Cyclic peptides as antagonist of interleukin-17b receptor (il-17RB) and use thereof

Abstract

The present invention relates to a cyclic peptide as an antagonist of interleukin-17B receptor (IL-17RB) which features interruption of the interaction of IL-17RB and MLK4. The present invention also relates to use of the cyclic peptide for treatment of diseases or disorders associated with IL-17RB activation.

Description

Attorney Docket No. 5992-0517PWO1 TITLE OF THE INVENTION CYCLIC PEPTIDES AS ANTAGONIST OF INTERLEUKIN-17B RECEPTOR (IL-17RB) AND USE THEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of the filing dates of U. S. Provisional Application 63 / 742,072, filed January 6. 2025, the entire content of which is incorporated herein by reference.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in. XML format and is hereby incorporated by reference in its entirety. Said. XML copy, created on January 2, 2026, is name “20260102-ACA0160WO-seqlist' and is 51 KB in size. The sequence listing contained in this. XML file is part of the specification and is hereby incorporated by reference herein in its entirety.TECHNOLOGY FIELD

[0003] The present invention relates to a cyclic peptide acting as an antagonist of interleukin-17B receptor (IL-17RB) by interruption of the interaction of IL-17RB and MLK4. The present invention also relates to use of the cyclic peptide for treatment of diseases or disorders associated with IL-17RB activation.BACKGROUND OF THE INVENTION

[0004] The IL- 17 (Cytokine interleukin- 17) receptor, identified through homology and receptor cloning, comprises five sub-clones (1L-17R A-E) and six ligands (1L17A-F), forming homo- or hetero-dimers upon binding1. IL-17RA-E exhibit high diversity, with IL-17RA and IL-17RF sharing the greatest similarity at 56%2. How ever, their SEFIR domain, especially compared to the protein interactive SEFIR domain, shows only 20% similarity among IL-17R A-E. indicating distinct signaling upon ligand binding. For instance, IL17-RA's extended SIFIR domain sequence, linked to the activation domain of C / EBP-b (CCAAT / enhancer binding protein b), sets it apart from IL-17RB3. In contrast to IL-17RA, IL17RB displays a unique topology' and knot structure in its SEFIR domain, suggesting different mechanisms for downstream events4,5. Our previous research indicates that IL-17RB's SEFIR domain includes an intrinsic disorderAttorney Docket No. 5992-0517PWO1 loop at V403-S416, providing flexibility crucial for interacting with the dual kinase mixed-lineage kinase 4 (MLK4)6.

[0005] IL-17RB was overexpressed in nearly 40% of pancreatic ductal adenocarcinoma (PDAC) and its signal pathway plays an essential role in promoting tumorigenesis and metastasis7'9. Our previous work elucidated the proximal mechanistic process of IL-17RB oncogenic signaling in PDAC6, revealing that IL-17RB forms a homo-dimer upon IL-17B binding, recruiting MLK.4 to phosphorylate IL-17RB’s Y447. This phosphorylation initiates downstream oncogenic signaling, leading to the release of cytokines and chemokines (e.g., CCL20, CXCL1, and TFF1) and eventually resulting in cancer cell metastasis. Disrupting IL-17RB-MLK4 interaction using aTAT-conjugated loop peptide (IL17RB403-416) reduces tumorigenesis and metastasis in PDAC, representing a promising strategy for treatment. However, the standalone loop peptide (IL17RB 403-416) faces challenges in penetrating cells, prompting the conjugation of the loop region with a TAT sequence to facilitate cellular entry. Nonetheless, the long TAT-loop peptide exhibits poor stability.

[0006] There is a need to provide an improved IL-17RB antagonist for treatment of diseases or disorders associated with IL-17RB activation.SUMMARY OF THE INVENTION

[0007] In this present invention, it is unexpectedly found that a shorten peptide from positions 403-416 of IL-17RB in a cyclic form, acting as an IL-17RB antagonist, exhibit improved effects in inhibition of tumor growth and metastasis via disrupting the interaction between IL-17RB and MLK4.

[0008] In one aspect, the present invention provides a cyclic peptide comprising the amino acid sequence of positions 406-412 of IL-17RB.

[0009] In some embodiments, the cyclic peptide of the present invention comprises the amino acid sequence ofX1-X2-X3-X4-X5-X6-X7 Formula Iwherein XI is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S). lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). The cyclic peptideAttorney Docket No. 5992-0517PWO1 targets the interaction between IL-17RB and mixed-lineage kinase 4 (MLK4).

[0010] In some embodiments. XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G) or aspartic acid (D). In some embodiments, XI is glycine (G); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G). In some embodiments, X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, X1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

[0011] In some particular embodiments, the cyclic peptide comprises the amino acid sequence selected from the group consisting of GTCGKSE (SEQ ID NO: 17), STCDKSE (SEQ ID NO: 21). GTCGKTE (SEQ ID NO: 22). GTCGKSD (SEQ ID NO: 23) and GTCGAAA (SEQ ID NO: 24).

[0012] In some embodiments, the cyclic peptide of the present invention is of Formula IIY1 -XI -X2-X3-X4-X5-X6-X7-Y2 Formula IIwhereinX1 is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A);Y 1 comprises 1 -5 amino acid residues at the amino terminus;Y2 comprises 1-5 amino acid residues at the carboxyl terminus; and cyclization occurs via a linkage between one amino acid residue of Y1 and one amino acid residue of Y2the cyclic peptide targets the interaction between IL-17RB and mixed-lineage kinase 4 (MLK4).

[0013] In some embodiments, XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G) or aspartic acid (D). In someAttorney Docket No. 5992-0517PWO1 embodiments, XI is glycine (G); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G). In some embodiments, X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, X1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

[0014] In some particular embodiments, the cyclic peptide is selected from the group consisting of cyclo (Y1-GTCGKSE-Y2) (Formula Ila), cyclo (Yl-STCDKSE-Y2) (Formula lib), cyclo (Y1-GTCGKTE-Y2) (Formula lie), cyclo (Yl-GTCGKSD-Y2) (Formula lid) and cyclo (Y1-GTCGAAA-Y2) (Formula lie).

[0015] In some particular embodiments, the cyclic peptide comprises 9 to 17 amino acid residues.

[0016] In some particular embodiments, the linkage for cyclization is a disulfide bond or a peptide bond.

[0017] In some embodiments. Y1 in Formula II is selected from the amino acid residues / sequence C (Cys), GS (Gly-Ser), S (Ser). GC (Gly-Cys) and G (Gly). In some embodiments, Y2 in Formula II is selected from the amino acid residues / sequence C (Cys), S (Ser), and D (Asp). In some embodiments, cyclization occurs via a disulfide linkage between two terminal cysteine residues. In some embodiments, cyclization occurs via an amide linkage formed by G(Gly)-S(Ser). S(Ser)-S(Ser) and G(Gly)-D(Asp).

[0018] In some particular embodiments, the cyclic peptide is selected from the group consisting of those set forth in SEQ ID NOs: 25-49 of Table 4.

[0019] The present invention also provides a composition comprising a cyclic peptide as described herein and a physiologically acceptable carrier.

[0020] In some embodiments, the composition of the present invention is a pharmaceutical composition.

[0021] In another aspect, the present invention provides a method for inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with IL-17B / IL-17RB activation comprising administering to a subject in need thereof anAttorney Docket No. 5992-0517PWO1 effective amount of a cyclic peptide as described herein. The present invention also provides use of a cyclic peptide as described herein for manufacturing a medicament for inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with IL-17B / IL-17RB activation. Further provided is a cyclic peptide as described herein for use in inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with IL-17B / IL-17RB activation. Typically, the disease or disorder is IL-17B / IL-17RB-mediated proliferation disorder e.g. a cancer and a metastasis thereof.

[0022] In some embodiments, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary tract cancer, gallbladder and bile duct cancer, mammary cancer, ovarian cancer, cervical cancer, uterine body cancer, bladder cancer, prostate cancer, testicular tumor, osteogenic and soft-tissue sarcomas, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and plural malignant mesothelioma.

[0023] In one certain example, the cancer is pancreatic cancer.

[0024] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0026] In the drawings:

[0027] Figs. 1A-1F are graphs showing that the cyclic peptide exhibited better efficacy than the original loop peptide in suppressing tumor growth and metastasis in both in vitro and in vivo models. Fig. 1A show s that the main sequence of the human IL-17RB loop peptide is highlighted in yellow and underlined, whereas the corresponding control viral transducing sequence TAT is shown in black. SequenceAttorney Docket No. 5992-0517PWO1 alignment of IL-17RB loop peptides across different species reveals a conserved region within the flexible loop (indicated by the red mark in the table). Fig. IB shows topology of human IL-17RB SEFIR domain. The purple residue denotes the noncanonical tyrosine phosphorylation site Y447 phosphorylated by MLK4 kinase. Yellow-red-yellow and red helical loop represent the corresponding region of the loop and the cyclic peptide in the SEFIR domain. Sequence of cyclic peptides are shown in the lower panel. The cyclic peptide is encircled by disulfide linkages from the two cysteines, as indicated. Fig. 1C shows the effects of loop peptide and cyclic peptide treatment on spheroid formation in CFPAC1 cells. Fig. ID shows the effects of loop peptide and cyclic peptide treatment on soft agar colony formation in CFPAC1 cells, “n” denotes the number of independent experiments, and each dot represents one such experiment. An orthotopic mouse model was established using GFP-Luc CFPAC 1 cells. One-week post implantation, mice were randomized into three groups (vehicle, TAT-loop peptide, or cyclic peptide) and treated thrice weekly. After 4 weeks, the mice were euthanized, and tumors and lungs were harvested for analysis. Fig. IE shows representative tumor images (lower panel) and quantification of tumor weight (upper panel) of different peptide treatment groups. Bar: 1 cm. Each dot indicates one mouse. *P < 0.05 (two-tailed Student’s t test). Fig. IF shows that lung metastasis was assessed using IVIS imaging (left panel), and the percentage of lung metastasis is presented in the right panel.

[0028] Figs. 2A-2E are graphs showing that the kinase domain of MLK4 alone is sufficient for interaction with IL-17RB, whereas the SKL domain of MLK4 is necessary for the IL-17B-induced increase in interaction. Fig. 2A is a diagram illustrating various truncated MLK4 domains. HEK293T cells stably expressing GFP-IL-17RB were transfected with His-MLK4 plasmids with different truncated domains. They were treated with or without the IL17B ligand (50 ng / mL) for co-IP using GFP-Trap to assess the interaction between IL-17RB and various truncated MLK4 domains. Fig. 2B are some representative images of IP-western blot analysis. Fig. 2C are quantitative results of IP-western blot analysis of Fig. 2B. The upper panel is normalized to wild-type (WT) MLK4 in the presence of IL17B. The lower panel shows different truncated MLK4 domains normalized to the signal without IL17B treatment, indicating the fold change upon IL17B induction. Fig. 2D are additional representative images of IP-western blot analysis. Fig. 2E are quantitative results of IP-western blot analysis of Fig. 2D. The upper panel is normalized to wild-type (WT)Attorney Docket No. 5992-0517PWO1 MLK4 in the presence of IL17B. The lower panel shows different truncated MLK4 domains normalized to the signal without IL17B treatment, indicating the fold change upon IL17B induction. Data are presented as mean ± SD, with each dot representing an independent experiment (n = 3).

