Ubiquitin high-affinity cyclic peptides and methods of using the same

Abstract

The present invention provides a cyclic peptide and a method of using the cyclic peptide for, among other things, ameliorating or treating K63Ub-related diseases in a subject in need thereof.

Description

[Technical Field] 【0001】 Reference to Electronic Sequence Listing The contents of the electronic sequence listing (TECH-TOK-P-0269-PCT ST26.xml; size: 15,910 bytes; created on June 5, 2023) are incorporated herein by reference in their entirety. 【0002】 CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 349,366, filed June 6, 2022, entitled "UBIQUITIN HIGH AFFINITY CYCLIC PEPTIDES AND METHODS OF USE THEREOF," the contents of which are incorporated herein by reference in their entirety. 【0003】 The present invention relates to the fields of peptide engineering and drug screening. [Background technology] 【0004】 Ubiquitination is a complex post-translational modification (PTM) involved in various cellular processes. In ubiquitination, the C-terminal glycine of ubiquitin (Ub) attaches mainly to the ε-amino side chain of a lysine residue of the substrate protein. This process is achieved by the action of three enzymes known as E1–E3. Poly-Ub chains with different linkages (e.g., Lys63-linked Ub chains) can be formed by adding another Ub to one of the seven lysine residues (e.g., Lys63) or to the N-terminus to extend the Ub. Importantly, Ub chains with different linkage types have different topologies and dynamics, and each Ub chain is recognized by a specific subset of cellular proteins. As a result, each chain can lead to specific cellular signaling such as proteasomal degradation (e.g., Lys48-linked Ub chains), mitophagy, cell cycle regulation, protein transport, autophagy, DNA repair (e.g., Lys63-linked Ub chains), and immune response. Similar to most other PTMs, ubiquitination is a reversible process in which an enzyme family known as deubiquitinating enzymes (DUBs) cleaves Ub chains from ubiquitinated proteins or completely detaches them. 【0005】 The major components of the Ub system (e.g., DUBs, E1–E3, and 26S proteasome) are well-known targets in drug development, and some of them have become approved anticancer drugs (e.g., bortezomib). Most approaches focus on interfering with the activity of specific enzymes involved in the Ub system, but another approach has emerged that targets the Ub chains themselves as signaling codes. In recent years, the inventors have discovered a class of cyclic peptides that specifically bind to Lys48-linked Ub chains, interfere with specific DUBs, and cause proteasomal degradation of ubiquitinated proteins. These cyclic peptides, selected using the Random Non-standard Peptides Integrated Discovery (RaPID) approach against synthetic Lys48-linked Ub chains, showed high levels of apoptosis in vivo and attenuated tumor growth. 【0006】 Since successful use of cyclic peptide modulators on Lys48-linked chains, the inventors aimed to push the limits of this approach and attempt to target Lys63-linked chains. These chains are known to be the second most dominant class intracellularly after Lys48-linked Ub chains. By finding selective modulators of Lys63-linked chains, it may be possible to interfere with other biological pathways involved in non-proteolytic cellular processes such as DNA damage repair (DDR). The main challenge of this approach is due to the structural characteristics of Lys63-linked Ub chains, which have been shown to adopt an open structure in the crystal and a series of conformations in solution. This is very different from the more defined closed structure of Lys48-linked Ub chains. Furthermore, in Lys63-linked Ub chains, the hydrophobic patches of proximal Ub monomers and distal Ub are not on the same surface, so each Ub within this chain likely operates as a separate unit. Therefore, the development of specific cyclic peptide modulators that can interact with both Ub units and interfere with the function of these chains is very difficult and remains an uncharted area. 【0007】 The RaPID method has attracted particular attention because it can generate diverse libraries, each with its own chemical space composed of up to 1 trillion thioether macrocyclic peptides, using in vitro translation of the protein of interest (POI). By combining this feature with the inventors' ability to synthesize any Ub chain with a defined length, linkage, and high purity, it becomes possible to selectively target any of the desired chains. 【0008】 There remains a great need for novel macrocyclic peptides with specific binding affinity for Lys63-linked Ub chains. SUMMARY OF THE INVENTION 【0009】 In some embodiments, the present invention is at least partially based on the characteristics of cyclic peptides and their chemically modified analogs, resulting in very potent compounds that are cell permeable. 【0010】 The present invention relates to cyclic peptides and methods of using cyclic peptides, including but not limited to, for the amelioration or treatment of Lys63Ub-related diseases such as cancer, in a subject in need thereof. 【0011】 The present invention is at least partially based on the finding that cyclic peptides bind to Lys63-linked Ub chains with nanomolar affinity. The present invention is further partially based on the surprising finding that cyclic peptides disclosed herein, such as Lys-63Ub binders, inhibit DNA repair in cancer cells, resulting in induction of cell cycle arrest, apoptosis, or both in cancer cells. D According to one aspect, there is provided a cyclic peptide comprising the amino acid sequence LLIWIGSSKNPYILCG (SEQ ID NO: 1) or functional analogs thereof having at least 80% homology or identity thereto. 【0012】 According to another aspect, there is provided a dimeric cyclic peptide comprising a cyclic peptide disclosed herein. 【0013】 According to another aspect, there is provided a pharmaceutical composition comprising a cyclic peptide disclosed herein, or a dimeric cyclic peptide disclosed herein, and a pharmaceutically acceptable carrier. 【0014】 According to another aspect, there is provided a pharmaceutical composition comprising a cyclic peptide disclosed herein, or a dimeric cyclic peptide disclosed herein, and a pharmaceutically acceptable carrier. 【0015】 According to another aspect, there is provided a method for ameliorating or treating a K63Ub-related disease in a subject in need thereof, the method comprising administering to the subject (a) a cyclic peptide disclosed herein, (b) a dimeric cyclic peptide disclosed herein, (c) a pharmaceutical composition disclosed herein, and (d) a therapeutically effective amount of any one of (a) to (c), thereby ameliorating or treating the K63Ub-related disease in the subject. 【0016】 In some embodiments, the cyclic group comprises at least one cysteine residue substituted with the amino acid residue of SEQ ID NO: 1. 【0017】 In some embodiments, the cyclic peptide comprises an amino acid sequence selected from the group consisting of: CLIWIGSSKNPYILCG (SEQ ID NO: 2); LCIWIGSSKNPYILCG (SEQ ID NO: 3); LLCWIGSSKNPYILCG (SEQ ID NO: 4); LLICIGSSKNPYILCG (SEQ ID NO: 5); LLIWCGSSKNPYILCG (SEQ ID NO: 6); LLIWICSSKNPYILCG (SEQ ID NO: 7); LLIWIGCSKNPYILCG (SEQ ID NO: 8); LLIWIGSCKNPYILCG (SEQ ID NO: 9); LLIWIGSSKCPYILCG (SEQ ID NO: 10); LLIWIGSSKNCYILCG (SEQ ID NO: 11); LLIWIGSSKNPCILCG (SEQ ID NO: 12); and LLIWIGSSKNPYCLCG (SEQ ID NO: 13). 【0018】 In some embodiments, the cyclic peptide further comprises at least one arginine residue. 【0019】 In some embodiments, at least one arginine residue is located at the C-terminus of the cyclic peptide. 【0020】 In some embodiments, the cyclic peptide comprises an amino acid sequence selected from the group consisting of: CLIWIGSSKNPYILCGRR (SEQ ID NO: 15); CLIWIGSSKNPYILCRR (SEQ ID NO: 16); and CLIWIGSSKNPYILCR (SEQ ID NO: 17). 【0021】 In some embodiments, the cyclic peptide comprises 14 to 20 amino acid residues. 【0022】 In some embodiments, the amino acid at the 1st position of the N-terminus is conjugated to the cyclized molecule. 【0023】 In some embodiments, the cyclized molecule contains a halogen. 【0024】 In some embodiments, the cyclized molecule is: 【Chemical formula】 including any one of them, wherein each wavy bond represents an attachment point to the first amino acid residue or the C-terminal amino acid, respectively. 【0025】 In some embodiments, the cyclic peptide is chemically modified. 【0026】 In some embodiments, the chemical modification is selected from the group consisting of alkylation, arylation, addition of a thiol protecting group, and any combination thereof. 【0027】 In some embodiments, the cyclic peptide is characterized by having cell penetration ability, ubiquitin (Ub) binding ability, or any combination thereof. 【0028】 In some embodiments, the Ub is polymeric Ub. 【0029】 In some embodiments, the polymeric Ub contains Ub monomers (K63Ub) linked at lysine 63. 【0030】 In some embodiments, the cyclic peptide has an increased affinity for the Lys63-linked Ub chain as compared to the control Ub chain. 【0031】 In some embodiments, the increased affinity is a binding affinity having a dissociation constant (K D ) in the range of 0.05 to 150 nM. 