50 is comparable to the effects observed with GSM- 192, a highly selective anti-MMP7 inhibitory antibody.

[0332] Some of the sequences herein disclosed and / or claimed are provided in Table 3.

[0333] Table 3: Exemplified sequences.

[0334] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references referring to well-known procedures are provided throughout this document for the convenience of the reader.

[0335] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Claims

CLAIMS1. A method for identifying a cyclic peptide comprising D-amino acids capable of binding to a target polypeptide, the method comprising:(i) synthesizing a D-amino acid analog of the target polypeptide (D-polypeptide analog), or of a binding domain thereof;(ii) preparing an mRNA display library encoding random cyclic peptides, wherein each mRNA display library molecule comprises, from 5’ to 3’: a first cyclization codon; a series of at least 6 randomized codons; a second cyclization codon, a spacer encoding 2-10 amino acids; a translation stop element; a linker; and puromycin, and wherein the randomized codons and the spacer codons do not include the first or second cyclization codons;(iii) preparing an in vitro translation system comprising aminoacyl-tRNAs in which a tRNA that responds to the first cyclization codon and a tRNA that responds to the second cyclization codon are each aminoacylated with amino acids capable of reacting together to form a cyclic peptide; and wherein the in vitro translation system does not include any other aminoacyl-tRNAs that respond to the first cyclization codon, to the second cyclization codon, or whose amino acid can interfere with the cyclization reaction;(iv) translating the mRNA display library molecules obtained in step (ii) in the in vitro translation system of step (iii) and providing conditions for the amino acids incorporated in response to the cyclization codons to cyclize, to produce a library of cyclic peptides wherein each peptide is conjugated to its progenitor mRNA;(v) incubating the library obtained in step (iv) with the D-polypeptide analog of step (i) to allow binding of cyclic peptides to the D-polypeptide analog;(vi) isolating at least one cyclic peptide binder and identifying its amino acid sequence by the reverse transcribed DNA of its conjugated progenitor mRNA;(vii) synthesizing a cyclic peptide having inverse stereochemistry to the at least one isolated cyclic peptide binder and confirming its binding to the target polypeptide.

2. The method of claim 1, wherein each mRNA display library molecule comprises between 6 and 15 randomized codons and wherein the cyclic peptide comprises between 8 and 17 amino acid residues.

3. The method of any one of claims 1 or 2, wherein the first cyclization codon is AUG and the second cyclization codon is UGG, wherein the randomized codons, and the spacer codons do not comprise AUG and UGG, and wherein the translation system does not comprise L-Met, L-Trp, or their cognate aminoacyl-tRNA synthetases (aaRS).

4. The method of any one of claims 1 to 3, wherein randomized codons comprise NNU codons.

5. The method of claim 1, wherein the spacer encodes the amino acid sequence Ser-Ser- Asn-Val-Ser-Ala (SEQ ID NO: 3).

6. The method of any one of claims 1-5, wherein the translation stop element is a stop codon and wherein the translation system does not include a catalytically active release factor (RF).

7. The method of claim 6, wherein the stop codon is UAG.

8. The method of any one of claims 1-7, wherein the linker comprises between 10-40 nucleotides.

9. The method according to any one of claims 1-8, wherein the in vitro translation system is a purified E. coli translation system.

10. The method of any one of claims 1-9, wherein the aminoacyl-tRNAs that respond to the first and second cyclization codons are provided to the in vitro translation system as precharged aminoacyl-tRNAs.

11. The method of any one of claims 1-10, wherein the amino acid encoded by one of the cyclization codons comprise a reactive thiol and the amino acid encoded by the other cyclization codon comprises an N-terminal 2-halogen-acetyl group, wherein the halogen is selected from chloro, bromo, and iodo.

12. The method of claim 11, wherein the amino acids capable of reacting together to form a cyclic peptide are D-Cys and A-chloroacctyl-D-tyrosinc (ClAc-D-Tyr).

13. The method of any one of claims 1-12, wherein the aminoacyl-tRNAs that respond to the cyclization codons are ClAc-D-Tyr-tRNA0^61, and D-Cys-tRNAftolE2ccA.

14. The method of any one of claims 1-13, wherein the cyclic peptide comprises an at least one additional unnatural amino acid.

15. The method of claim 14, wherein the at least one additional unnatural amino acid is a P-amino acid.

16. The method of claim 15, wherein the P-amino acid is aminoacylated onto a tRNA that responds to a cysteine codon.

17. The method of claim 15 or 16, wherein the peptide comprises at least one additional 0- amino acid, aminoacylated onto a tRNA that responds to an AUU, AUC, CUU, or CUC codons.