[0029] Figs. 3A-3D are graphs showing that the cyclic peptide disrupts the IL-17RB-MLK4 interaction by directly binding to the kinase domain of MLK.4. Fig. 3A shows that HEK293T cells stably expressing GFP-IL17RB were transfected with FLAG-tagged full-length MLK4, treated with varying concentrations of cyclic peptide for 2 h, and stimulated with the rhIL17B ligand for 5 min. Cell lysates coimmunoprecipitated using GFP-Trap Magnetic Agarose. Fig.3B shows that HEK293T cells stably expressing GFP-IL17RB were transfected with His-tagged MLK4-SKL truncated domain, treated with varying concentrations of cyclic peptide for 2 h, and stimulated with the rhIL17B ligand for 5 min. Cell lysates coimmunoprecipitated using GFP-Trap Magnetic Agarose. The IL-17RB-MLK4 interaction was assessed by immunoblotting with the indicated antibodies. The left panel displays representative IP-western blot images, whereas the right panel shows quantification of the protein-protein interaction. Each dot represents an individual experiment (n = 2). Data are represented as mean ± SD. Fig. 3C shows that HEK293T cells stably expressing GFP-IL17RB were transfected with His-MLK4 kinase domain or His-MLK4-SKL domain, treated with various concentrations of cyclic peptide for 2 h, and harvested for co-IP. The upper panel shows representative IP-western blot images, whereas the lower panel presents quantification results of the His-MLK4-SKL-GFP-IL17RB interaction. Each dot represents an individual experiment (n = 4). Data are represented as mean ± SD. **** < 0.0001 (two-tailed Student’s t test). Fig.3D shows that biotinylated MLK.4 kinase domain proteins were immobilized on the Super Streptavidin (SSA) biosensor for biolayer interferometry (BLI). The upper panel illustrates the experimental procedure of the BLI assay for measuring the kinetic binding of MLK4 kinase domain and IL-17RB SEFIR domain in the presence and absence of different concentrations of the cyclic peptide. The lower panel depicts the kinetic results of BLI when various concentrations of cyclic peptide were used.

[0030] Figs. 4A-4F are graphs showing that the cyclic peptide disrupts the IL-17RB-MLK4 interaction by forming a cysteine-mediated bond with arginine 216 in the kinase domain of MLK4. Fig. 4A shows the sequences of different synthetic peptides compared to the original cyclic peptide. Red color with underline denotesAttorney Docket No. 5992-0517PWO1 mutated residues corresponding to the original cyclic peptide. Fig. 4B shows that cells were harvested for co-IP. Representative IP-western blot images are shown (upper panel). Quantitative results depict the effects of different mutated synthetic peptides in disrupting the IL-17RB-MLK4 interaction (lower panel). All peptide results were compared to the vehicle treatment result. Each dot represents an individual experiment (n = 4). Data are represented as mean ± SD. Cotransfection of His-MLK4 kinase domain and the indicated FLAG-IL-17RB in HEK293T cells for co-IP using anti-FLAG M2 beads. Fig. 4C shows the effects of C408S IL-17RB (SEQ ID NO: 50) in interactions with the His-MLK4 kinase domain. Fig.4D shows the effects of K410R IL-17RB (SEQ ID NO: 52) in interactions with the His-MLK4 kinase domain. In the quantification results, each dot represents an individual experiment (n = 3). Data are represented as mean ± SD. Fig. 4E shows upper panel, docking analysis of MLK kinase domain and cyclic peptide. Pink color indicates the cyclic peptide, whereas blue color indicates the region of the MLK4 kinase domain bound with the cyclic peptide. Lower panel, the insets illustrate the chemical bonds between MLK4 kinase domain and cyclic peptide, revealing only the site-chained residues corresponding to the loop region of the SEFIR domain. Fig. 4F shows that HEK293T cells stably expressing GFP-IL17RB were transfected with wild-ty pe (WT) His-MLK4 kinase domain or R216A mutated His-MLK4 kinase domain and treated with 10 pM of cyclic peptide for 2 h for co-IP using GFP-Trap. The effects of the cyclic peptide in disrupting the interaction between GFP-IL17RB and different MLK4 variants were compared with the GFP-IL-17RB-WT-MLK4 interaction upon vehicle treatment. Each dot represents an individual experiment (n = 2). Data are represented as mean ± SD.

[0031] Figs. 5A-5G are graphs showing that the cyclic peptides, despite linker variations, disrupt the IL-17RB-MLK4 interaction and inhibit PDAC growth.HEK293T cells stably expressing GFP-IL17RB were transfected with FLAG-tagged full-length MLK4, treated with varying concentrations of the indicated cyclic peptide for 2 h, and stimulated with the rhIL17B ligand for 5 min. Cell lysates coimmunoprecipitated using GFP-Trap Magnetic Agarose. The IL-17RB-MLK4 interaction w as assessed by immunoblotting with the indicated antibodies. Fig.5A shows the molecular dynamics simulation of the cyclic peptide with GSS linker, GSGTCGKSES (SEQ ID No: 26), and Fig. 5B shows the results of immunoblotting of the cyclic peptide. Fig. 5C shows the molecular dynamics simulation of the cyclicAttorney Docket No. 5992-0517PWO1 peptide with SS linker, SGTCGKSES (SEQ ID No: 27), and Fig. 5D shows the results of immunoblotting of the cyclic peptide. Fig. 5E shows the molecular dynamics simulation of the cyclic peptide with GCS linker, GCGTCGKSES (SEQ ID No: 28), and Fig.5F shows the results of immunoblotting of the cyclic peptide. The left panel displays representative IP-western blot images, whereas the right panel shows quantification of protein-protein interactions. Fig.5G shows the influence of various cyclic peptides on the spheroid-forming capacity of CFPAC1 cells was evaluated.c'n” denotes the number of independent experiments, and each dot represents one such experiment.

[0032] Fig. 6 shows a proposed model for the disruption of the IL-17RB-MLK4 interaction by the cyclic peptide. The binding of IL17-B to IL17RB induces the IL17RB receptor to form a homodimer and recruit MLK4. MLK4 is released from autoinhibition, exposing the kinase domain to interact with the SEFIR domain of IL17RB, especially the dynamic loop region (knot structure) that corresponds with the sequence of the cyclic peptide. The configuration of binding is mainly through R216 on MLK4-K and C408 on IL17RB SEFIR. However, K410 on SEFIR could also maintain the structure of the loop region for the MLK4-K-IL17RB SEFIR interaction. After MLK4 binds to the loop region of IL17RB SEFIR, the loop region rotates to bring the MLK4-K domain and SEFIR domain in proximity. In this scenario, the interaction between the two proteins becomes strong. The active site of MLK.4 would turn to be sorely closed to allow the phosphorylation of SEFIR Y447 to be occurred. However, in the presence of the cyclic peptide, the cyclic peptide competes with the IL17RB SEFIR loop region to bind MLK4-K with a similar configuration as the IL17RB SEFIR loop region. Therefore, once the cyclic peptide binds MLK4. it prevents further binding of MLK.4-K to IL17RB SEFIR and subsequently terminates the phosphorylation of SEFIR Y447.

[0033] Figs. 7A-7B are graphs showing that cyclic peptide treatment suppresses the growth of pancreatic tumor progression. Fig. 7A shows that the progression of PDAC tumor growth in mice under various treatments, including vehicle, TAT-loop peptide, or cyclic peptide, was monitored weekly using IVIS signal. Fig. 7B shows the results plotted over time. Data represented as mean±SD. (n=4-5 for each group). *, P<0.05 (Two-tailed student t-test).

[0034] Fig. 8 shows that the activity of the serine / threonine kinase MLK4 isAttorney Docket No. 5992-0517PWO1 partially inhibited by the cyclic peptide. The various concentration of cyclic peptide was pre-incubated with recombinant human MLK4 (amino acids 114-420) for 30 minutes. The serine / threonine MLK4 activity was measured by incubating MLK4 kinase with the myelin basic protein (MBP) substrate at room temperature for 30 min. The reaction was subsequently ended and luminescence was measured by ADP-Glo™ reagent and Promega kinase detection kit, respectively. Data represented as mean±SD. (n=3)

[0035] Fig. 9 shows purification of the human IL-17RB SEFIR domain.Assessment of IL17RB SEFIR domain purity via SDS- and Native PAGE analysis revealed the formation of dimeric structures under native conditions.

[0036] Fig. 10 shows molecular dynamics simulations of different mutated synthetic cyclic peptides. Molecular dynamics simulations were employed to investigate the conformational dynamics and stability7of a series of synthetic cyclic peptides incorporating site-specific mutations.

[0037] Fig. 11 shows sequencing results of IL17RB C408S, K410R and MLK.4 kinase domain R216Amutants. Upper panel: Mutant of IL17RB C408S and K410R; Lower panel: Mutant of MLK4 R216.

[0038] Fig. 12 shows molecular dynamics simulations of new cyclic peptides with different linkers and compared to original cyclic peptide. Molecular dynamics simulations were conducted to evaluate the structural dynamics and stability of newly designed cyclic peptides incorporating various linkers. These results were subsequently compared to the original cyclic peptide to assess the impact of linker modifications on peptide conformation.DETAILED DESCRIPTION OF THE INVENTION

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0040] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component” includes a plurality of such components and equivalents thereof known to those skilled in the art.Attorney Docket No. 5992-0517PWO1

[0041] The term “comprise"’ or “comprising’" is generally used in the sense of include / including which means permiting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”

[0042] As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues linked via peptide bonds. The term “peptide” refers to a relatively short polypeptide composed of linked amino acids e.g., 200 amino acids or less, 175 amino acids or less, 150 amino acids or less e.g. 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less or 40 or less amino acids in length.

[0043] As used herein, "corresponding to," refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.

[0044] IL-17RB is one of the IL-17 receptors which are single-pass transmembrane proteins. IL-17RB as described herein can include human IL-17RB and its homologues from vertebrates, and particularly those homologues from mammals. Specifically, IL-17RB as described herein includes the IL-17RB amino acid sequences from human (SEQ ID NO: 1), and the IL-17RB amino acid sequences from other mammals (SEQ ID NOs: 2 to 9).

[0045] In structure, IL-17RB includes an extracellular domain, a transmembrane domain and an intracellular cytoplasmic tail. Specifically, the extracellular domain is located at positions corresponding to positions 18-289 of SEQ ID NO: 1, the transmembrane domain is located at positions corresponding to positions 290-312 of SEQ ID NO: 1, the intracellular cytoplasmic tail is located at positions corresponding to positions 313-502 of SEQ ID NO: 1, in which a flexible loop for MLK4 binding is located at positions 403-416.