【0032】 In some embodiments, the pharmaceutical composition is for use in the treatment of K63Ub-related diseases. 【0033】 In some embodiments, the K63Ub-related disease is a cell proliferation-related disease. 【0034】 In some embodiments, the cell proliferation-related disease includes cancer. 【0035】 In some embodiments, ameliorating or treating includes increasing the amount of fragmented DNA in the cells of the subject, increasing the amount, rate, or both of cell apoptosis in the subject, or any combination thereof. 【0036】 In some embodiments, the cell is a cancer cell or a cancerous cell. 【0037】 In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an anti-cancer agent. 【0038】 Unless defined otherwise, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, but exemplary methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. 【0039】 Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description shown below. However, while the detailed description and specific examples represent preferred embodiments of the present invention, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, and it should be understood that these are shown by way of illustration only. 【Brief Description of the Drawings】 【0040】 【FIG. 1A - 1B】 A schematic workflow image explaining the development strategy of macrocyclic peptides against Lys63-linked Ub chains is shown. (1A) is a scheme of the general process for discovering macrocyclic peptides of Di-Ub chains that affect specific biological functions using chemical protein synthesis and the RaPID (Random Non-standard Peptides Integrated Discovery) approach. (1B) is a schematic diagram of the RaPID method for identifying a novel binder 1 (CP1) against Lys63-linked Di-Ub. 【FIG. 2A - 2E】 Synthetic scheme images and graphs showing the chemical synthesis of cyclic peptides and affinity screening against Lys63-linked Di-Ub. (2A) is a schematic diagram of the screening of a cyclopeptide library chemically prepared for using Fmoc-SPPS. (2B) and (2C) show synthetic scheme images showing the preparation of cyclic peptides having Cys residues at various positions. (2D) is a bar graph showing the binding affinity of cyclic peptides against Lys63-linked Di-Ub, normalized to the affinity of 1 (CP1; SEQ ID NO: 1). (2E) is a scatter plot of the binding curve of FITC-labeled cyclic peptide 1 (CP1-FITC). The KD (95.8 ± 2.3 nM) of CP1-FITC is determined. All measurements were performed in triplicate, and at least three biological replicates were performed. Error bars represent the standard deviation. 【FIG. 3A - 3B】Chemical synthesis of a modified cyclic peptide containing Cys at the 1-position (Cys1) and a synthetic scheme, table, and graph showing affinity screening against Lys63-linked Di-Ub. (3A) is a schematic diagram showing alkylation and arylation of Cys1. The alkylation and arylation substituents of cyclic peptide 2 are shown in the table. (3B) is a bar graph showing the binding affinity of derivatives of cyclic peptide 2 to Lys63-linked Di-Ub, normalized to the affinity of 1 (CP1). All measurements were performed in triplicate and at least three biological replicates were performed. Error bars represent the standard deviation. 【FIG. 4A - 4K】Synthetic schemes, immunofluorescence images, Western blot images, and graphs showing the interference of DNA damage repair activity by cyclic peptides. (4A) is a diagram of the chemical composition of cyclic peptide 2 and its TAMRA-labeled 26. (4B) shows representative confocal images of 26 in living U2OS cells. Scale bar 20 μm. (4C) is a Western blot analysis of lysates of U2OS cells treated with 33 (scrambled sequence of cyclic peptide 2) and 2 (upper panel). H2AX was used as a loading control (lower panel). Representative of three independent experiments. (4D) is a bar graph showing the quantified relative γ-H2AX signal of C. (4E) is a Western blot analysis of lysates of U2OS cells treated with 2 and 20 (upper panel). H2AX was used as a loading control (lower panel). Representative image of three independent experiments. (4F) is a bar graph showing the quantified relative γ-H2AX signal of E. (4G) shows immunofluorescence images of DNA damage visualized by the "comet-like" bis-tagged green signal from the DNA of individual cells, analyzing over 100 cells in two independent experiments. (4H) is a bar graph showing the quantified relative tail moment (mean ± SEM) of the images from G. (4I) is an image of a Western blot using anti-Flag immunoprecipitation of lysates of 293T cells transfected with wt or mutant of RFN168, treated with or without 2, probed with anti-Flag antibody (upper panel) and anti-ubiquitin (lower panel). (4J) is a bar graph showing the cell cycle distribution of HeLa cells treated with 2 or untreated with 2 after 72 hours and 96 hours. By two independent flow cytometry experiments (for each condition, >15,000 cells). (4K) is a bar graph showing the relative population of annexin V-FITC (apoptosis) cells after 96 hours, obtained from two independent flow cytometry experiments (for each condition, >20,000 cells). Data are plotted as mean ± SD (unless otherwise specified), * indicates P<0.05, ** indicates P<0.005, *** indicates P<0.0005, NS indicates not significant. 【FIG. 5A - 5D】Schematic diagram, Western blot image, and graph showing the identification of ubiquitinated proteins that bind to peptides 31 and 38. (5A) is a schematic workflow of sample preparation and analysis by proteomics using biotin-conjugated cyclic peptides. (5B) is a Western blot analysis showing Ub chains pulled down by peptides 31 and 38 and detected by antibodies against Lys63- and Lys48-linked Ub chains. (5C) is a volcano plot showing proteins at different enrichments. Proteins involved in processes mediated by Lys63-linked Ub are shown as protein transport (blue), DNA repair (red), histone modification (green), and cell cycle (purple). (5D) is a horizontal bar graph showing gene ontology (GO) analysis of genes from C with at least 3-fold enrichment compared to scrambled control 38. The experiment was repeated 3 times. 【FIG. 6A - 6B】 Synthetic scheme image and histogram showing the synthesis of biotinylated Lys63-linked Di-Ub. (6A) is a schematic diagram of the synthesis of biotin-Lys63-linked Di-Ub. (6B) is the analytical HPLC and mass of purified biotinylated Lys63-linked Di-Ub, observed mass 17592.7 ± 0.2 Da (calculated value 17592.8, average isotope). 【FIG. 7】 Bar graph showing the RaPID selection of three new libraries with mClBz as the initiator and the results of its real-time PCR. (Negative = 50% bare M280 streptavidin beads + 50% Ub1-attached M280 streptavidin beads; Positive = Lys63-linked Di-Ub-attached M280 streptavidin beads). 【FIG. 8】 Schematic diagram showing a general presentation for screening peptides using a fluorescence-based competition assay. 【FIG. 9A - 9B】 Synthetic scheme image and histogram showing the synthesis of CP1. (9A) is a schematic diagram of cyclic peptide synthesis. (9B) is a histogram of HPLC and mass spectrometry of cyclic peptide 1, CP1. Observed mass 1891.5 ± 0.1 Da (calculated value 1891.6 Da, average isotope). 【FIG. 10A - 10B】 Synthetic schemes showing the synthesis of different analogs of 1 and histograms are shown. (10A) is a schematic diagram of the cysteine - mutated cyclic peptide. (10B) shows histograms of high - performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses of each cyclic peptide analog. 【FIG. 11】 A histogram of HPLC - MS analysis of cyclic peptide 15, CP1 - L1C - CH3 is shown. Observed mass 1895.5 ± 0.1 Da (calculated value 1895.3 Da, average isotope). 【FIG. 12】 A histogram of HPLC - MS analysis of cyclic peptide 16, CP1 - L1C - CH2C6H5 is shown. Observed mass 1971.5 ± 0.2 Da (calculated value 1971.3 Da, average isotope). 【FIG. 13】 A histogram of HPLC - MS analysis of cyclic peptide 17, CP1 - L1C - CH2CONH2 is shown. Observed mass 1938.5 ± 0.2 Da (calculated value 1938.3 Da, average isotope). 【FIG. 14】 A histogram of HPLC - MS analysis of cyclic peptide 18, CP1 - L1C - CH2C10H7 is shown. Observed mass 2021.5 ± 0.2 Da (calculated value 2021.3 Da, average isotope). 【FIG. 15】 A histogram of HPLC - MS analysis of cyclic peptide 19, CP1 - L1C - CH2C10H7 is shown. Observed mass 2069.6 ± 0.2 Da (calculated value 2069.3 Da, average isotope). 【FIG. 16】 A histogram of HPLC - MS analysis of cyclic peptide 20, CP1 - L1C - C6F5 is shown. Observed mass 2047.5 ± 0.2 Da (calculated value 2047.3 Da, average isotope). 【FIG. 17】 A histogram of HPLC - MS analysis of cyclic peptide 21, CP1 - L1C - C10F9 is shown. Observed mass 2195.6 ± 0.2 Da (calculated value 2195.3 Da, average isotope). 【FIG. 18A - 18B】A synthetic scheme image showing the synthesis of CP1-FITC cyclic peptide and a histogram are presented. (18A) shows a schematic diagram of the synthesis of CP1-FITC. (18B) (i) HPLC-MS analysis of the FITC-labeled cyclic peptide CP1-FITC, observed mass 2492.7 ± 0.1 Da (calculated value 2492.5 Da, average isotope). (ii) HPLC-MS analysis of the TAMRA-labeled cyclic peptide CP1-TAMRA. Observed mass 2546.5 ± 0.1 Da (calculated value 2546.5 Da, average isotope). 