18. The method of claim 17, wherein the in vitro translation system does not comprise L- Cys, L-Leu, L-Ile, or their cognate aminoacyl-tRNA synthetases (aaRS).

19. The method of any one of claims 15-18, wherein the 0-amino acid is selected from (lS,2S)-2-aminocyclohexane carboxylic acid, (lR,2S)-2-aminocyclopentane carboxylic acid, and (lR,2R)-2-aminocyclopentane carboxylic acid.

20. The method of any one of claims 1-19, wherein the library of cyclic peptides each conjugated to its progenitor mRNA comprises at least 1012molecules.

21. The method of any one of claims 1-20, wherein the D-polypeptide analog of the target polypeptide, or of a binding domain thereof, comprises a biotin tag.

22. The method of any one of claims 1-21, wherein the reverse transcribed DNA is prepared before performing step (v).

23. The method of any one of claims 1-22, wherein the reverse transcribed DNA of step (vi) is used to enrich the cyclic peptide binder.

24. The method of claim 23, wherein the method comprises at least two enrichment rounds.

25. The method of any one of claims 1-24, wherein the target polypeptide is a human matrix metalloproteinase or a catalytically active variant thereof.

26. The method of claim 25, wherein the target polypeptide is human matrix metalloproteinase 7 (MMP7) comprising the catalytic domain sequence set forth in SEQ ID NO: 2, or a catalytically active variant thereof having at least 80% sequence identity.

27. A cyclic peptide that binds to human matrix metalloproteinase 7 (MMP7) and inhibits its activity comprising the amino acid sequence (D-Ser)-((7R,2R)-2- aminocyclohexylcarboxy)-(D-Arg)-(D-Tyr)-(D-Thr)-(D-Val)-(D-Phe)-(D-Val)-(D- Ala)-(D-Val)-(D-Tyr)-(D-Val)-(D-Ser) (SEQ ID NO: 8); or a derivative thereof comprising 1-3 modifications to SEQ ID NO: 8.

28. The cyclic peptide of claim 27, further comprising an N-terminal and a C-terminal residue that form a cyclic peptide.

29. The cyclic peptide of claim 27 or 28, wherein the N-terminal and a C-terminal residue react to form a thioether bond.

30. The cyclic peptide of claim 29, wherein the residues that react to form a thioether bond are N-chloroacetyl-L-tyrosine and L-cysteine.

31. The cyclic peptide of any one of claims 27-30, comprising the structure:

32. The cyclic peptide of any one of claims 27-31, comprising a C-terminus that is PEGylated, amidated, carboxylated, or substituted with an alcohol moiety.

33. A conjugate comprising the cyclic peptide of any one of claims 27-32 and a moiety that increases solubility, stability and / or permeability.

34. The cyclic peptide or conjugate of any one of claims 27-33, comprising 4-30 linear PEG units attached to its C-terminus.

35. The cyclic peptide or conjugate of claim 34 having a structure selected from:

36. A pharmaceutical composition comprising a cyclic peptide or conjugate according to any one of claims 27-35, and an acceptable salt, diluent or carrier.

37. The pharmaceutical composition of claim 36, for use in inhibiting an activity of MMP7.

38. The pharmaceutical composition of claim 36, for use in preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7.

39. The pharmaceutical composition according to claim 37 or 38, wherein the disease or disorder is selected from a cancer or a tumor, a kidney disease, disorder or injury, angiogenesis related disorders, and biliary atresia.

40. The pharmaceutical composition for use of any one of claims 37-39 wherein the use involves at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

41. A method of preventing, inhibiting, delaying, or treating a disease or disorder associated with the activity of MMP7, comprising administering to a subject in need a pharmaceutical composition according to claim 36.

42. The method of claim 41, wherein the disease or disorder is selected from a cancer or a tumor, a kidney disease, disorder or injury, an angiogenesis related disorder, and biliary atresia.

43. The method of claim 42, wherein the cancer is a solid cancer or a metastatic cancer.

44. The method of claim 43, wherein the solid cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, gastric cancer, esophageal cancer, gallbladder cancer, and brain cancer.

45. The method of claim 42, wherein the cancer is hematologic cancer.

46. The method of any one of claims 41-45, wherein the pharmaceutical composition is administered before, together with, or following, an additional anti-cancer treatment.

47. The method of claim 46, wherein the additional anti-cancer treatment is selected from the group consisting of surgery, chemotherapy, radiotherapy, and immunotherapy.

48. The method of any one of claims 41-47, wherein the method results in at least one of: preventing, reducing, or inhibiting metastases formation, migration, adhesion, spread, and / or growth, pushing metastatic cells into a dormant state, eliminating metastatic cells, and preventing metastatic cells outbreak into big metastatic lesions.

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