[0046] Table 1 below shows the amino acid sequence of IL17RB from human and other mammals (SEQ ID NOs: 1 to 9).Table 1IL-17RB; interleukin- 17 receptor B [ Homo sapiens (human)]NCBI-GenelD: 55540Source: www.genome.jp / dbget-bin / www bget?hsa:55540Attorney Docket No. 5992-0517PWO1 Amino Acid Sequence (502 aa) (SEQ ID NO: 1) MSLVLLSLAALCRSAVPREPTVQCGSETGPSPEWMLQHDLIPGDLRDLRVEPVTTSVATG DYS I LMNVS WVLRADAS I RLLKATKI C VTGKSNFQS YSCVRCNYTEAFQTQTRPSGGKWT FSYIGFPVELNTVYFIGAHNIPNANMNEDGPSMSVNFTSPGCLDHIMKYKKKCVKAGSLW DPNITACKKNEETVEVNFTTTPLGNRYMALIQHSTIIGFSQVFEPHQKKQTRASVVIPVT GDSEGATVQLTPYFPTCGSDCIRHKGTVVLCPQTGVPFPLDNNKSKPGGWLPLLLLSLLV ATWVLVAGIYLMWRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCR SEVILEKWQKKKIAEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQ DLFPLAFNLFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELL HVKQQVSAGKRSQACHDGCCSLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 403- 416) _IL-17rb; interleukin- 17 receptor B [Pan troglodytes (Chimpanzee))!NCBI-GenelD: 460451S ource: https: / / www. ncbi. nlm, nih, gov / gene / 460451 _Amino Acid Sequence (505 aa) (SEQ ID NO: 2 ) MSLVLLSLAALCRSAVPREPTVQCGSETGPSPEWMLQHDLIPGDLRDLRVELVTTSVATG DYS I LMNVS WVLRADAS I RLLKATKI C VTGKSNFQS YSCVRCNYTEAFQTQTRPSGGKWT FSYVGFPVELNTVYFIGAHNIPNANMNEDGPSMPVNFTSPGCLDHIMKYKKKCVKAGSLW DPNITACKKNEETVEVNFTTTPLGNRYMALIQHSTHSTVIGFSQVFEPHQKKQTRASVVI PVTGDSEGAMVQLTPYFPTCGSDCIRHKGIVVLCPQTGVPFPLDNNKSKLGGWLPLLLLS LLVATWVLVAGIYLMWRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQN HCRSEVILEKWQKKKIAEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSE NSQDLFPLAFNLFCSDLRSQIHLHKYVVVYFRETDTKDDYNALSVCPKYHLMKDATAFCA ELLHVKQQVSAGKRSQACHDGCCSLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 406- 419) _IL-17rb; interleukin- 17 receptor B [Gorilla (Western lowland gorilla)]NCBI-GenelD: 101142225Source: https: / / www.ncbi.nlm.nih.gov / gene / 101142225 _Amino Acid Sequence (502 aa) (SEQ ID NO: 3) MSLVLLSLAALCRSAVPREPTIQCGSETGPSPEWMLQHDLIPGDLRDLRVEPVKTSVATG DYS I LMNVS WVLRADAS I RLLKATKI C VTGKSNFQS YSCVRCNYTEAFQTQTRPSGGKWT FSYIGFPVELNTVYFIGAHNIPNANMNEDGPSMSVNFTSPGCLDHIMKYTKKCVKAGSLW DPNITACKKNEETVEVNFTTTPLGNRYMALIQHSTI IGFSQVFEPHQKKQTRASVVIPVT GDSEGAMVQLTPYFPTCGSDCIRHKGTVVLCPQTGVPFPLDNNKSKPGGWLPLLLLSLLV ATWVLVAGIYLMWRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCR SEVILEKWQKKKIAEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQ DLFPLAFNLFCSDLRSQIHLHKYVVVYFRETDTKDDYNALSVCPKYHLMKDATAFCAELL HVKQQVSAGKRSQACHDGCCSLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 403- 416) _IL-I7rb: interleukin- 17 receptor B [Pteropus alecto (Black flying fox)]NCBI-GenelD: 102891713Attorney Docket No. 5992-0517PWO1 Source: htps: / / www.ncbi.nlm.nih.gov / gene / 102891713 _Amino Acid Sequence (454 aa) (SEQ ID NO: 4) MGGDDLAMSLMLLSLAALCWGAVSPEPTIQCGPETGPSPEWMVRHTLTPGDLRDLRVEPV KSSVASEDYSILMNISWILRADASIRLLKATKICVTGKSGFQSYGCVRCNYTEVFQTQSR PSGGKWMFFYIGFPVELNTLYFIGAHNIPNANMNEDSPSMSVNFTSPGSLWDPNITACKK NENMVEVNFTISPLGNRYMVLILKNTVIGTSVSEEKLTRTSVMVPVTGESEGAVVQLTPY FHTCGNDCIRRKGMVVLCPQTGVPFSPDNKKIKMSLSSTMLLPITVLVVYPSEICFHHTV CYFAEFLQNHCRSQVILDKWQKKKIAEMGPVQWLTTQKKAADKVIFLLSNDVNTiiiiti GKSEGSPCENSQDLFPLAFNLFCSDLRSQTHQHKYIVVYFREADTKDDYNALNVCPKYCL MKDATSFCMELLHVEQQVSTGKRLRACHNRCSSLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 355- 368)IL- 17rb: interleukin- 17 receptor B [Rattus norvegicus (Rat)]NCBI-GenelD: 306247Source: htps: / / www.ncbi.nlm.nih.gov / gene / 306247 _Amino Acid Sequence (354 aa) (SEQ ID NO: 5) MNEDSPSLSVNFTSPGCLNHVMKYKKQCIEAGSLWDPNITACKKNEKTVEVNFTTNSLGN RYMVLIRRDTMLGVSIVLENKLTRTSVVIPVNDESEGALVELTPYLHTCDNDCIRRKGTV VLCSETSAPFPPDDNRSMLRGWLPLLLVLLVATWVLAVGIYLTWRQGRSTKTSFPITAML LPLVKVLVVYPSEICFHHTVCRFTDFLQNYCRSEVILEKWQKKKIAEMGPVQWLTTQKQA ADKVVFLLPSDAPSLCDSACGHKEGSATENSQDLFPLAFNLFCSDFSSQTHLHKYLVVYL GGADLKGDYNALRVCPQYHLMKDAPAFHTELLKATQSMPLKKRPQACHGSCSPLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 255- 268)1L-I7rb; interleukin- 17 receptor B [Mus musculus (Mouse)]NCBI-GenelD: 50905Source: htps: / / www.ncbi.nlm.nih.gov / gene / 50905 _Amino Acid Sequence (499 aa) (SEQ ID NO: 6 ) MLLVLLILAASCRSALPREPTIQCGSETGPSPEWMVQHTLTPGDLRDLQVELVKTSVAAE EFSILMNISWILRADASIRLLKATKICVSGKNNMNSYSCVRCNYTEAFQSQTRPSGGKWT FSYVGFPVELSTLYLISAHNIPNANMNEDSPSLSVNFTSPGCLNHVMKYKKQCTEAGSLW DPDITACKKNEKMVEVNFTTNPLGNRYTILIQRDTTLGFSRVLENKLMRTSVAIPVTEES EGAVVQLTPYLHTCGNDCIRREGTVVLCSETSAPIPPDDNRRMLGGWLPLFLVLLVAVWV LAAGIYLTWRQGRSTKTSFPISTMLLPLIKVLVVYPSEICFHHTVCRFTDFLQNYCRSEV ILEKWQKKKIAEMGPVQWLTTQKQAADKVVFLLPSDVPTLCDSACGHNEGSARENSQDLF PLAFNLFCSDFSSQTHLHKYLVVYLGGADLKGDYNALSVCPQYHLMKDATAFHTELLKAT QSMSVKKRSQACHDSCSPLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 400- 413) _IL-17RB; interleukin- 17 receptor B [Equus caballus (Horse)]NCBI-GenelD: 100059421Source: htps: / / www.ncbi.nlm.nih.gov / gene / 100059421Attorney Docket No. 5992-0517PWO1 Amino Acid Sequence (499 aa) (SEQ ID NO: 7) MSLVLLSLAALCWGAVPREPTIQCGSEAGPSPEWMVQHALTPGDLRDLQVEPVKSRVATE DYSVLMNISWILRADASIRFLKATKICVTGKSNFQSYSCVRCNYTEAFQTQTRPSGGKWT FSYIGFPVELDTLYFIGAHNIPNANMNGDGPSLSVNFTSPGCLDHVMKYKKKCIEAGSLW DPNITACKKNEKMVEVNFTTSPLGNRYMALIQNNTVIGLSNVLENKLTRTSVVIPVTGES EGAVVQLTPYFHTCGSDCIRRRGIVVLCPHTGASSPPDNSRSVLGGWLPFLLPALLVATW VLAVGIYLTWRHERIKKTSFPTTALLSPIKVLVVYPSEICFHHTVCYFTKFLQNHCRTEV ILEKWQKKKIAEMGPVQWLTTQKKAADKVIFLLSNNVNNVCDGTCGKSEGSPHENSQDLF PLAFNLFCSDLRSQNHLHKYMVVYFREADTKDDYDALHVCPKYCLMKDAAAFCTELLHVE QH VS VGKRWRACHNRC SALNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 400- 413)IL-17rb; interleukin- 17 receptor B [Sus scrofa (Pig)]NCBI-GenelD: 100156014Source: https: / / www.ncbi.nlm.nih.gov / gene / 100156014 _Amino Acid Sequence (477 aa) (SEQ ID NO: 8) MLLVLLSLAALCWGAMPPEPTIQCGSEPGLSPEWMVRHALTPGDLRDLRVEPIKSSVAVE D YS I LMN I S W I LRADAS I RLLKATKI C VTGKSQKQT YS C VRCN YTEAFQTQTRPSGGKWM FSYVGFPVELNTRYFIGAHNIPNANMNEDGPSLAVNFTSPGCLDRIMKYKKKCIEAGSLW DPNITACKKSENTVEVNFTTSPLGNRYMALIQNSTVIGTSYVSELTPYFRTCGNDCIRRR GTVVRCPHTGVPFPQDQSRSMLSGWLPLLLLALLVAIWVLAGGIYLTRRHERIKKTSFSA TILLPPIKVLVVYPSEICFHHTVCYFTRFLQNHCRSEVILEKWQKKKIAEMGPVQWLTTQ KAAADKVIFLLSNDGNTACDGTCSNSEGGPHENSQDLFPLAFNLFCSDLRSQTHLHKYVV VYFREVDIKDDYSALSVCPTYHLMKDAPAFCKELLHAEQHVSVGRRLQACHYSCSSLNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 378- 391)IL-17rb; interleukin- 17 receptor B [Felis catus (Cat) (Felis silvestris catus)]NCBI-GenelD: 101080437,S ource: htps: / / www. ncbi.nlm, nih.gov / gene / 101080437 _Amino Acid Sequence (494 aa) (SEQ ID NO: 9) MSLALLSLAALCWGLVSTEPTIQCGSEPGPSPEWMVQHTLTPGDLRDLRVEPVRSRVAMD YSILMNVSWVLRADASIRLLKATKICVTGKSNLQSYSCVRCNYTEAFQTQTRPSGGRWTF SYVGFPVELNTVYFIGAHNIPNANINEDSPSMSVNFTSPGCLDHIMKYQKKCIKAGSLWD PNVTACKKNKTVVEVNFTTSPLGNKYMALIQNRIVIGFSNVLENKPPTRTSVVIPVTGES EGAMVQLTPYFHTCGNDCIRRKGTVVLCPQTGISFPLDGRRSMMGGWQPFLLPALLGATA LLAAGIYVIWRHKRIKKASFPTATLLSPIKVLVVYPSEICFHHTVCHFTEFLQNHCRSEV ILEEWQKKKIAEMGPVQWLTTQKKAVDKIIFLLSNDVNTMCDSTCDKSEGSPHENSQDLF PLAFNLFCSDLRNQTPLRKYMVVYFREADTKDEYSALSVCPKYRLMKDAPAFCTELLRVE QHMSAGKRLTACSMNote: The area with gray background indicates the flexible loop for MLK4 binding (aa 400-Attorney Docket No. 5992-0517PWO1

[0047] In function, IL-17RB can be activated upon stimulation by the cytokine IL-17B. IL-17RB forms ahomo-dimer upon IL-17B binding and recruits MLK4 through the flexible loop to phosphorylate it at position Y447, and the phosphorylated IL-17B in turn recruits TRIM56, an ubiquitin ligase, to add K63-linked ubiquitin chains on position K470. Activation of the IL-17B / IL-17RB signaling confers oncogenic activities. IL-17RB also can be recognized by IL-17E. However, unlike IL-17B, the binding of IL-17E to IL-17RB induces hetero-dimerization of IL-17RB with IL-17RAto activate Th2 immune responses which is not mediated by MLK4 phosph orylati on. An IL-17RB antagonist which targets the interaction between IL-17RB and MLK4, Y447 phosphorylation and / or K470 ubiquitination is useful for inhibiting IL-17B / IL-17RB activation and thus treating a disease or disorder associated therewith with. PCT application PCT / US2021 / 063226 describes a peptide based on the loop sequence of positions 403-416 of IL-17RB as IL-17RB antagonists, which is incorporated herein by reference in its entirety.

[0048] Table 2 below shows the amino acid sequences corresponding to positions 403-416 of IL-17RB in view of SEQ ID NO: 1.Table 2VCDGTCGKSEGSPS human aa 403-416 of SEQ ID NO: 1(SEQ ID NO: 10)VCDGTCGKSEGSPS chimpanzee aa 406-419 of SEQ ID NO: 2(SEQ ID NO: 10)VCDGTCGKSEGSPS gorilla aa 403-416 of SEQ ID NO: 3(SEQ ID NO: 10)ICDGTCGKSEGSPC fox aa 355-368 of SEQ ID NO: 4(SEQ ID NO: 11)LCDSACGHKEGSAT rat aa 255-268 of SEQ ID NO: 5(SEQ ID NO: 12)LCDSACGHNEGSAR mouse aa 400-413 of SEQ ID NO: 6(SEQ ID NO: 13)VCDGTCGKSEGSPH horse aa 400-413 of SEQ ID NO: 7(SEQ ID NO: 14)ACDGTCSNSEGGPH Pig aa 378-391 of SEQ ID NO: 8(SEQ ID NO: 15)MCDSTCDKSEGSPH cat aa 400-413 of SEQ ID NO: 9(SEQ ID NO: 16)

[0049] Table 3 below shows the amino acid sequences corresponding to positions 406-412 of IL-17RB in view of SEQ ID NO: 1.Table 2Attomey Docket No. 5992-0517PWO1 GTCGKSE human aa 406-412 of SEQ ID(SEQ ID NO: 17) NO: 1GTCGKSE chimpanzee aa 409-415 of SEQ ID(SEQ ID NO: 17) NO: 2GTCGKSE gorilla aa 406-412 of SEQ ID(SEQ ID NO: 17) NO: 3GTCGKSE fox aa 358-364 of SEQ ID(SEQ ID NO: 17) NO: 4SACGHKE rat aa 258-264 of SEQ ID(SEQ ID NO: 18) NO: 5SACGHNE mouse aa 403-409 of SEQ ID(SEQ ID NO: 19) NO: 6GTCGKSE horse aa 403-409 of SEQ ID(SEQ ID NO: 17) NO: 7GTCSNSE Pig aa 481-487 of SEQ ID(SEQ ID NO: 20) NO: 8STCDKSE cat aa 403-409 of SEQ ID(SEQ ID NO: 21) NO: 9

[0050] In the present invention, a peptide comprising the amino acid sequence corresponding to positions 406-412 of IL-17RB in view of SEQ ID NO: 1 in a cyclic form is developed, which acts as an IL-17RB antagonist, exhibiting improved effects in inhibition of tumor growth and metastasis via disrupting the interaction between IL-17RB and MLK4.