【FIG. 19】 A histogram showing the HPLC-MS analysis of cyclic peptide 23 is presented. Observed mass 2214.5 ± 0.1 Da (calculated value 2214.5 Da, average isotope). 【FIG. 20】 A histogram showing the HPLC-MS analysis of cyclic peptide 24 is presented. Observed mass 2126.6 ± 0.2 Da (calculated value 2126.5 Da, average isotope). 【FIG. 21】 A histogram showing the HPLC-MS analysis of cyclic peptide 25 is presented. Observed mass 2607.5 ± 0.1 Da (calculated value 2607.5 Da, average isotope). 【FIG. 22】 A histogram showing the HPLC-MS analysis of cyclic peptide 26 is presented. Observed mass 2536.9 ± 0.2 Da (calculated value 2537.1 Da, average isotope). 【FIG. 23A - 23B】 A synthetic scheme image showing the synthesis of TAMRA-labeled CP1-L1C-C6F5 cyclic peptide and a histogram are presented. (23A) is a schematic diagram of the synthesis of TAMRA-labeled CP1-L1C-C6F5. (23B) HPLC-MS analysis of the cyclic peptide corresponding to the following peaks. (i) 26, observed mass 2537.1 ± 0.2 Da (calculated value 2537.2 Da, average isotope), (ii) 27, observed mass 2702.6 ± 0.2 Da (calculated value 2702.5 Da, average isotope). Here, TAMRA = tetramethylrhodamine-5-maleimide. 【FIG. 24】Graphs showing the binding affinities of cyclic peptides 1 and 2 to Ub chains of different linkages and lengths are shown. (i) Binding of cyclic peptide 1 to Lys11-linked Di-Ub. No binding was observed by SPR. (ii) Binding of cyclic peptide 1 to Lys29-linked di-Ub. (Red: original trace, black: fitting curve) (iii) Relative binding of cyclic peptides 1 and 2 to linear di-Ub. (iv) Relative binding of cyclic peptides 1 and 2 to Lys48-linked Di-Ub. (v) Relative binding of cyclic peptides 1 and 2 to Lys48-linked Tetra-Ub. 【FIG. 25】 Histogram of HPLC-MS analysis of cyclic peptide 28 is shown. Observed mass 2553.7 ± 0.1 Da (calculated value 2553.5 Da, average isotope). 【FIG. 26A - 26B】 (26A) Synthetic scheme image and histogram showing the synthesis of FITC-labeled cyclic peptide 29, and (26B) HPLC-MS analysis of cyclic peptide 29 are shown. Observed mass 2482.6 ± 0.1 Da (calculated value 2482.5 Da, average isotope). 【FIG. 27】 Graph showing the binding of cyclic peptide 1 to Lys63-linked Di-Ub by SPR is shown. 【FIG. 28】 Curve representing the binding of CP1-FITC to Lys63-linked Di-Ub is shown. The KD value determined using the equation Y = Bmax*X / (KD+X) is 95.8 ± 2.3 nM. All measurements were performed in triplicate. 【FIG. 29】 Curve representing the binding of CP1-TAMRA to Lys63-linked Di-Ub is shown. The KD value determined using the equation Y = Bmax*X / (KD+X) is 101.9 ± 3.6 nM. All measurements were performed in triplicate. 【FIG. 30】 Curve representing the binding of 2-FITC to Lys63-linked Di-Ub is shown. The KD value determined using the equation Y = Bmax*X / (KD+X) is 43.2 ± 4 nM. All measurements were performed in triplicate. 【FIG. 31】The curve representing the binding of 2-TAMRA to Lys63-linked Di-Ub is shown. The KD value determined using the equation Y = Bmax*X / (KD+X) is 47.4 ± 5.9 nM. All measurements were performed in triplicate. 【FIG. 32A - 32B】 A synthetic scheme image showing the synthesis of biotin-PEG6-coupled cyclic peptide 31 and a histogram are shown (32A). (32B) HPLC-MS analysis of cyclic peptide 31. Observed mass 2757.5 ± 0.1 Da (calculated value 2757.6 Da, average isotope). 【FIG. 33A - 33C】 A synthetic scheme image, a histogram, and a bar graph showing the synthesis of scrambled cyclic peptide 33 are shown. (33A) is a schematic diagram of the synthesis of cyclic peptide 33. (33B) is a histogram of the HPLC-MS analysis of cyclic peptide 33. Observed mass 1881.4 ± 0.1 Da (calculated value 1881.3 Da, average isotope). (33C) is a bar graph showing the relative binding of cyclic peptide 33 and mJ08-L8W on Lys63-linked Di-Ub. 【FIG. 34A - 34B】 A synthetic scheme image and a histogram showing the synthesis of biotin-PEG6-coupled scrambled cyclic peptide 38 are shown. (34A) is a schematic diagram of the synthesis of biotinylated cyclic peptide 38. (34B) is a histogram of the HPLC-MS analysis of cyclic peptide 38. Observed mass 2757.1 ± 0.1 Da (calculated value 2757.6 Da, average isotope). 【FIG. 35A - 35L】Fluorescence representative images showing the delivery of cyclic peptides 26 (2-TAMRA) and 29 (2-FITC) to live U2OS cells are shown. CLSM images of cells treated with 26, DMSO, and 29 are (Figure 35A - Figure 35D), (Figure 35E - Figure 35H), and (Figure 35I - Figure 35L), respectively. Figure 35A, Figure 35E) (TAMRA, red channel). Figure 35I) (FITC, green channel). Figure 35B, Figure 35F, Figure 35J) Hoechst (blue channel). Figure 35C, Figure 35G) A combination of the TAMRA channel and the Hoechst channel. Figure 35K) A combination of the FITC channel and the Hoechst channel. Figure 35D, Figure 35H, Figure 35L) Brightfield channel. (Scale bar 20 μm). The experiments were repeated twice at 2 μM each. 【FIG. 36A - 36B】 The following Western blot images and graphs are shown: (36A) Western blot analysis of lysates of HeLa cells treated with 2 and 20 (upper panel). H2AX was used as a loading control (lower panel). It was representative of three independent experiments. (36B) is a bar graph showing the quantified relative γ-H2AX signal of A. Data were plotted as mean ± SD, with * being P < 0.05 and ** being P < 0.005. 【FIG. 37A - 37B】 Dot plot graphs showing flow cytometry analysis are shown. Cells were double-stained with annexin V-FITC and PI and then analyzed by a CYTEK Aurora flow cytometer. Two independent experiments were performed. HeLa cells were treated with DMSO (37A) and cyclic peptide 2 (37B) for 96 hours, and representative dot plots are shown here (for each condition, cells > 20,000). 【FIG. 38A - 38C】 Histograms showing flow cytometry analysis are shown. Cell cycle distributions of HeLa cells treated with DMSO (38A) and cyclic peptide 2 for 72 hours (38B) and 96 hours (38C). Representative heat plots show the relative populations of cells in the G1, S, and G2 / M phases at the green, red, and blue boundaries, respectively (for each condition, cells > 15,000). Representative histograms of two independent experiments. 【FIG. 39】Shown are images of proteomics analysis after pulldown of Lys63-linked Ub-modified protein components from U2OS cell lysates with streptavidin beads. STRING network of DDR proteins (identified by gene name) enriched at 31. Data showed an enrichment of at least 3-fold compared to scramble control 38. Experiments were performed in triplicate. 【FIG. 40A - 40C】 Shown are a peptide diagram, vertical bar graph, and photograph showing arginylation of cyclic peptide sequences for improvement of the physical and biological properties of the parent peptide (2). (40A) A chemical library of arginine conjugates 2 (39 - 42) was chemically prepared by using Fmoc-SPPS. (40B) Binding affinity of cyclic peptides for Lys63-linked Di-Ub. Normalized to the affinity of 1. Data were plotted as mean ± SEM of n = 3 biologically independent experiments, repeated three times each. (40C) Western blot analysis of HeLa cell lysates treated with 2 and its arginylated derivatives (39 - 42). H2AX was used as a loading control performed on a separate blot. Shown are representative images of n = 4 independent experiments and quantified relative γ-H2AX signals. Data are shown as mean values ± SD. 【FIG. 41A - 41E】Figures, vertical bar graphs, and photographs showing cyclic peptides having different linkers for cyclization to improve the biological properties of 40 are presented. (41A) Chemical libraries of 43 - 50 were chemically prepared using Fmoc - SPPS. (41B) Binding affinity of cyclic peptides for Lys63 - linked Di - Ub. Normalized to the affinity of 1. Data are plotted as mean ± SD of n = 2 biologically independent experiments, repeated 3 times each. (41C) Western blot analysis of lysates of HeLa cells treated with 40 and its derivatives having different linkers 43 - 46. H2AX was used as a loading control performed on a separate blot. Representative images of n = 4 independent experiments and quantified relative γ - H2AX signals are shown. Data are represented as mean ± SEM. (41D) Western blot analysis of lysates of HeLa cells treated with 43 (the most excellent compound in the previous biological screening in c) and other derivatives having different linkers 47 - 49. H2AX was used as a loading control performed on a separate blot. Representative images of n = 4 independent experiments and quantified relative γ - H2AX signals are shown. Data are shown as mean ± SEM. Western blot analysis of lysates of HeLa cells treated with 43 and cleavable derivatives in the presence of eGSH 50. 【FIG. 42A - 42H】 Exemplary synthetic schemes showing the synthesis of cyclic peptides of the present invention such as peptide 39 - 42 (42A), peptide 43 (42B), peptide 44 (42C), peptide 45 (42D), peptide 46 (42E), peptide 50 (42F), peptide 48 (42G), and peptide 49 (42H) are presented. 【FIG. 43】 An exemplary synthetic scheme showing the synthesis of peptide 47 and a general synthetic scheme for solid - phase synthesis of an amino acid sequence containing propargylglycine, a precursor of peptides 47 - 49, are presented. 