[0051] As used herein, the amino acid sequences of peptides disclosed herein are indicated from the N-terminus at the left side to the C-terminus at the right side of the respective depicted amino acid sequence. For cyclic peptides, the corresponding sequences are indicated from the side corresponding to the left side of the structural formula to the side corresponding to the right side of the structural formula.

[0052] As used herein, a “cyclic peptide” or “cyclopeptide” or a peptide in a cyclic form is a polypeptide chain forming a cyclic ring structure. A cyclic peptide can be a peptide fragment in which one amino acid residue is linked to another amino acid residue in the peptide fragment. The linking or cyclization can take place between head-to-tail, head-to-side chain, side chain-to-tail, and side chain-to-side chain. In particular, a cyclic peptide can be formed upon formation of a peptide bond between the amino and carboxy termini of a linear peptide. Side chains of two amino acid residues in the peptide may also combine to form rings, for example, through an ester, amide, disulfide or lanthionine bond. Specifically, an amide bond can be formed between the side-chain carboxyl group of an acidic amino acid residue and the side-Attorney Docket No. 5992-0517PWO1 chain amino group of a basic amino acid residue; an ester bond can be formed between the side-chain carboxyl group of an acidic amino acid residue and the sidechain hydroxyl group of a hydroxyl-containing amino acid residue; a disulfide bond can be formed between amino acid residues containing side chain sulfhydryl groups; and a lanthionine bridge can be formed by desulfurization of the corresponding disulfide. As a particular example, Cys-Cys cyclization utilized resulting from the formation of a disulfide bond between two cysteine residues of the peptide. In some embodiments, cyclization occurs by a linkage such as a Gly-Ser amide bond, a Ser-Ser amide bound and a Gly-Asp amide bond.

[0053] In some embodiments, a cyclic peptide as described herein comprises the amino acid sequences ofXI -X2-X3-X4-X5-X6-X7 Formula Iwherein XI is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). The cyclic peptide targets the interaction between IL-17RB and mixed-lineage kinase 4 (MLK4).

[0054] In some embodiments. XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G) or aspartic acid (D). In some embodiments, XI is glycine (G); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G). In some embodiments, X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, X1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

[0055] In some particular embodiments, the cy clic peptide comprises the amino acid sequence selected from the group consisting of GTCGKSE (SEQ ID NO: 17), STCDKSE (SEQ ID NO: 21), GTCGKTE (SEQ ID NO: 22), GTCGKSD (SEQ IDAttorney Docket No. 5992-0517PWO1 NO: 23) and GTCGAAA (SEQ ID NO: 24).

[0056] In some embodiments, a cyclic peptide as described herein is of formula IIY1-X1-X2-X3-X4-X5-X6-X7-Y2 Formula IIwhereinX1 is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A);Y 1 comprises 1 -5 amino acid residues at the amino terminus;Y2 comprises 1-5 amino acid residues at the carboxyl terminus; and cyclization occurs via a linkage between one amino acid residue of Y1 and one amino acid residue of Y2.

[0057] In some embodiments. XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G) or aspartic acid (D). In some embodiments, XI is glycine (G); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G). In some embodiments, X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, XI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A). In some embodiments, X1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

[0058] In some embodiments, Y1 in Formula II is selected from the amino acid / sequence C (Cys), GS (Gly-Ser), S (Ser), GC (Gly-Cys) and G (Gly). In some embodiments, Y2 in Formula II is selected from the amino acid / sequence C (Cys), S (Ser), and D (Asp). In some embodiments, Y1 comprises a terminal C (Cys) residue and Y2 comprises a terminal C (Cys) where a disulfide bond is formed between the two terminal cysteine residues. In some embodiments, Y1 comprises terminal GS (Gly-Ser) residues and Y2 comprises a terminal S (Ser) residue where an amide bond is formed between the amine group of the N-terminal glycine residue and the carboxy group of the C -terminal serine. In some particular embodiments, Y1 comprises aAttorney Docket No. 5992-0517PWO1 terminal S (Ser) residue and Y2 comprises a terminal S (Ser) residue where an amide bond is formed between the two terminal serine residues. In some embodiments, Y 1 comprises terminal GC (Gly-Cys) residues andY2 comprises a terminal S (Ser) residue where an amide bond is formed between the amine group of the N-terminal glycine residue and the carboxy group of the C-terminal serine. In some embodiments, Y 1 comprises terminal G (Gly) and Y2 comprises a terminal D (Asp) residue where an amide bond is formed between the amine group of the N-terminal glycine residue and the carboxy group of the C-terminal aspartic acid residue.

[0059] In some embodiments, a cyclic peptide of formula I comprises the amino acid sequence selected from the group consisting of cyclo (Y1-GTCGKSE-Y2) (Formula Ila), cyclo (Y1-STCDKSE-Y2) (Formula lib), cyclo (Y1-GTCGKTE-Y2) (Formula lie), cyclo (Y1-GTCGKSD-Y2) (Formula lid) and cyclo (Yl-GTCGAAA-Y2) (Formula lie) wherein Y1 and Y2 are as defined above.

[0060] Table 4 below shows the exemplified cyclic peptide of the present invention 1. cyclo (Y1-GTCGKSE-Y2) Formula Ila1.1 cyclo (CGTCGKSEC) a disulfide bond is formed between the two (SEQ ID NO: 25) terminal cysteine (C) residues1.2 cyclo (GSGTCGKSES) an amide bond is formed betw een the amine group (SEQ ID NO: 26) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S)1.3 cyclo (SGTCGKSES) an amide bond is formed betw een the two terminal (SEQ ID NO: 27) serine (S) residues1.4 cyclo (GCGTCGKSES) an amide bond is formed between the amine group (SEQ ID NO: 28) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S)1.5 cyclo (GGTCGKSED) an amide bond is formed betw een the amine group (SEQ ID NO: 29) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal aspartic acid (D) residue2. cyclo (Yl- STCDKSE-Y2) Formula lib2.1 cyclo (CSTCDKSEC) a disulfide bond is formed between the two (SEQ ID NO: 30) terminal cysteine (C) residues2.2. cyclo (GSSTCDKSES) an amide bond is formed between the amine group (SEQ ID NO: 31) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S)2.3 cyclo (SSTCDKSES) an amide bond is formed betw een the two terminal (SEQ ID NO: 32) serine (S) residues2.4 cyclo (GCSTCDKSES) an amide bond is formed between the amine group (SEQ ID NO: 33) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S)2.5 cyclo (GSTCDKSED) an amide bond is formed between the amine group(SEQ ID NO: 34) of the N-terminal glvcine (G) residue and theAttorney Docket No. 5992-0517PWO1 carboxy group of the C-terminal aspartic acid (D) residuecyclo (Y1-GTCGKTE-Y2) Formula liecyclo (CGTCGKTEC) a disulfide bond is formed between the two (SEQ ID NO: 35) terminal evsteine (C) residues. cyclo (GSGTCGKTES) an amide bond is formed between the amine group (SEQ ID NO: 36) of the N-terminal glycine (G) residue and the carboxv group of the C-terminal serine (S) cyclo (SGTCGKTES) an amide bond is formed between the two terminal (SEQ ID NO: 37) serine (S) residuescyclo (GCGTCGKTES) an amide bond is formed between the amine group (SEQ ID NO: 38) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S) cyclo (GGTCGKTED) an amide bond is formed between the amine group (SEQ ID NO: 39) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal aspartic acid (D) residuecyclo (Y1-GTCGKSD-Y2) Formula lidcyclo (CGTCGKSDC) a disulfide bond is formed between the two (SEQ ID NO: 40) terminal evsteine (C) residuescyclo (GSGTCGKSDS) an amide bond is formed between the amine group (SEQ ID NO: 41) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S) cyclo (SGTCGKSDS) an amide bond is formed between the two terminal (SEQ ID NO: 42) serine (S) residuescyclo (GCGTCGKSDS) an amide bond is formed between the amine group (SEQ ID NO: 43) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S) cyclo (GGTCGKSDD) an amide bond is formed between the amine group (SEQ ID NO: 44) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal aspartic acid (D) residuecyclo (Y1-GTCGAAA-Y2) Formula liecyclo (CGTCGAAAC) a disulfide bond is formed between the two (SEQ ID NO: 45) terminal evsteine (C) residuescyclo (GSGTCGAAAS) an amide bond is formed between the amine group (SEQ ID NO: 46) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal serine (S) cvelo (SGTCGAAAS) an amide bond is formed between the two terminal (SEQ ID NO: 47) serine (S) residuescyclo (GCGTCGAAAS) an amide bond is formed between the amine group (SEQ ID NO: 48) of the N-terminal glycine (G) residue and the carboxv group of the C-terminal serine (S) cyclo (GGTCGAAAD) an amide bond is formed between the amine group (SEQ ID NO: 49) of the N-terminal glycine (G) residue and the carboxy group of the C-terminal aspartic acid (D)residueAttorney Docket No. 5992-0517PWO1

[0061] In some embodiments, Y1 comprises 1-4 amino acid residues at the amino terminus; and / or Y2 comprises 1-4 amino acid residues at the carboxyl terminus. In some embodiments, Y1 comprises 1-3 amino acid residues at the amino terminus; and / or Y2 comprises 1-3 amino acid residues at the carboxyl terminus. In some embodiments, Y1 comprises 1-2 amino acid residues at the amino terminus; and / or Y2 comprises 1-2 amino acid residues at the carboxyl terminus. In some embodiments, Y1 comprises one amino acid residue at the amino terminus; and / or Y2 comprises one amino acid residue at the carboxyl terminus.

[0062] In some embodiments, a cyclic peptide as described herein comprises 9 to 17 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 16 amino acid residues in length. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 15 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 14 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 13 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 12 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 11 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 to 10 amino acid residues. In some particular embodiments, a cyclic peptide as described herein comprises 9 amino acid residues.

[0063] In particular, a cyclic peptide as described herein does not include a cyclic peptide comprising the amino acid sequence set forth in Table 2.

[0064] The cyclic peptides of the present invention may be prepared by conventional procedures known in the art. In general, the preparation of the cyclic peptides is carried out by first synthesizing a linear peptide of the desired sequence followed by a cyclization step. In some embodiments, the cyclic peptides of the present invention may be synthesized by solution phase procedure which permits a condensation between the free alpha amino group of an amino acid residue having its carboxyl group and other reactive groups protected and the free primary carboxyl group of another amino acid residue having its amino group or other reactive groups protected. In some embodiments, the cyclic peptides of the present invention may be synthesized by solid-phase synthesis which can be carried out by sequentially incorporating the desired amino acid residues one at a time into thegrowing peptide chain according to the general principles of solid phase methods.Attorney Docket No. 5992-0517PWO1

[0065] In additional embodiments, the IL-17RB inhibitory peptide as described herein may be a variant thereof with one or more mutations. It is understandable that a polypeptide may have a limited number of changes or modifications that may be made within a certain portion of the polypeptide irrelevant to its activity or function and still result in a functionally equivalent variant with an acceptable level of equivalent or similar biological activity or function. In some examples, the amino acid residue mutations are conservative amino acid substitution, which refers to the amino acid residue of a similar chemical structure to another amino acid residue and the polypeptide function, activity or other biological effect on the properties smaller or substantially no effect. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skills in the art such as those found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (i) A, G; (ii) T, S; (iii) Q, N; (iv) D, E; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

[0066] As used herein, the term “substantially identical” refers to two sequences having 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, still even more preferably 90% or more, and most preferably 95% or more or 100% identity.

[0067] To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first nucleotide sequence for optimal alignment with a second nucleotide sequence). In calculating percent identity, typically exact matches are counted. The determination of percent homology or identity between two sequences can be accomplished using a mathematical algorithm known in the art, such as BLAST and Gapped BLAST programs, the NBLAST and XBLAST programs, or the ALIGN program.

[0068] In some embodiments, the peptide of the present invention can be said to be “purified” if it is substantially free of chemical precursors or other chemicals that may be involved in the process of peptide preparation. It is understood that the term “purified” does not necessarily reflect the extent to which the peptide has been “absolutely” purified e.g. by removing all other substances (e.g., impurities or cellularAttorney Docket No. 5992-0517PWO1 components). In some cases, for example, a purified peptide includes a preparation containing the peptide having less than 50%, 40%, 30%, 20% or 10% (by weight) of other proteins (e.g. cellular proteins), having less than 50%, 40%, 30%, 20% or 10% (by volume) of culture medium, or having less than 50%, 40%, 30%, 20% or 10% (by weight) of chemical precursors or other chemicals involved in synthesis procedures. The term “isolated'’ or “purified” can also apply in the nucleic acid of the present invention.

[0069] In particular, the peptide of the present invention is capable of disrupting the interaction between IL-17RB and MLK4. In some embodiments, the peptide of the present invention is effective in disrupting the interaction between IL-17RB and MLK.4 by 50% or more, 55% or more. 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% (complete disruption), compared with IL-17RB and MLK4 interaction without treatment of the peptide of the present invention.

[0070] According to the present invention, an effective amount of a cyclic peptide as described herein may be formulated with a physiologically acceptable carrier into a composition of an appropriate form for the purpose of delivery and absorption. The composition of the present invention particularly comprises about 0.1% by weight to about 100% by eight of the active ingredient, wherein the percentage by weight is calculated based on the weight of the whole composition. In some embodiments, the composition of the present invention can be a pharmaceutical composition or medicament for treatment.

[0071] As used herein, “phy siologically acceptable” means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize said active ingredient and is safe to the receiving individual. Said earner may be a diluent, vehicle, excipient, or matrix to the active ingredient. Some examples of appropriate excipients include lactose, sucrose, dextrose, sorbose, mannose, starch, Arabic gum, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, sterilized water, syrup, and methylcellulose. The composition may additionally comprise lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxy benzoates; sweeteners; and flavoring agents. The composition of the present invention can provide the effect of rapid, continued, or delayed release of the active ingredient after administration to theAttorney Docket No. 5992-0517PWO1 patient.

[0072] According to the present invention, the form of the composition may be tablets, pills, powder, lozenges, packets, troches, elixers, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, and packaged powder.

[0073] The composition of the present invention may be delivered via any physiologically acceptable route, such as oral, parenteral (such as intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. Regarding parenteral administration, it is preferably used in the form of a sterile water solution, which may comprise other substances, such as salts or glucose sufficient to make the solution isotonic to blood. The water solution may be appropriately buffered (e.g. with a pH value of 3 to 9) as needed. Preparation of an appropriate parenteral composition under sterile conditions may be accomplished with standard pharmacological techniques well known to persons skilled in the art.

[0074] In this present invention, a cyclic peptide as described herein, acting as a IL-17RB antagonist, exhibits superior effects in inhibition of tumor growth and metastasis via disrupting the interaction between IL-17RB and MLK4 and inhibiting IL-17RB / IL-17RB activation. Therefore, the present invention provides a method for inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with IL-17B / IL-17RB activation via administering an effective amount of a cyclic peptide as described herein to a subject in need thereof.

[0075] To practice the method disclosed herein, an effective amount of a composition such as a pharmaceutical composition described herein, comprising a cyclic peptide as described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route. The term ‘'effective amount” used herein refers to the amount of an active ingredient to confer a desired biological effect in a treated subject or cell. The effective amount may change depending on various reasons, such as administration route and frequency, body weight and species of the individual receiving said pharmaceutical, and purpose of administration. Persons skilled in the art may determine the dosage in each case based on the disclosure herein, established methods, and their own experience.

[0076] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to. farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subjectAttorney Docket No. 5992-0517PWO1 who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease / disorder, such as cancer. A subject suspected of having any of such target disease / disorder might show one or more symptoms of the disease / disorder. A subject at risk for the disease / disorder can be a subject having one or more of the risk factors for that disease / disorder.

[0077] In some embodiments, abnormal IL-17RB activation is associated with a proliferation disorder e.g. a cancer and a metastasis thereof.

[0078] In certain embodiments, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary tract cancer, gallbladder and bile duct cancer, mammary cancer, ovarian cancer, cervical cancer, uterine body cancer, bladder cancer, prostate cancer, testicular tumor, osteogenic and soft-tissue sarcomas, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and pleural malignant mesothelioma.

[0079] In another particular example, the cancer is pancreatic cancer.

[0080] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0081] Examples

[0082] The IL-17B / IL-17RB oncogenic signaling axis promotes pancreatic cancer progression through interaction with mixed-lineage kinase 4 (MLK4). Here, we improved the effectiveness of a therapeutic peptide that disrupted MLK4 binding by converting its linear structure into a cyclic form. The modified cyclic peptide inhibited pancreatic cancer cell growth and metastasis, outperforming the original linear peptide both in vitro and in an orthotopic mouse model. At the molecular level, cysteine residues, particularly C408 in IL-17RB, were important for mediating interactions with arginine 216 within MLK4 kinase domain. This interaction was fundamental to the efficacy of the cyclic peptide. Additionally, lysine 410 in IL-17RB was essential for maintaining the structural integrity7of the cyclic peptide as a protein-protein disruptor These findings provide a deeper understanding of the IL-17RB-MLK4 interaction, offering insights for developing therapeutic agents targeting this pathway in pancreatic cancerAttorney Docket No. 5992-0517PWO1

[0083] 1. Material and Methods

[0084] 1.1 Experimental model and study participant details of modeling the structure of the human IL-17RB SEFIR domain

[0085] The initial three-dimensional (3D) molecular structural model of the SEFIR domain in interleukin 17 receptor B (IL-17RB) was created by homology modeling applications in SWISS-MODEL29and refined by using the Prime module of the Schrodinger suite (Schrodinger, LLC, New York) with OPLS_2005 force field.Because SEFIR domains can structurally resemble each other, we applied the crystal structure of IL-17RB-SEFIR (PDB ID: 3VBC)4and the crystal structures of IL17RA (PDB ID: 4NUX) and a prokaryotic SEFIR domain (PDB ID: 5Y8F)30as templates for the structural model of the segment (amino acids 403-416) in the IL-17RB-SEFIR domain (refer to 3VBC).

[0086] 1.2 Cyclic peptide synthesis

[0087] The cyclic peptide amide was manually synthesized using standard Fmoc solid-phase synthesis protocols. The peptide was assembled on a 0.1 mmol scale Fmoc-Rink-amide polystyrene resin with a loading of 0.69 mmol / g. Side chains of Fmoc-amino acids were protected as follows: Cys(Acm), Cys(Trt), Glu(tBu), Lys(Boc), Ser(tBu), and Thr(tBu).

[0088] A peptide containing two Cys(Acm) residues was converted to the corresponding disulfide bridge cystine peptide by thallium trifluoroacetate oxidation. For direct disulfide bond formation on the solid phase, peptidyl resin was suspended in DMF and thallium trifluoroacetate (1.2 eq. relative to peptide) at 0°C for 80 min and washed with DMF.

[0089] The peptidyl resin was cleaved with TFA / triisopropylsilane (TIPS) / H₂O (98:1:1) for 2 h with stirring at room temperature. The product was precipitated and washed with cold diethyl ether and lyophilized from 0.05% TFA in ACN / H₂O (1:1). The crude peptide with correct mass was purified using preparative RP-HPLC.

[0090] 1.3 Cell culture

[0091] Human 293T cells (CRL-3216) and CFPAC1 cells (CRL-1918) were purchased from ATCC. Human 293T cells (CRL-3216) were cultured in high glucose DMEM medium. CFPAC1 (CRL-1918) cells were cultured in IMDM. All medium was supplemented with 10% FBS, 2 mM L-glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10 μg / mL antibiotics / antimycotics or penicillin and streptomycin (100 IU / mL and 100 μg / mL, respectively). Cell subculture wasAttorney Docket No. 5992-0517PWO1 performed by using StemPro Accutase (Stemcell Technologies) or Accutase solution (Sigma Aldrich). All cells were maintained in a 37°C incubator containing 5% CO2 and were regularly checked for mycoplasma infection.

[0092] 1.4 Soft agar colony formation assay

[0093] Soft agar colony formation assay for measuring the anchorage-independent growth of cancer cells was performed as described6. In brief, 500 cells were seeded in a layer of 0.35% agar or complete growth medium over a layer of 0.5% agar or complete growth medium in a 96-well plate. The cell medium, containing varying concentrations of the cyclic peptide, was replenished daily for 1 week. On day 7, Hoechst 33342 (1:200), diluted in PBS, was added to each well of a 96-well plate (20 μL / well), and automated images were captured using ImageXpress Micro High-Content Imaging System. Subsequently, the number of colonies was determined using an automated analysis program with a size cut-off of >50 μm.

[0094] 1.5 Spheroid formation assay

[0095] For the spheroid (organoid) formation assay, 600 CFPAC1 and 5000 BxPC3 cells were seeded on a Coming 96-well transparent flat bottom ultra-low attachment microplate. IMDM or RPMI, containing 0.5% Matrigel (Coming) and various concentrations of the cyclic peptide, was replenished daily over 6 d. Spheroids >50 μm in size were counted and analyzed using light microscopy on day 6.

[0096] 1.6 Orthotopically xenografted animal model

[0097] To assess the antitumorigenic effects of the loop and cyclic peptides, an orthotopically xenografted animal model was established by injecting GFP / luciferase-labeled CFPAC1 cells into the pancreatic tail. In detail, NOD-SCID gamma mice were anesthetized with 3.5% isoflurane. Their abdominal area was sterilized with 75% ethanol before conducting laparotomy. Mice were locally shaved, and a small wound (5-10 mm) was made at the left upper quadrant of the abdomen, where the spleen was approximately beneath. The pancreas was exteriorized and injected with 2.5 x 105CFPAC1 cells. To prepare cells for injection, 8 x 106GFP / luciferase-labeled CFPAC1 cells were suspended in 800 pL Matrigel (BD-Bioscience), and only 25 μL was injected into the pancreatic tail of mice using a 29-gauge needle (BD-Bioscience). The wound was closed with a 5-0 PDS II violet suture (Ethicon) and the AutoClip system (Braintree Scientific). One week after surgery, tumor growth in all xenografted mice was assessed using an in vivo image system (intraperitoneal injection of 150 mg / kg D-luciferin). Only mice with baseline signal at 1 x 104Attorney Docket No. 5992-0517PWO1 (photon / s / cm2 / sr) were divided into three groups on the basis of treatment received as follows: 1) vehicle, 2) TAT-loop peptide, and 3) cyclic peptide, respectively. Mice were intraperitoneally injected twice a week with vehicle (PBS 10 mL / kg), TAT-loop peptide (160 mg / kg), or cyclic peptide (160 mg / kg) for 4 weeks. Tumor volume was monitored using an in vivo image system (intraperitoneal injection of 150 mg / kg D-luciferin). In week 5. the mice were euthanized and tumor weight was measured. Collected lungs were immersed in PBS containing D-luciferin (150 pg / mL) to visualize metastatic cancer cells.