【Modes for Carrying Out the Invention】 【0041】 In some embodiments, the present invention relates to cyclic peptides and methods of using cyclic peptides for binding to Ub of cells, for improving or treating diseases in a subject in need of improvement or treatment, and the like. In some embodiments, the present invention combines a powerful RaPID (random non-standard peptides integrated discovery) method with the ability of the inventor to synthesize specific Ub chains such as Ub chains linked via lysine 63 residues (Lys63-linked Ub), and discovers new cyclic peptides that specifically bind to Lys63-linked Ub with high affinity at the nanomolar level. In some embodiments, the present invention is further based, at least in part, on the ability of these new cyclic peptides to affect various cellular processes such as DNA repair and apoptosis, and highlights these cyclic peptides as a treatment strategy for diseases in which Lys63 ubiquitination, such as cancer, is dysregulated. 【0042】 Cyclic peptide According to some embodiments, the present invention relates to peptides. 【0043】 In some embodiments, the peptide can permeate cells (e.g., cancer cells), bind to ubiquitin, or a combination thereof. 【0044】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSSKNPYILCG (SEQ ID NO: 1). 【0045】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: CLIWIGSSKNPYILCG (SEQ ID NO: 2). 【0046】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LCIWIGSSKNPYILCG (SEQ ID NO: 3). 【0047】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLCWIGSSKNPYILCG (SEQ ID NO: 4). 【0048】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLICIGSSKNPYILCG (SEQ ID NO: 5). 【0049】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWCGSSKNPYILCG (SEQ ID NO: 6). 【0050】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWICSSKNPYILCG (SEQ ID NO: 7). 【0051】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGCSKNPYILCG (SEQ ID NO: 8). 【0052】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSCKNPYILCG (SEQ ID NO: 9). 【0053】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSSKCPYILCG (SEQ ID NO: 10). 【0054】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSSKNCYILCG (SEQ ID NO: 11). 【0055】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSSKNPCILCG (SEQ ID NO: 12). 【0056】 In some embodiments, the peptide of the present invention comprises or consists of the following amino acid sequence: LLIWIGSSKNPYCLCG (SEQ ID NO: 13). 【0057】 In some embodiments, the peptide of the present invention comprises a functional analog of any one of SEQ ID NOs: 1-17. 【0058】 In some embodiments, the functional analog is characterized by having at least 70%, 80%, 85%, 90%, 95% or 99%, or any value and range therebetween of homology or identity to any one of SEQ ID NOs: 1-17. Each possibility represents a separate embodiment of the present invention. In some embodiments, the functional analog is characterized by having 70-100%, 80-100%, 85-100%, 90-100%, 95-100% or 97-100% to any one of SEQ ID NOs: 1-17. Each possibility represents a separate embodiment of the present invention. 【0059】 As used herein, the term "functional analog" generally refers to any peptide that is functionally essentially the same as the cyclic peptides disclosed herein. In some embodiments, the functional analogs disclosed herein bind to Lys63Ub with at least 50%, 60%, 70%, 80%, ninety%, 95%, or 99%, or any value and range therebetween of binding affinity of the peptides of the present invention disclosed herein. Each possibility represents a separate embodiment of the present invention. In some embodiments, the functional analog is characterized by having 70-100%, 80-100%, or 90-100% homology or identity to any one of SEQ ID NOs: 1-17. Each possibility represents a separate embodiment of the present invention. 【0060】 As used interchangeably herein, the terms "homology" or "identity" refer to sequence identity between two amino acid sequences or two nucleic acid sequences, and identity refers to a more stringent comparison. The phrases "percent identity or percent homology" and "identity % or homology %" refer to the percentage of sequence identity found by comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be 0 to 100% identical, or any value in between. Identity can be determined by comparing the positions within each sequence that can be aligned for purposes of comparison to a reference sequence. If a position within the sequences being compared is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. The degree of identity of an amino acid sequence corresponds to the number of identical amino acids at positions shared by the amino acid sequences. The degree of identity between nucleic acid sequences corresponds to the number of nucleotides that are identical or match at positions shared by the nucleic acid sequences. The degree of homology of an amino acid sequence corresponds to the number of amino acids at positions shared by the polypeptide sequences. 【0061】 The following is a non-limiting example for calculating homology or sequence identity (these terms are used interchangeably herein) between two sequences. The sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for purposes of comparison). The optimal alignment is determined as the highest score using the GAP program of the GCG software package with a Blossum62 scoring matrix where the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position within the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position within the second sequence, then the molecules are identical at that position. The percent identity between two sequences corresponds to the number of identical positions shared by the sequences. 【0062】 In some embodiments, the percent homology or percent identity described herein is calculated or determined using BLAST (basic local alignment search tool). In some embodiments, the percent homology or percent identity described herein is calculated or determined using the Blossum62 scoring matrix. 【0063】 As used herein, the term "analogue" refers to a polypeptide that is similar but not identical to a peptide of the invention and that still has the ability to bind to Ub, such as a Lys63Ub dimer, oligomer, or polymer. An analogue may have deletions or mutations that result in an amino acid sequence different from that of the peptide of the invention. It should be understood that all analogues of the peptide of the invention can still bind to Ub. Further, an analogue may be similar to a fragment of the peptide of the invention, but in such a case, the fragment must contain at least 14 contiguous amino acids of the peptide of the invention. 【0064】 In some embodiments, the peptides of the invention are linear or cyclic. 【0065】 As defined herein, the amino acid sequence of a peptide of the invention is recited from the N-terminus to the C-terminus. In some embodiments, the amino acid residue located at the N-terminus of the peptide is placed at the first position of the peptide. In some embodiments, the recited position of a particular amino acid residue within a cyclic peptide is referenced based on the position of the amino acid residue in the linear form of the peptide. 【0066】 In some embodiments, the N-terminus of a cyclic peptide disclosed herein is defined as an "in-ring terminus or position". 【0067】 In some embodiments, the N-terminal amino acid of a cyclic peptide disclosed herein is located at an in-ring position of the cyclic peptide. 【0068】 In some embodiments, the C-terminus of the cyclic peptides disclosed herein is defined as an "extracellular terminus or position". 【0069】 In some embodiments, the C-terminal amino acid of the cyclic peptides disclosed herein is located at an extracellular position of the cyclic peptide. 【0070】 In some embodiments, the peptide further comprises a positively charged amino acid residue. In some embodiments, the positively charged amino acid residue is positively charged under physiological conditions such as, but not limited to, human cells and / or the body, e.g., pH, temperature, osmotic pressure, etc. 【0071】 In some embodiments, the peptide further comprises at least one amino acid residue selected from arginine, lysine, histidine, or any combination thereof. 【0072】 In some embodiments, the at least one amino acid residue comprises a plurality of amino acid residues. In some embodiments, the plurality comprises any integer of 2 or more. In some embodiments, the plurality comprises 2 to 8, 2 to 6, or 2 to 4 amino acid residues. Each possibility represents a separate embodiment of the present invention. 【0073】 In some embodiments, the plurality of amino acid residues are covalently bonded to each other. In some embodiments, the plurality of amino acid residues are bonded to each other by peptide bonds. 【0074】 In some embodiments, the at least one amino acid residue or the plurality thereof is disposed at an intracellular position or terminus of the peptide. 【0075】 In some embodiments, the at least one amino acid residue or the plurality thereof is disposed at an extracellular position or terminus of the peptide. 【0076】 In some embodiments, at least one amino acid residue comprises or consists of arginine. 【0077】 In some embodiments, the peptide further comprises at least one arginine residue. 