[0098] 1.7 Co-IP assay and immunoblot analysis

[0099] HEK293T stably expressing GFP-IL-17RB, CFPAC-1 cells, BxPC3 were starved for 2 h and treated with 50 ng / mL rIL-17B (R& D systems) for 5 min. During starvation, 10 μM cyclic peptide with 4 x diluted series was added to the culture dishes. Cells were lysed by adding 1 mL of lysis 125 buffer (50 mM Tris [pH 7.4], 125 mM NaCl, 5 mM ethylenediaminetetraacetic acid [EDTA], 5 mM EGTA, 0.1% Nonidet P-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 x PhosSTOP, and 1 x proteinase inhibitor cocktail [Roche, Indianapolis, IN]) and subjected to three freeze-thaw cycles using liquid nitrogen. Protein concentration was determined by the Bradford method. For immunoprecipitation, 1.5 or at least 0.8 mg protein was used. GFP-Trap Magnetic Agarose (Chromotek) and anti-MLK4 antibody (E-ll) conjugated agarose (Santa Cruz) were used for IP GFP-IL-17RB and endogenous MLK4, respectively. Protein lysate underwent a pulldown process for 2 h (GFP-Trap) or overnight (anti-MLK4 Agarose) at 4°C, followed by western blotting with respective antibodies to detect Flag-MLK4, GFP-IL-17RB, pY447 of IL-17RB, IL-17RB, and p84.[000100] HEK293T cells stably expressing GFP-IL-17RB underwent transfection with MLK4-His truncated domains SK (SH3 + kinase), K (kinase only), or KL (kinase + doublet leucine zipper) using TransIT-LTl Transfection Reagent (Mirus Bio). Briefly. 3 pg of plasmid DNA was mixed with 500 pL of opti-MEM and 9 pL of TransIT-LT 1 reagent. The mixture was incubated at room temperature for 20 min and added to cultured cells. After 24 h, the cells were starved for 2 h with the addition of 10 μM of either the loop or cyclic peptide and treated with 50 ng / mL rIL-17B (R& D Systems) for 5 min. The cell lysates were collected using lysis 125 buffer. In the overexpressed system, GFP-Trap Magnetic Agarose (Chromotek) was employed to pulldown GFP-IL-17RB, followed by western blotting with respective antibodies toAttorney Docket No. 5992-0517PWO1 detect MLK4-His, GFP-IL-17RB, β-actin, and GAPDH.[000101] 1.8 Assessment of MLK4 Kinase activity[000102] The influence of the cyclic peptide on MLK4 serine / threonine kinase activity was assessed utilizing MLK4 Kinase Enzyme System (Promega, Cat# VA7504) in accordance with the manufacturer’s protocol. The assay utilized recombinant human MLK4 (amino acids 114-420) as the kinase, with myelin basic protein serving as the substrate. Kinase activity was quantified through bioluminescence using ADP-Glo Kinase Assay.[000103] 1.9 Protein purification and biolayer interferometry[000104] For purification of the His-tagged IL-17RB SEFIR domain, the pET15b-IL-17RB SEFIR-His vector was transformed in BL21(DE3) bacteria, which were cultured in Terrific-Broth medium. Protein induction was performed under conditions of OD₆₀₀ = 0.5-0.8 and 0.1 mM isopropyl-thio-galactopyranoside at 16°C for 12 h. The cells were lysed in lysis buffer (50 mM HEPES, 500 mM NaCl, 20 mM imidazole, 5 mM DTT. 0.5 mM CHAPS, and protease inhibitor cocktail [pH 7.4]). The homogeneity of the cell lysate was enhanced through microfluidization, and the supernatant was obtained by centrifugation at 4°C and 12,000 x g for 20 min. The supernatant was applied to a Ni-NTA column pre-equilibrated with lysis buffer. The column underwent three washes with a buffer that gradually increased in imidazole concentrations (20-30-50 mM, containing 50 mM HEPES, 500 mM NaCl, 0.5 mM DTT, 0.5 mM CHAPS, and 30% glycerol). Proteins were eluted using the elution buffer (50 mM HEPES, 500 mM NaCl, 300 mM imidazole, 0.5 mM DTT, 0.5 mM CHAPS, and 30% glycerol) and dialyzed in storage buffer (50 mM HEPES, 500 mM NaCl, 0.5 mM DTT, 0.5 mM CHAPS, and 30% glycerol), preparing them for biolayer interferometry.[000105] Biolayer interferometry kinetic assay was conducted using Octet HTX system. In brief, GST-MLK4 kinase domain was biotinylated at a 1: 1 molar ratio and immobilized on a high-capacity Super Streptavidin biosensor. Excess biotinylated reagent remaining after labeling was removed through desalting columns with an appropriate molecular weight cut-off.[000106] 1.10 Plasmid generation and site-directed mutagenesis[000107] Flag-tagged MLK4 (NM_032435) and Flag-tagged IL-17RB(NM_018725.3) were purchased from OriGene. To construct the truncated MLK4Attorney Docket No. 5992-0517PWO1 domains, the coding regions of various MLK4 truncated domains were PCR-amplified using primers designed with Hind III (New England Biolabs) and Xhol (New England Biolabs) cutting sites. The open reading frame of the truncated MLK4 domains was then inserted into pcDNA3.1 myc-His A, resulting in the fusion of all truncated MLK4 domains with a myc-His (6×) tag at the C-terminal. Mutations in IL-17RB-tagged-Flag (C408S and K410R) and MLK4-kinase-domain-His (6×) (R216A) were introduced using site-directed mutagenesis. In brief, primers containing the mutated sites were PCR-amplified with the vector using Herculase II Fusion Enzyme with dNTPs Combo Kit (Agilent Technologies). The methylated strand was digested with Dpnl for 1 h (New England Biolabs) and transformed. For lentivirus infection and generation of a HEK293T stable clone overexpressing IL-17RB, the viral plasmid pLenti-C-mGFP-P2A-Puro with IL-17RB-ORF (NM 018725) was purchased from OriGene. The human SEFIR (L328-K483) domain of IL-17RB was cloned into pET-15b using a PCR-based method and used for protein purification. All vectors involving PCR amplification were verified by DNA sequencing.[000108] 1.11 Structure simulation[000109] MD simulation of MLK4 kinase domain-peptide complex was conducted using GROMACS with the CHARMM27 force field in a 100 cm’ simulation box. TIP3P water molecules and salt concentration were included to mimic the bioassay conditions. The temperature was set to 310 K. and the calculated binding free energy of the complex was -29.26 kcal / mol.[000110] 1.12 Native gel[000111] The native gel was run without SDS in all steps at 4°C. The running buffer comprised 30 mM Tris, 300 mM glycine, and 2 mM EDTA at pH 8.3. The 4% and 10% acrylamide gels were prepared under typical PAGE conditions for the stacking gel (pH 6.8) and separating gel (pH 8.8), respectively.[000112] 1.13 Statistics analysis[000113] A two-tailed Student’s t test was performed to compare two groups. (*), (**), (***). and (****) indicate / * < 0.05, P < 0.01, P < 0.001, and / < 0.0001, respectively. The data are shown in the figure legends with the indicated number of independent experiments (n) and presented as means ± SD. The analyses were performed using Excel, SigmaPlot 10, and GraphPad Prism 10.0.3 (GraphPad Software, USA).[000114] 2. ResultsAttorney Docket No. 5992-0517PWO1[000115] 2.1 The cyclic peptide exhibited enhanced therapeutic efficacy in suppressing tumor growth and metastasis in both in vitro cell culture systems and an orthotopic xenograft mouse model of pancreatic cancer[000116] The standalone loop peptide (IL-17RB403-416) cannot penetrate cells, and the extended TAT-loop peptide is inherently unstable. To improve the therapeutic effectiveness of the loop peptide, we developed a cyclic peptide based on the alignment of flexible loops from diverse species within IL-17RB and the structure of the human IL-17RB SEFIR domain, encompassing the loop sequence IL-17RB403-416 (Figs. lAand IB). The cyclic peptide sequence (IL-17RB406-412, GTCGKSE, SEQ ID NO: 17, plus two terminal cysteine (C) residues as a linker, shown in yellow-red, CGTCGKSEC, SEQ ID NO: 25) features a coil-to-alpha-tum structure and is generated to cyclic form by disulfide bonds between two C residues (Fig. IB).[000117] To evaluate the therapeutic potential of these peptides, we employed spheroid formation and soft agar colony formation assays to assess tumor growth inhibition in CFPAC1 pancreatic cancer cells, which are characterized by high IL-17RB expression9. The cyclic peptide demonstrated markedly superior efficacy compared with the TAT-loop peptide, exhibiting approximately 26-fold and 196-fold lower IC50 values in suppressing spheroid and colony formation, respectively (Figs.1C and ID). To validate the in vitro findings, we utilized an orthotopic xenograft model to investigate the in vivo antitumorigenic and antimetastatic effects of the cyclic peptide. In this model, the cyclic peptide significantly outperformed the TAT-loop peptide, demonstrating a marked delay in tumor growth (Figs. 7A and 7B), reduction in tumor weight (Fig. IE), and potent suppression of lung metastasis (Fig. IF). Collectively, these results underscore the enhanced therapeutic efficacy of the cyclic peptide in inhibiting pancreatic tumor growth and metastasis in vitro and in vivo.[000118] 2.2 MLK4 kinase domain binds IL-17RB, but the SH3-kinase-leucine zipper domains are the minimum MLK4 region required for this interaction following IL-17B stimulation[000119] To investigate how the cyclic peptide disrupts the IL-17RB-MLK4 interaction, we initially performed coimmunoprecipitation (co-IP) assays to identify the MLK4 domain responsible for IL-17RB binding. We expressed various His-tagged truncated MLK.4 constructs (Fig. 2A) in 293T cells stably expressing GFP-IL-17RB. Our results revealed that all truncated MLK4 fragments containing both SH3Attorney Docket No. 5992-0517PWO1 and kinase domains interacted with GFP-IL-17RB (Figs. 2B and 2C). Interestingly, the smallest fragment, encompassing only the SH3-kinase-leucine zipper (SKL) domains, showed the strongest binding affinity, whereas the fragment containing the proline-rich domain displayed the weakest. Furthermore, the SKL domains formed the minimal region within MLK4 necessary for the IL-17B-stimulated interaction with IL-17RB.[000120] To confirm these findings and assess the independent binding ability of the kinase domain, we generated truncated MLK4-K and MLK4-KL plasmids for co-IP (Figs. 2D and 2E). These results confirmed the finding that MLK4 kinase domain alone is sufficient for binding to IL-17RB, with the adjacent SH3 and leucine zipper domains promoting this interaction to a lesser extent.[000121] In conclusion, MLK4 kinase domain is essential for its interaction with IL-17RB, and the MLK4-SKL region is the minimal functional unit required for IL-17B-induced binding between IL-17RB and MLK4.[000122] 2.3 The cyclic peptide disrupted the IL-17RB-MLK4 interaction by directly targeting and binding to MLK4 kinase domain[000123] To elucidate how the cyclic peptide disrupts the IL-17RB-MLK4 interaction, we employed HEK293T cells stably expressing GFP-IL-17RB. The cells were further transfected to coexpress full-length Flag-tagged MLK4 or a truncated variant containing only the His-MLK4 SKL domain. Treatment with the cyclic peptide abrogated the interaction between IL-17RB and full-length and SKL domaincontaining MLK4 (Figs. 3A and 3B). Notably, the cyclic peptide disrupted the MLK4-K-IL-17RB and MLK4-SKL-IL-17RB complexes even without IL-17B stimulation (Fig. 3C). This suggests that the cyclic peptide directly interacted with MLK4 kinase domain, thereby impeding its association with IL-17RB.[000124] To confirm that the cyclic peptide disrupted the IL-17RB-MLK4 interaction through direct binding to MLK4 kinase domain, we used biolayer interferometry (Octet system). Commercially available GST-MLK4-K and purified His-IL-17RB SEFIR (confirmed as a dimer in its native state. Fig. 8) were employed. Kinetic biolayer interferometry analysis revealed that the cyclic peptide disrupted the IL-17RB-MLK4 kinase domain interaction in a dose-dependent manner, with an EC50 value of 6.752 pM (Fig. 3D). This was observed when the immobilized MLK4 kinase domain on the biosensor was pretreated with the cyclic peptide, followed by incubation with the His- IL-17RB-SEFIR domain. Additionally, the cyclic peptideAttorney Docket No. 5992-0517PWO1 displayed only a minor inhibitory effect on the serine / threonine kinase activity of MLK4 (Fig. 9), suggesting its high selectivity as an IL-17RB and MLK4 disruptor.[000125] In conclusion, the cyclic peptide disrupted the IL-17RB-MLK4 interaction by binding to MLK4 kinase domain, with minimal impact on its serine / threonine kinase activity7.[000126] 2.4 Interaction between the central C residue of the cyclic peptide and R216 of MLK4 is crucial for disrupting the IL-17RB-MLK4 complex [000127] To understand how the cyclic peptide interacts with MLK4 kinase domain, we synthesized various mutated cyclic peptides by altering the conserved, charged, and polar residues in the original cyclic peptide (Fig. 4A and Fig. 10). Subsequently, we evaluated the disruptive efficacy of the synthetic cyclic peptides using co-IP. This involved expressing His-MLK4-K in 293T cells stably expressing GFP-IL-17RB. The objective was to identify the key residues responsible for binding to MLK4 kinase domain.[000128] We identified C4 (C residue at position 4) and K6 (K residue at position 6) within the cyclic peptide as critical for binding to MLK4 kinase domain (Fig. 4B). Because the cyclic peptide is derived from the loop region of the SEFIR domain of IL-17RB, which is essential for MLK4 interaction, we introduced site-directed mutagenesis in Flag-IL-17RB, replacing C408 (corresponding to C4) with serine and K.410 (corresponding to K6) with the R residue (Fig. 11). co-IP using His-tagged MLK4-K and wild-type or mutant Flag-IL-17RB demonstrated a significant reduction in binding affinity7between MLK4-K and the C408S mutant, but not the K410R mutant, compared with wild-type IL-17RB (Figs. 4C and 4D). These findings suggest that C408 in IL-17RB is essential for its interaction with MLK4 kinase domain.[000129] To identify the residues within MLK4 kinase domain that interact with the cyclic peptide, we employed molecular simulation using the structural information of MLK4 kinase domain (PDB: 4YAU) and the cyclic peptide (Fig. 4E). The simulations revealed that R216 in MLK4 kinase domain forms strong chemical bonds with C4, S7, and E8 residues in the cyclic peptide (Fig. 4E). To further validate this interaction, we generated an R216A mutant of MLK4 kinase domain (Fig. 11) and assessed its binding affinity to IL-17RB. The mutant exhibited decreased binding affinity7to GFP-IL-17RB compared with the wild-type. The cyclic peptide could not disrupt the interaction between the R216A mutant MLK4 kinase domain and IL-17RB (Fig. 4F), emphasizing the crucial role of R216 in mediating cyclic peptide binding.Attorney Docket No. 5992-0517PWO1[000130] Although molecular simulations suggested a strong interaction between C4 of the cyclic peptide and R216 of MLK4, no predicted interactions were observed between the kinase domain and K6 of the cyclic peptide, despite its functional importance. Intriguingly, molecular dynamics (MD) simulation data indicated that mutating K6 to R could indirectly affect the side chains of C4, S7, and E8 in the cyclic peptide, leading to a loss of binding to R216 of MLK4 (Fig. 10). These findings collectively highlight the essential role of the C4 residue in the cyclic peptide for its direct interaction with R216 in MLK4 kinase domain, whereas K6 appears to be critical for maintaining the overall structural integrity of the cyclic peptide, enabling its function as a disruptor of the IL-17RB-MLK4 interaction.[000131] 2.5 Even with sequence variations, maintaining the cyclic peptide’s structure effectively disrupted the IL-17RB-MLK4 interaction and suppressed tumor cell growth[000132] To examine if peptide cyclization with varied sequences and an amide bond linkage method impacted functionality, we generated cyclic peptides with differing sequences but similar structures compared with the original cyclic peptide (Figs. 5 A, 5C, 5E, and Fig. 12). We found that various cyclic peptides disrupted the IL-17B-induced IL-17RB-MLK4 interaction (Figs. 5B, 5D, and 5F) and inhibited tumor sphere formation. Notably, the cyclic peptide variant with amide bond linkage between GS residues and high structural similarity to the original cyclic peptide was most effective in disrupting the interaction, suggesting its potential as a candidate for pancreatic cancer treatment (Figs. 5A-5G and Fig. 12). These findings underscore the importance of cyclic peptide structure in biological activity.[000133] 3. Discussion[000134] In this study, we have developed a novel cyclic peptide that has better therapeutic efficacy than the TAT-loop peptide. The cyclic peptide effectively suppressed the growth and metastasis of pancreatic cancer cells (Figs. 1D-1F). We identified MLK4 kinase domain as sufficient for interaction with IL-17RB (Figs. 2A-2E) and elucidated how the cyclic peptide disrupts the IL-17RB-MLK4 interaction (depicted in the proposed model in Fig. 6). In the initial step of IL- 17B / IL- HRB-mediated oncogenic signaling, the interaction between R216 of MLK4 kinase domain and C408 within the loop region of the IL-17RB SEFIR domain induces conformational changes that reorient the knot structure, bringing the two domains into close proximity. This positioning places the Y447 site on the IL-17RB SEFIR domainAttorney Docket No. 5992-0517PWO1 close to the active site of MLK4 kinase domain, facilitating Y447 phosphorylation and activation of downstream oncogenic signaling pathways. Conversely, when a cyclic peptide is present prior to the IL-17B-induced IL-17RB-MLK4 interaction, C4, S7, and E8 of the cyclic peptide can preemptively interact with R216 of MLK4 kinase domain. Consequently, the interaction between the cyclic peptide and MLK4 prevents further binding of IL-17RB induced by IL-17B, inhibiting oncogenic signaling and thereby suppressing tumorigenesis and metastasis.[000135] The C4 residue within the cyclic peptide, aligned with C408 in the loop region of IL-17RB, assuming a pivotal role in disrupting the IL-17RB-MLK4 interaction and directly engaging with R216 in MLK4 kinase domain (Figs. 4A-4F). Additionally, the S7 and E8 residues within the cyclic peptide demonstrated a modest contribution to interacting with R216 in MLK4 kinase domain, enhancing its disruptor function (Figs. 4B and 4E). Notably, the K6 residue on the cyclic peptide did not directly interact with MLK4 kinase domain. However, mutating K to R significantly diminished the efficacy of the cyclic peptide, contrary to the expectation that replacing a charged residue with another of the same charge should not drastically reduce efficacy (Figs. 4 B, 4D, and 4E). Silicon analysis revealed that replacing K6 with R6 led to the formation of polar contacts or ionic bonds with adjacent G5, potentially altering the backbone structure of the cyclic peptide and its cyclized configuration. This led to different orientations of key residues (C4, S7, and E8) and consequently altered the chemical bonds formed between MLK4 and the cyclic peptide (Fig. 10). Conversely, S7 and E8 residues on the cyclic peptide primarily interacted with MLK4 kinase domain through backbone interactions.Therefore, substituting all three residues (K6, S7, and E8) with alanine (AAA mutant) did not significantly reduce the efficacy of the cyclic peptide in disrupting the IL-17RB-MLK4 interaction (Fig. 4B). However, changing KSE to RSD or replacing K with R dramatically reduced the binding efficacy of the cyclic peptide due to stereostructural alterations induced by the formation of an ionic bond between R6 and G5 (Fig. 10). Further supporting the importance of structural integrity, cyclic peptide variants with different sequences and amide bond linkage demonstrated varying efficacy in disrupting the IL-17RB-MLK4 interaction and inhibiting tumor growth. The variant that maintained high structural similarity to the original was the most effective. In summary, our findings emphasize the importance of both specific residue interactions, such as C4-R216, and the overall structural conformation of the cyclicAttorney Docket No. 5992-0517PWO1 peptide, particularly the role of K6, in disrupting the IL-17RB-MLK4 interaction. This knowledge provides insights for the design and development of therapeutic agents that target this pathway, potentially leading to new treatment options for pancreatic cancer.[000136] The development of MLK4 kinase inhibitors may face challenges due to the dual kinase properties of MLK4. wherein both tyrosine and serine / threonine kinase activities may play normal physiological roles in maintaining tissue homeostasis. Studies have indicated that MLK4 is a negative regulator of events related to carcinogenesis and drug resistance13’14. In our previous study6, tumorigenesis induced by IL-17B / IL-17RB signaling was primarily driven by the tyrosine kinase activity of MLK.4. and not its serine / threonine kinase activity. This observation aligns with the established role of tyrosine kinase activity in diseases, such as cancer and cell transformation15'17. Although the tyrosine and serine / threonine activities of MLK4 may have distinct roles in the regulation of MAPK signaling to suppress carcinogenesis in PDAC13,14,18'23, the cyclic peptide predominantly disrupts the IL-17B-induced IL-17RB-MLK4 interaction and downstream oncogenic signaling, with only minimal impact on inhibiting the serine / threonine activity of MLK4 (Fig. 3 and Fig. 8). Furthermore, to validate the selectivity of the cyclic peptide in protein-protein interactions, we conducted BLAST analysis to identify proteins with sequences similar to the cyclic peptide. Our analysis revealed that no proteins in Homo sapiens possessed identical sequences to the cyclic peptide found in the loop region of the SEFIR domain on the IL-17RB receptor, suggesting high selectivity of the binding mediated by the cyclic peptide toward MLK4. The mechanism of action of the cyclic peptide may distinguished from the corresponding CC' loop decoy peptide found in IL17RA24that could disrupt the Actl-IL17RA interaction. Overall, these findings suggest that the cyclic peptide exhibits high selectivity in suppressing PDAC growth by disrupting the IL-17RB-MLK4 interaction.References1. Iwakura, Y., Ishigame, EL, Saijo, S., andNakae, S. (2011). Functional specialization of interleukin- 17 family members. Immunity 34, 149-162.10, 1016 / i.immuni,2011.02,012.2. Gu, C., Wu, L., and Li, X. (2013). IL-17 family: cytokines, receptors and signaling.Cytokine 64, 477-485. 10.1016 / i.cyto.2013.07.022.Attorney Docket No. 5992-0517PWO1i, R. M., Park, S. J.. Hanel, W.. Ho, A. W., Maitra, A., and Gaffen. S. L. (2010). SEF / IL-17R (SEFIR) is not enough: an extended SEFIR domain is required for il-17RA-mediated signal transduction. J Biol Chem 285, 32751-32759.10.1074 / ibc. M110.121418.g, B., Liu, C., Qian, W., Han, Y., Li, X., and Deng, J. (2013). Crystal structure of IL- 17 receptor B SEFIR domain. J Immunol 190, 2320-2326.10.4049 / iimmunol.1202922.g. B., Liu, C., Qian, W., Han, Y., Li, X., and Deng, J. (2014). Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved alpha-helix for Actl binding and IL-17 signaling. Acta Crystallogr D 70, 1476-1483.10.1107 / S1399004714005227.H. H., Tsai, L. H., Huang, C. K., Hsu, P. H., Chen, M. Y., Chen, Y L, Hu, C M., Shen. C. N.. Lee, C. C., Chang, M. C.. et al. (2021). Characterization of initial key steps of IL- 17 receptor B oncogenic signaling for targeted therapy of pancreatic cancer. Sci Transl Med 13. 10.1126 / scitranslmed.abc2823. g, C. K., Yang, C. Y., Jeng, Y. M., Chen, C. L., Wu, H. H., Chang, Y. C., Ma, C., Kuo, W. H., Chang, K. J., Shew, J. Y., and Lee, W. H. (2014).Autocrine / paracrine mechanism of interleukin- 17B receptor promotes breast tumorigenesis through NF-KB-mediated antiapoptotic pathway. Oncogene 33, 2968-2977. 10, 1038 / onc.2013.268.g, S. C., Wei, P. C., Hwang-Verslues, W. W., Kuo, W. H., Jeng, Y. M., Hu, C. M., Shew, J. Y., Huang, C. S., Chang, K. J., Lee, E. Y., and Lee, W. H. (2017). TGF-betal secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB. EMBO Mol Med 9, 1660-1680.10, 15252 / emmm,201606914.H. H., Hwang-Verslues, W. W.. Lee, W. H.. Huang. C. K.. Wei, P. C., Chen. C. L., Shew, J. Y., Lee, E. Y., Jeng, Y. M., Tien, Y. W., et al. (2015). Targeting IL-17B-IL-17RB signaling with an anti-IL-17RB antibody blocks pancreatic cancer metastasis by silencing multiple chemokines. J Exp Med 212, 333-349.10.1084 / iem.20141702.J., Wu, X., Schiffmann, L., MacVicar, T., Zhou, C., Wang, Z., Li, D., Camacho, O. V., Heuchel, R., Odenthal, M., et al. (2021). IL-17B / RB activation in pancreatic stellate cells promotes pancreatic cancer metabolism and growth. Cancers (Basel) 13. 10, 3390 / cancersl 3215338.Attorney Docket No. 5992-0517PWO1aco, M., Valle. J., Flores, I., Andreu, D.. and A R B Castanho, M. (2021). Estimating peptide half-life in serum from tunable, sequence-related physicochemical properties. Clin Transl Sci 14, 1349-1358.10.1111 / cts.12985.lensky, L. D., Kung, A. L., Escher, I., Malia, T. J., Barbuto, S., Wright, R D., Wagner, G., Verdine, G. L., and Korsmeyer, S. J. (2004). Activation of apoptosis in vivo by a hydrocarbon-stapled BEI3 helix. Science 305, 1466- 1470. 10, 1126 / science.1099191.usiak, A. A., Stephenson, N. L., Baik, H., Trotter, E. W., Li, Y., Blyth, K, Mason, S., Chapman, P., Puto, L. A., Read, J. A., et al. (2016). Recurrent MLK4 loss-of-function mutations suppress JNK signaling to promote colon tumorigenesis. Cancer Res 76, 724-735. 10.1158 / 0008-5472. CAN-15-0701-T. Saab, W. F., Brown, M. S., and Chadee, D. N. (2012). MLK4beta functions as a negative regulator of MAPK signaling and cell invasion. Oncogenesis 1, e6.10, 1038 / oncsis.2012,6.me-Jensen, P., and Hunter, T. (2001). Oncogenic kinase signalling. Nature 411, 355-365. 10,1038 / 35077225.ter, T. (2014). The genesis of tyrosine phosphorylation. Cold Spring Harb Perspect Biol 6, a020644. 10, 1101 / cshperspect.a020644.ter. T. (2009). Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol 21, 140-146. 10.1016 / i.ceb.2009.0L028.huba, V. I., Grigorieva, E. V., Kvasha, S. M., Pavlova, T V., Grigoriev, V., Protopopov, A., Kharchenko, O., Gizatullin, R., Rynditch, A. V., and Zabarovsky, E. R. (2011). Cloning and initial functional characterization of Mlk4a and Mlk4p. Genomics Insights 4, 1-12. 10,4137 / GEI. S6092.E., and Brognard, J. (2019). Orange is the new black: kinases are the new- master regulators of tumor suppression. IUBMB Life 71, 738-748.10.1002 / iub.l981.usiak, A. A., Prelowska, M. K., Mehlich, D., Lazniewski, M., Kaminska, K., Gorczynski, A., Korwat, A., Sokolow^ska, O., Kedzierska, H., Golab, J., et al. (2019). Upregulation of MLK4 promotes migratory and invasive potential of breast cancer cells. Oncogene 38, 2860-2875. 10, 1038 / s41388-018-0618-0., S. H., Ezhilarasan, R., Phillips, E., Gallego-Perez, D., Sparks, A., Taylor, D., Ladner, K., Furuta, T.. Sabit, H., Chhipa, R.. et al. (2016). Serine / threomneAttorney Docket No. 5992-0517PWO1kinase MLK4 determines mesenchymal identity in glioma stem cells in an NF-KB-dependent manner. Cancer Cell 29, 201-213. 10, 1016 / i.ccell.2016,01, 005. -Nebi, A., Cheng, W., Xu, H., and Han, J. (2012). MLK4 has negative effect on TLR4 signaling. Cell Mol Immunol 9, 27-33. 10.1038 / cmi, 2011,15. lo, K. A., and Johnson, G. L. (2002). Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat Rev Mol Cell Biol 3, 663-672. 10, 1038 / nrm906., C.. Swaidani, S., Qian, W., Kang. Z., Sun, P.. Han, Y., Wang, C., Gulen, M. F., Yin, W., Zhang, C., et al. (2011). A CC’ loop decoy peptide blocks the interaction between Actl and IL-17RA to attenuate IL-17- and IL-25-induced inflammation. Sci Signal 4, ra72. 10,1126 / scisignal.2001843.overn, S. L., Caselli, E., Grigorieff, N., and Shoichet, B. K. (2002). A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem 45, 1712-1722. 10.1021 / im010533y. overn. S. L.. and Shoichet, B. K. (2003). Kinase inhibitors: not just for kinases anymore. J Med Chem 46, 1478-1483. 10.1021 / im020427b.a, B., Gomy, M., Konieczny, L., Piekarska, B., Rybarska, J., Skowronek, M., and Roterman, I. (1998). Supramolecular ligands: monomer structure and protein ligation capability'. Biochimie 80, 963-968. 10.1016 / s0300-9084(99)80001-7.lsen, C. E., and Carroll, KS. (2013). Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery'. Chem Rev 113, 4633-4679.10.1021 / cr300163e.terhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., de Beer, T. A., Rempfer, C., Bordoli, L., et al. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46, W296-W303. 10.1093 / nar / gkv427.g, H., Zhu. Y., Chen, X.. Li, X., Ye. S., and Zhang, R. (2018). Structure of a prokaryotic SEFIR domain reveals two novel SEFIR-SEFIR interaction modes. J Struct Biol 203, 81-89. 10.1016 / i.isb.2018.03.005.