【0078】 In some embodiments, the peptide further comprises a plurality of arginine residues. 【0079】 In some embodiments, the peptide further comprises two arginine residues. In some embodiments, the peptide further comprises two arginine residues linked to each other by a peptide bond. 【0080】 In some embodiments, at least one arginine or a plurality thereof is located at an in-loop position or terminus of the peptide. 【0081】 In some embodiments, at least one arginine or a plurality thereof is located at an out-of-loop position or terminus of the peptide. 【0082】 In some embodiments, the peptide further comprises two arginine residues that are linked to each other by a peptide bond and are located at an in-loop position of the peptide. 【0083】 In some embodiments, the peptide further comprises two arginine residues that are linked to each other by a peptide bond and are located at an out-of-loop position of the peptide. 【0084】 In some embodiments, the peptide of the present invention comprises or consists of the amino acid sequence: CRIWIGSSKNPYILCG (SEQ ID NO: 14). 【0085】 In some embodiments, the peptide of the present invention comprises or consists of the amino acid sequence: CLIWIGSSKNPYILCGRR (SEQ ID NO: 15). 【0086】 In some embodiments, the peptide of the present invention comprises or consists of the amino acid sequence: CLIWIGSSKNPYILCRR (SEQ ID NO: 16). 【0087】 In some embodiments, the peptide of the present invention comprises or consists of the amino acid sequence: CLIWIGSSKNPYILCR (SEQ ID NO: 17). 【0088】 In some embodiments, the peptide comprises 14 to 20 amino acids. In some embodiments, at least 14 amino acid residues include at least 14, 15, 16, 17, 18, 19, or 20 amino acid residues, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the 14 to 20 amino acid residues include 14 to 19, 14 to 18, 14 to 17, 14 to 16, 14 to 15, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 15 to 16, 16 to 20, 16 to 19, 16 to 18, 16 to 17, 17 to 20, 17 to 19, 17 to 18, 18 to 20, 18 to 19, or 19 to 20 amino acid residues. Each possibility represents a separate embodiment of the present invention. 【0089】 In some embodiments, the peptide of the present invention comprises at least one amino acid substitution as compared to SEQ ID NO: 1. 【0090】 In some embodiments, the amino acid substitution includes substitution to a cysteine residue. 【0091】 In some embodiments, the substituted cysteine residue is chemically modified. In some embodiments, the substituted cysteine residue is functionalized. 【0092】 In some embodiments, the peptide of the present invention comprises at least one chemical modification. 【0093】 In some embodiments, the chemical modification includes a protecting or protective group. In some embodiments, the protecting or protective group includes a thiol protecting group. In some embodiments, the thiol protecting group includes an acetamidomethyl (Acm) group. 【0094】 "Thiol protecting groups" are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999, which are hereby incorporated by reference in their entirety. Examples of protected thiol groups include, but are not limited to, thioesters, carbonates, sulfonates, allyl thioethers, thioethers, silyl thioethers, alkyl thioethers, arylalkyl thioethers, and alkyloxyalkyl thioethers. Examples of ester groups include formate esters, acetate esters, propionate esters, pentanoate esters, crotonate esters, and benzoate esters. Specific examples of ester groups include formate esters, benzoylformate esters, chloroacetate esters, trifluoroacetate esters, methoxyacetate esters, triphenylmethoxyacetate esters, p-chlorophenoxyacetate esters, 3-phenylpropionate esters, 4-oxopentanoate esters, 4,4-(ethylenedithio)pentanoate esters, pivaloyl esters (trimethylacetate esters), crotonate esters, 4-methoxycrotonate esters, benzoate esters, p-benzylbenzoate esters, 2,4,6-trimethylbenzoate esters. Examples of carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonates. Examples of silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ethers, and other trialkylsilyl ethers. Examples of alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ethers, or derivatives thereof.Examples of arylalkyl groups include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ether, and the like. 【0095】 In some embodiments, at least one chemical modification is selected from alkylation, arylation, oxidation, or any combination thereof. 【0096】 In some embodiments, the functionalized cysteine disclosed herein is conjugated to a carbon chain. In some embodiments, the peptide of the invention comprises a functionalized cysteine residue conjugated to a carbon chain. In some embodiments, the carbon chain contains one or more carbons. In some embodiments, the carbon chain containing one or more carbons contains at least 2, at least 3, at least 4, or at least 5 carbons, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the carbon chain containing one or more carbons contains 2 - 3, 2 - 4, 2 - 5, 3 - 4, 3 - 5, or 4 - 5 carbons. Each possibility represents a separate embodiment of the invention. In some embodiments, the amino acid residues of the polypeptides of the invention described above are functionalized by conjugation to a methyl group, an ethyl group, a propyl group, a butyl group, or any combination thereof. 【0097】 In some embodiments, the alkylation or arylation comprises a conjugate of one of the following groups: CH3, CH2C6H5, CH2CONH2, CH2C 10 H7, CH2C9H5O2, CH2C 10 H7O3, C6F5, C 10 F9, or a combination thereof. In some embodiments, the alkylation or arylation is any conjugate: CH3, CH2C6H5, CH2CONH2, CH2C 10 H7, CH2C9H5O2, CH2C 10H7O3, C6F5, C 10 comprises a conjugate to the first residue of SEQ ID NO: 2 (Cys1) of H7O3, C6F5, C, F9, or a combination thereof. 【0098】 In some embodiments, the peptide is conjugated to a fluorophore. In some embodiments, the peptide is conjugated to a fluorophore selected from: fluorescein-5-isothiocyanate (FITC) and carboxytetramethylrhodamine (TAMRA). In some embodiments, the conjugated peptide fluorophore is used to perform intracellular tracking, evaluate cell permeability, or screen for additional unlabeled peptides that compete with the peptide fluorophore for Ub binding. 【0099】 In some embodiments, the peptides of the invention can bind to ubiquitin (Ub). In some embodiments, the peptides of the invention have specific binding affinity for ubiquitin (Ub). In some embodiments, the peptides of the invention have increased binding affinity for ubiquitin molecules compared to a control. 【0100】 As used herein, the term "ubiquitin" refers to a regulatory protein that is added to other proteins by post-translational modification. In some embodiments, Ub is dimeric Ub (Di-Ub). In some embodiments, Ub is polymeric Ub (poly-Ub). In some embodiments, polymeric Ub comprises at least 2, at least 3, at least 4, or at least 5, or any value and range therebetween of Ub monomers. Each possibility represents a separate embodiment of the invention. In some embodiments, polymeric Ub comprises 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 Ub monomers. Each possibility represents a separate embodiment of the invention. In some embodiments, the Ub dimer comprises two Ub monomers linked to each other at the Lys residue at position 63 (Lys63Di-Ub). 【0101】 In some embodiments, the peptides of the invention have a high binding affinity for Lys63-linked Ub as compared to other Ub dimers, oligomers, or polymers such as, but not limited to, Lys11-linked Ub, Lys29-linked Ub, and Lys48-linked Ub. In some embodiments, the peptides of the invention have a high binding affinity for Lys63-linked Di-Ub as compared to any of the following: Lys11-linked Di-Ub, Lys29-linked Di-Ub, and Lys48-linked Di-Ub. 【0102】 In some embodiments, the control Ub chain does not have Lys63Ub. In some embodiments, the control Ub chain does not contain Lys63Ub. In some embodiments, the control Ub chain contains a Ub chain (e.g., a polymer) in which the Ub monomers of the Ub chain are not linked to each other via Lys63. In some embodiments, the control Ub chain contains a Ub chain (e.g., a polymer) in which the Ub monomers of the Ub chain are linked to each other via Lys11, Lys29, Lys48, or any combination thereof. 【0103】 In some embodiments, Ub is further conjugated to a biotin tag. In some embodiments, Lys-63Di-Ub conjugated to biotin is used to screen for peptides that selectively bind to Lys-63Di-Di-Ub. In some embodiments, the screened peptide(s) comprise cyclic peptide(s). 【0104】 In some embodiments, the peptides of the invention can bind to Lys63 Di-Ub in vitro, in vivo, ex vivo, or any combination thereof. In some embodiments, the peptides of the invention can permeate cells. In some embodiments, the peptide does not require additional elements to permeate cells. In some embodiments, the peptide may be further formulated with other elements to enhance cell permeation. In some embodiments, the peptide may be used as a carrier or vehicle for delivering other elements into cells. 【0105】 In some embodiments, the peptide or functional analog thereof of the invention has a Lys63Di-Ub binding affinity with a KD of 0.05 - 1 nM, 0.5 - 5 nM, 1 - 10 nM, 5 - 15 nM, 10 - 20 nM, 15 - 30 nM, 20 - 40 nM, 35 - 50 nM, 45 - 60 nM, 55 - 70 nM, 65 - 80 nM, 75 - 90 nM, 85 - 95 nM, 90 - 120 nM, 100 - 500 nM, 250 - 750 nM, 0.7 - 1.5 μM, 1 - 5 μM, 4 - 10 μM, 8 - 20 μM, 1 nM - 1 μM, or 15 - 40 μM. Each possibility represents a distinct embodiment of the invention. In some embodiments, the peptide or functional analog thereof of the invention has a Ub binding affinity with a KD of at most 0.1 nM, at most 0.5 nM, at most 1 nM, at most 5 nM, at most 10 nM, at most 20 nM, at most 30 nM, at most 40 nM, at most 50 nM, at most 60 nM, at most 70 nM, at most 80 nM, at most 90 nM, at most 100 nM, at most 110 nM, at most 150 nM, at most 250 nM, at most 500 nM, at most 750 nM, at most 1,500 nM, at most 1 μM, at most 5 μM, at most 10 μM, at most 15 μM, at most 20 μM, or at most 30 μM, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. 【0106】 The present invention encompasses derivatives of the peptides of the present invention. The term "derivative" or "chemical derivative" includes any chemical derivative of a peptide having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, molecules in which the free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. The free carboxyl group can be derivatized to form a salt, amide (e.g., -CONH2), methyl ester and ethyl ester, or other types of esters or hydrazides. The free hydroxyl group can be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Chemical derivatives also include peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acid residues. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine (O) can be substituted for lysine. 【0107】 Furthermore, the peptide derivatives can differ from the native sequence of the peptides of the present invention by chemical modifications such as, but not limited to, terminal NH2 acylation, acetylation, or thioglycolic acid amidation, and also by chemical modifications such as terminal carboxyl amidation, for example by ammonia, methylamine, etc. The peptide can have any arbitrary three-dimensional structure, such as linear, cyclic, or branched, and this can be obtained using methods known in the art. In some embodiments, the peptides of the present invention further comprise any PTMs except post-translational modifications that occur naturally in mammals. 【0108】 As used herein, the terms "peptide", "polypeptide" and "protein" include native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications), as well as peptide analogs peptoids and semipeptoids, or any combination thereof. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid. 【0109】 As used herein, the term "amino acid" means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are naturally occurring amino acids, modified amino acids, abnormal amino acids, non-natural amino acids, and amino acids that are known to exist either free or in combined form biologically, but that do not normally occur within proteins. This term includes modified and abnormal amino acids such as those disclosed, for example, in Roberts and Vellaccio(1983)The Peptides.5:342-429. Modified, abnormal, or non-natural amino acids include, but are not limited to, D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz protected aminovaleric acid (Nva), ornithine (O), aminooctanoic acid (Aoc), 2,4-diaminobutyric acid (Abu), homoarginine, norleucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, 1-phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-norleucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid, trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminopentanoic acid, norvaline (Nva), 4-O-methyl-threonine (TMe), 5-O-methyl-homoserine (hSM), tert-butyl-alanine (tBu), cyclopentyl-alanine (Cpa), 2-amino-isobutyric acid (Aib), N-methyl-glycine (MeG), N-methyl-alanine (MeA), N-methyl-phenylalanine (MeF), 2-thienyl-alanine (2Th), 3-thienyl-alanine (3Th), O-methyl-tyrosine (YMe), 3-benzothienyl-alanine (Bzt), and D-alanine (DAl). 【0110】 One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a peptide or protein sequence which alter, add or delete a single amino acid or a low percentage of amino acids in the encoded sequence are conservatively modified variants where the alteration results in substitution of an amino acid with similar charge, size, and / or hydrophobic characteristics, for example, substitution of glutamic acid (E) with aspartic acid (D). 【0111】 As used herein, the term "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such a peptide exhibits the requisite function as defined herein. 【0112】 Peptide derivatives according to the principles of the present invention may include side chain linkage modifications including, but not limited to, -CH2-NH-, -CH2-S-, -CH2-S=O, OC-NH-, -CH2-O-, -CH2-CH2-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. The peptide bond (-CO-NH-) within a peptide may be substituted, for example, by an N-methylated bond (-N(CH3)-CO-); an ester bond (-C(R)H-C-O-O-C(R)H-N); a ketomethylene bond (-CO-CH2-); an a-aza bond (-NH-N(R)-CO-), where R is any alkyl group, for example, methyl; a carba bond (-CH2-NH-); a hydroxyethylene bond (-CH(OH)-CH2-); a thioamide bond (-CS-NH); an olefmic double bond (-CH=CH-); and a peptide derivative (-N(R)-CH2-CO-), where R is a "normal" side chain naturally present on a carbon atom. These modifications can occur at one or more bonds along the peptide chain and can occur simultaneously at multiple (e.g., 2-3) bonds. 【0113】 The present invention further includes peptides and derivatives thereof that may contain one or more D isomeric forms of amino acids. The production of retro-inverso D-amino acid peptides, where at least one amino acid, and perhaps all amino acids, are D-amino acids, is well known in the art. When all of the amino acids in a peptide are D-amino acids and the N-terminus and C-terminus of the molecule are reversed, a molecule is obtained that has the same structural groups in the same positions as the L-amino acid form of the molecule. However, this molecule is more stable to proteolysis and is thus useful in many of the applications described herein. Diastereomer peptides can be highly advantageous over all L-amino acid peptides or all D-amino acid peptides having the same amino acid sequence because they are highly water-soluble, have low immunogenicity, and are less sensitive to proteolysis. As used herein, the term "diastereomer peptide" refers to a peptide that contains both L-amino acid residues and D-amino acid residues. The number and position of D-amino acid residues in the diastereomer peptides of the present invention can be variable as long as the peptide can exhibit the functions of the disclosed chimeras of the present invention. 【0114】 In some embodiments, the peptides of the present invention are cyclized. In some embodiments, cyclization is effected via the linkage of the first amino acid residue at the N-terminus of the peptide of the present invention with the C-terminal amino acid. In some embodiments, the C-terminal amino acid is located at a position in the range of 3 to 20, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 10 to 20, 10 to 18, 10 to 16, 10 to 14, or any range therebetween. In some embodiments, the linkage is a covalent bond. In some embodiments, the linkage is formed (i) between the N-terminus of the first amino acid residue and the side chain of the C-terminal amino acid, or (ii) between the side chain of the first amino acid residue and the side chain of the C-terminal amino acid. In some embodiments, the first amino acid residue is a cysteine residue and the linkage is effected via the thiol group of the first amino acid. In some embodiments, the covalent bond is effected via direct conjugation of the side chains (i.e., without a spacer such as an S-S bond between cysteine residues). In some embodiments, the covalent bond includes a linkage between a cysteine residue (e.g., its N-terminal amino group or thiol group) and the C-terminal amino acid via a cyclizing molecule. In some embodiments, the first amino acid residue at the N-terminus (intra-ring position) of the peptide of the present invention is conjugated to the cyclizing molecule. In some embodiments, the first amino acid residue of the peptide (located at the N-terminus or intra-ring position) binds to another amino residue of the peptide (located at the C-terminus), thereby obtaining a cyclic peptide. In some embodiments, the cyclizing molecule binds to both the first amino acid residue and another amino acid residue located at the C-terminus. In some embodiments, the cyclizing molecule facilitates the binding of the first amino acid residue (located at the N-terminus or intra-ring position) to the C-terminal amino acid residue. In some embodiments, the cyclizing molecule is conjugated to both the first amino acid residue (located at the N-terminus or intra-ring position) and the C-terminal amino acid residue. As defined herein, the "C-terminal amino acid residue" refers to an amino acid residue that is closer to the C-terminus (extra-ring position) of the linear peptide compared to the N-terminus of the peptide.In some embodiments, the C-terminal amino acid residue is the eighth, seventh, sixth, fifth, fourth, third, second, or first from the last, or is the last amino acid residue of the linear peptide. Each possibility represents a distinct embodiment of the invention. By way of non-limiting example, the ninth amino acid residue of a peptide containing 16 amino acid residues is considered the C-terminal amino acid residue. For example, as discussed by White and Yudin (2011), various methods are available for cyclizing (e.g., macrocyclizing) polypeptides. 