Claims

Attorney Docket No. 5992-0517PWO1 CLAIMSWhat is claimed is:

1. A cyclic peptide comprising the amino acid sequence ofX1-X2-X3-X4-X5-X6-X7 Formula Iwherein X1 is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A); and the cyclic peptide targets the interaction between IL-17RB and mixed-lineage kinase 4 (MLK.4).

2. The cyclic peptide of claim 1, whereinXI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); orXI is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); or X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A); orXI is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S). threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A); orX1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

3. The cyclic peptide of claim 1 or 2, wherein the cyclic peptide comprises the amino acid sequence selected from the group consisting of GTCGKSE (SEQ ID NO: 17), STCDKSE (SEQ ID NO: 21), GTCGKTE (SEQ ID NO: 22), GTCGKSD (SEQ ID NO: 23) and GTCGAAA (SEQ ID NO: 24).

4. A cyclic peptide of Formula II:Attorney Docket No. 5992-0517PWO1Y1-X1-X2-X3-X4-X5-X6-X7-Y2 Formula IIwhereinX1 is glycine (G) or serine (S); X2 is threonine (T) or alanine (A); X3 is cysteine (C); X4 is glycine (G), serine (S) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), lysine (K), asparagine (N), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A);Y1 comprises 1-5 amino acid residues at the amino terminus;Y2 comprises 1 -5 amino acid residues at the carboxyl terminus;cyclization occurs via a linkage between one amino acid residue ofYl and one amino acid residue of Y2; andthe cyclic peptide targets the interaction between IL-17RB and mixed-lineage kinase 4 (MLK4).

5. The cyclic peptide of claim 4, whereinX1 is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G) or aspartic acid (D); orX1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); and X4 is glycine (G); orX5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A); orX1 is glycine (G) or serine (S); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G) or aspartic acid (D); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A); orX1 is glycine (G); X2 is threonine (T); X3 is cysteine (C); X4 is glycine (G); X5 is lysine (K) or alanine (A); X6 is serine (S), threonine (T) or alanine (A); and X7 is glutamic acid (E), aspartic acid (D) or alanine (A).

6. The cyclic peptide of claim 4 or 5, wherein the cyclic peptide is selected from the group consisting of Y1-GTCGKSE-Y2 (Formula Ila), Y1-STCDKSE-Y2 (Formula lib), Y1-GTCGKTE-Y2 (Formula lie), Y1-GTCGKSD-Y2 (Formula lid) and Y1-GTCGAAA-Y2 (Formula lie).Attorney Docket No. 5992-0517PWO17. The cyclic peptide of any one of claims 4 to 6. wherein the cyclic peptide comprises 9 to 17 amino acid residues.

8. The cyclic peptide of any one of claims 4 to 7, wherein the linkage is a disulfide bond or a peptide bond.

9. The cyclic peptide of any one of claims 4 to 8. whereinY1 is selected from the amino acid residues / sequence C (Cys). GS (Gly-Ser), S (Ser), GC (Gly-Cys) and G (Gly); and / orY2 is selected from the amino acid residues / sequence C (Cys), S (Ser), and D (Asp).

10. The cyclic peptide of any one of claims 4 to 9, whereinY1 comprises a terminal cysteine residue and Y2 comprises a terminal cysteine residue where a disulfide bond is formed between the two terminal cysteine residues;Y1 comprises terminal GS (Gly-Ser) residues and Y2 comprises a terminal S (Ser) residue where an amide bond is formed between the amine group of the N-terminal glycine residue and the carboxy group of the C-terminal serine;Y1 comprises a terminal S (Ser) residue and Y2 comprises a terminal S (Ser) residue where an amide bond is formed between the two terminal serine residues;Y1 comprises terminal GC (Gly-Cys) residues and Y2 comprises a terminal S (Ser) residue where an amide bond is formed between the amine group of the N-terminal glycine residue and the carboxy group of the C-terminal serine; or Y1 comprises terminal G (Gly) and Y2 comprises a terminal D (Asp) residue where an amide bond is formed between the amine group of the N- terminal glycine residue and the carboxy group of the C-terminal aspartic acid residue.

11. The cyclic peptide of any one of claims 4 to 19, wherein the cyclic peptide is selected from the group consisting of those set forth in SEQ ID NOs: 25-49.

12. A composition, comprising a cyclic peptide of any one of claims 1 to 11, and a physiologically acceptable carrier.Attorney Docket No. 5992-0517PWO113. The composition of claim 12, which is a pharmaceutical composition.

14. A method for inhibiting interleukin- 17B (IL-17B) / interleukin-17 receptor B (IL-17RB) activation and / or treating a disease or disorder associated with IL-17B / IL-17RB activation comprising administering to a subject in need thereof an effective amount of a cyclic peptide of any of claims 1 to 11 and a composition of claim 12 or 13.

15. The method of claim 14, wherein the disease or disorder is IL- 17B / IL- HRB-mediated proliferation disorder.

16. The method of claim 14 or 15, wherein the disease or disorder is a cancer and a metastasis thereof.

17. The method of claim 16, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary tract cancer, gallbladder and bile duct cancer, mammary cancer, ovarian cancer, cervical cancer, uterine body cancer, bladder cancer, prostate cancer, testicular tumor, osteogenic and soft-tissue sarcomas, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and pleural malignant mesothelioma.

18. The method of claim 17, wherein the cancer is pancreatic cancer or lung cancer.

19. A cyclic peptide of any of claims 1 to 11 for use in inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with such activation in a subject in need thereof.

20. The cyclic peptide for use of claim 19, wherein the disease or disorder is IL-17B / IL-17RB-mediated proliferation disorder.

21. The cyclic peptide for use of claim 19 or 20, wherein the disease or disorder is a cancer and a metastasis thereof.Attorney Docket No. 5992-0517PWO122. The cyclic peptide for use of claim 21, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary' tract cancer, gallbladder and bile duct cancer, mammary cancer, ovarian cancer, cervical cancer, uterine body cancer, bladder cancer, prostate cancer, testicular tumor, osteogenic and soft-tissue sarcomas, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and pleural malignant mesothelioma.

23. The cyclic peptide for use of claim 22, wherein the cancer is pancreatic cancer or lung cancer.

24. Use of a cyclic peptide of any of claims 1 to 11 for manufacturing a medicament for inhibiting IL-17B / IL-17RB activation and / or treating a disease or disorder associated with such activation in a subject in need thereof.

25. The use of claim 24, wherein the disease or disorder is IL- 17B / IL- HRB-mediated proliferation disorder.

26. The use of claim 24 or 25, wherein the disease or disorder is a cancer and a metastasis thereof.

27. The use of claim 26, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary tract cancer, gallbladder and bile duct cancer, mammary cancer, ovarian cancer, cervical cancer, uterine body cancer, bladder cancer, prostate cancer, testicular tumor, osteogenic and soft-tissue sarcomas, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and pleural malignant mesothelioma.

28. The use of claim 27, wherein the cancer is pancreatic cancer or lung cancer.

Images