【0115】 In some embodiments, the cyclized molecule is a linker. In some embodiments, the linker comprises an optionally substituted C1-C10 alkyl, alkylaryl, haloaryl, halobiaryl, alkyl-C(O)-alkyl, heteroatom (e.g., O, N, NH, or S), -C(O)NH-, -C(O)O-, -C(O)-, -C(O)S-, -C(NH)NH-, -C(NH)O-, -C(NH)S-, -S-S-, -S-C(=O), Y-(C0-10)alkyl-X-(C0-10)alkyl-Y, or a click reaction product comprising any combination thereof; wherein X and Y are each independently absent or selected from heteroatom, oligomer, click reaction product, oxo, -CONR'-, -CNNR'-, -CSNR'-, -NC(=O)O-, -NC(=S)O-, -NC(=S)N-, -SO2-, -SO-, -SR', -C(=O)-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, and -OC(=S)N-. In some embodiments, the oligomer comprises 2-15 repeating units. In some embodiments, the repeating unit is selected from alkylene oxide, natural or unnatural amino acid residues, α-hydroxycarboxylic acid residues (including any copolymers and any combination thereof). 【0116】 Click reactions are well known in the art and include, inter alia, the Michael addition of maleimide and thiol (resulting in the formation of succinimide - thioether); azide - alkyne cycloaddition; Diels - Alder reaction (e.g., direct and / or inverse electron demand Diels - Alder); dibenzylcyclooctyne 1,3 - nitrone (or azide) cycloaddition; alkene - tetrazole photoclick reaction, etc. 【0117】 Accordingly, the term "click reaction product" includes moieties formed via a click reaction, where the click reaction is as described above. In some embodiments, the click reaction product includes a product formed by any of the following: the Michael addition of maleimide and thiol (resulting in the formation of succinimide - thioether); azide - alkyne cycloaddition; Diels - Alder reaction (e.g., direct and / or inverse electron demand Diels - Alder); dibenzylcyclooctyne 1,3 - nitrone (or azide) cycloaddition; alkene - tetrazole photoclick reaction, or any combination thereof. 【0118】 In some embodiments, the cyclized molecule includes 【Chemical formula】 any one of, wherein the wavy bonds each represent an attachment point to the first amino acid residue and the C - terminal amino acid, respectively. 【0119】 In some embodiments, the linkage is effected between the side chain of the first amino acid residue or the N - terminal amino group and the C - terminal amino acid via a cyclized molecule, where the cyclized molecule binds to the amino group of the C - terminal amino acid. Exemplary linkages are as shown below: 【Chemical formula】 In the formula, each X independently represents an amino acid, R is the side chain of the C-terminal amino acid, the arrow indicates the attachment point of the cyclized molecule to the amino group of the C-terminal amino acid, and the dashed arrow indicates the N-terminal amino group of the first amino acid residue. Those skilled in the art will understand that the cyclized molecules shown above are only exemplary cyclized molecules without limitation, and that the peptides of the present invention may include any cyclized molecule as disclosed herein. 【0120】 Additional exemplary cyclized molecules include, but are not limited to, the following: 【Chemical Structure】 In the formula, the wavy bond represents the attachment point to the first amino acid residue (e.g., its N-terminal amino group), and the dashed bond represents the attachment point to the amino group of the C-terminal amino acid. 【0121】 In some embodiments, the cyclized molecule contains one or more halogen atoms selected from: fluoride (F), chlorine (Cl), bromide (Br), iodine (I), and astatine (At), or any combination thereof. Non-limiting examples of cyclized molecules containing a halogen include, but are not limited to, the following. Chloroacetyl (ClAc), 3-(chloromethyl)benzoic acid (mCIBz), 4-(chloromethyl)benzoic acid (pCIBz), chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl (4-ClBz), or Cl2SAc. In one embodiment, the cyclized molecule containing a halogen group is conjugated to the first amino acid residue at the N-terminus of the polypeptide and nucleophilically attacks the thiol group of the cysteine residue located at the C-terminus of the peptide, thereby resulting in a cyclic peptide. 【0122】 In some embodiments, the cyclized molecule is selected from chloroacetyl (ClAc), 3-(chloromethyl)benzoic acid (mCIBz), 4-(chloromethyl)benzoic acid (pCIBz), chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl (4-ClBz) or Cl2SAc. 【0123】 In some embodiments, the cyclized molecules disclosed herein include cyclization precursor molecules. In some embodiments, the cyclization precursor molecules include at least one halogen atom. 【0124】 In some embodiments, the peptide is prepared using a cyclized molecule that includes at least one halogen. 【0125】 In some embodiments, the peptides of the present invention are represented by Formula 1: 【Chemical formula】 Wherein each X independently represents an amino acid residue, X1 is the C-terminal amino acid, L represents a cyclized molecule or a bond, and n is an integer in the range of 0-16, 1-16, 2-16, 3-16, 4-16, 5-16, and any range therebetween. 【0126】 In some embodiments, dimeric peptides comprising the peptides of the present invention are provided. In some embodiments, dimeric cyclic peptides comprising the cyclic peptides of the present invention are provided. In some embodiments, the dimeric peptide is a homodimer or a heterodimer. In some embodiments, the dimeric peptide is a homodimer. In some embodiments, the dimeric peptide comprises two monomeric peptides covalently linked to each other via cysteine residues. In some embodiments, the monomeric peptides are linked via a disulfide bond (-S-S-). In some embodiments, the monomeric peptides are linked via a linker, such as a carbon chain of one or more carbon atoms. In some embodiments, the monomeric peptides are linked via a CH2 linker. In some embodiments, the monomeric peptides are linked via the following bond -S-CH2-S-. 【0127】 Peptide synthesis According to one embodiment, the peptides of the present invention can be synthesized or prepared by any method and / or technique known in the field of peptide synthesis. According to another embodiment, the peptides can be synthesized by the solid-phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). According to another embodiment, the peptides of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, 1984). 【0128】 Generally, the synthesis method involves sequentially adding one or more amino acids or suitably protected amino acids to the growing peptide chain attached to a suitable resin. Usually, either the amino group or the carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid is then either attached to an inert solid support (resin) or can be utilized in solution by adding the next amino acid in a sequence having a complementary (amino or carboxyl) group that is suitably protected under conditions suitable for forming an amide linkage. Next, the protecting group is removed from this newly added amino acid residue, the next amino acid (suitably protected) is added, and this is repeated. After all the desired amino acids have been linked in the appropriate order, any remaining protecting groups are removed sequentially or simultaneously, and the peptide chain synthesized by the solid-phase method is cleaved from the solid support to obtain the final peptide. 【0129】 In solid-phase peptide synthesis, the α-amino group of an amino acid is protected by a group that is highly sensitive to acids or bases. Such a protecting group shall have the property of being stable to the conditions for peptide bond formation and being easily removable without breaking the growing peptide chain. Suitable protecting groups include t-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and the like. In solid-phase peptide synthesis, the C-terminal amino acid is attached to a suitable solid support. A suitable solid support useful for the above synthesis is a material that is inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and insoluble in the solvent medium used. Suitable solid supports include chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like. The coupling reaction is achieved in a solvent such as ethanol, acetonitrile, N,N-dimethylformamide (DMF). The coupling of successive protected amino acids can be carried out using an automated polypeptide synthesizer as is well known in the art. 【0130】 In another embodiment, the peptide of the present invention can be synthesized such that one or more of the bonds linking the amino acid residues of the peptide are non-peptide bonds. In another embodiment, non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, and these can be formed by reactions well known to those skilled in the art. 【0131】 The term "linker" refers to a molecule or polymer that serves to link different parts of a peptide or polypeptide. In one embodiment, the linker can also facilitate other functions including, but not limited to, maintaining biological activity and maintaining subunit and domain interactions. 【0132】 In some embodiments, the peptides of the present invention may be attached or linked to another molecule via a chemical linker. In some embodiments, being attached means occurring in a conjugation reaction. Chemical linkers are well known in the art and include, but are not limited to, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline (EEDQ), N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ). In another embodiment, the linker may be a monomeric entity such as a single amino acid. In another embodiment, an amino acid with a small side chain, or a small polypeptide chain, or a polymeric entity of several amino acids is particularly preferred. In another embodiment, the polypeptide linker is 15 amino acids or less in length, 10 amino acids or less in length, or 5 amino acids or less in length. In one embodiment, the linker can be a nucleic acid encoding a small polypeptide chain. In another embodiment, the linker encodes a polypeptide linker that is 15 amino acids or less in length, 10 amino acids or less in length, or 5 amino acids or less in length. 【0133】 Recombinant techniques can be used to express the peptides of the present invention, which are well known in the art. In another embodiment, the linker can be a cleavable linker, and when delivered to a selected tissue or cell, the peptide of the present invention is cleaved. In such embodiments, the cell or tissue has an endogenous enzyme (a naturally occurring enzyme, or an enzyme recombinantly engineered to express an enzyme) that can cleave the cleavable linker, or has an exogenous (e.g., by injection, absorption, etc.) enzyme. 【0134】 In another embodiment, the linker can be biodegradable, whereby the polypeptide of the present invention is further processed by hydrolysis and / or enzymatic cleavage within the cell. In one embodiment, a tumor-specific expression protease can be used for delivery of a prodrug of a cytotoxic agent in a state where the linker is selective for site-specific proteolysis. In some embodiments, readily cleavable groups include acetyl, trimethylacetyl, butanoyl, methylsuccinoyl, t-butylsuccinoyl, ethoxycarbonyl, methoxycarbonyl, benzoyl, 3-aminocyclohexylidenyl, and the like. 【0135】 The present invention further encompasses a polynucleotide sequence comprising a nucleic acid encoding any of the peptides of the present invention. In another embodiment, the nucleic acid sequence encoding the peptide is at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or any value and range therebetween, homologous to the nucleic acid sequence encoding the peptide or derivative thereof of the present invention. Each possibility represents a separate embodiment of the present invention. 【0136】 In some embodiments, the present invention provides a polynucleotide encoding the peptide of the present invention. 【0137】 In some embodiments, the polynucleotide of the present invention is ligated into an expression vector that includes transcriptional control of cis-regulatory sequences (e.g., promoter sequences). In some embodiments, the cis-regulatory sequences are suitable for directing constitutive expression of the peptide of the present invention. In some embodiments, the cis-regulatory sequences are suitable for directing tissue-specific expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequences are suitable for directing inducible expression of the peptide of the present invention. 【0138】 The term "polynucleotide" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains the coding sequence necessary for the production of a peptide. In one embodiment, a polynucleotide is a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above). 【0139】 In one embodiment, the term "complementary polynucleotide sequence" refers to a sequence obtained by reverse transcribing messenger RNA using reverse transcriptase or other RNA-dependent DNA polymerases. In one embodiment, the sequence can then be amplified in vivo or in vitro using DNA polymerase. 【0140】 In one embodiment, the term "genomic polynucleotide sequence" refers to a sequence derived from (or isolated from) a chromosome and thus represents a continuous portion of the chromosome. 【0141】 In one embodiment, the term "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, the composite sequence can include several exon sequences necessary for encoding the polypeptide of the present invention, as well as several intron sequences present therebetween. In one embodiment, the intron sequences can be from any source such as other genes and typically can include conserved splicing signal sequences. In one embodiment, the intron sequences include cis-acting expression regulatory elements. 【0142】 In some embodiments, the polynucleotides of the present invention are prepared using PCR technology or other methods or procedures known to those skilled in the art. 【0143】 In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to enable the expression of the recombinant peptides disclosed herein. In one embodiment, the expression vector contains additional sequences that make this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector contains additional sequences that make this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector contains a shuttle vector that makes this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector contains transcriptional and translational start sequences (e.g., promoters, enhancers) as well as transcriptional and translational terminators (e.g., polyadenylation signals). 【0144】 In one embodiment, various prokaryotic or eukaryotic cells can be used as the host expression system for expressing the peptide of the present invention. In some embodiments, these include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a polypeptide coding sequence; yeast transformed with a recombinant yeast expression vector containing a polypeptide coding sequence; plant cell lines infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus; CaMV, tobacco mosaic virus, TMV), a recombinant plasmid expression vector, e.g., a plant cell line transformed with a Ti plasmid containing a polypeptide coding sequence. 【0145】 In some embodiments, a non-bacterial expression system (e.g., a mammalian expression system) is used to express the peptide of the present invention. In one embodiment, the expression vector is used to express the polynucleotide of the present invention in mammalian cells. 【0146】 In some embodiments, in a bacterial system, multiple expression vectors can be advantageously selected according to the intended use for the expressed peptide. In one embodiment, a large amount of peptide is desired. In one embodiment, a vector that directs the expression of a high level of protein product, perhaps as a fusion with a hydrophobic signal sequence, and directs the expressed product to the bacterial periplasm or a culture medium where the protein product can be easily purified, is desirable. In one embodiment, a particular fusion protein is engineered with a specific cleavage site to aid in the recovery of the polypeptide. In one embodiment, vectors adaptable to such engineering include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)]. 【0147】 In one embodiment, a yeast expression system is used. In one embodiment, multiple vectors containing constitutive or inducible promoters in yeast can be used, as disclosed in U.S. Patent Application Publication No. 5,932,447. In another embodiment, a vector that promotes the integration of foreign DNA sequences into the yeast chromosome is used. 【0148】 In one embodiment, the expression vector may further contain an additional polynucleotide sequence that enables the translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES), for example. 【0149】 In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech), and derivatives thereof. 【0150】 In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses can be used. Examples of SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector capable of expressing proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoter shown to be effective for expression in eukaryotic cells. 【0151】 In some embodiments, recombinant viral vectors that provide advantages such as lateral infection and target specificity are used for in vivo expression of the peptides of the present invention. In one embodiment, lateral infection is, for example, inherent in the life cycle of retroviruses and is the process by which one infected cell produces many progeny virions that bud and infect adjacent cells. In one embodiment, as a result, a large area becomes rapidly infected, most of which were not initially infected by the original virus particles. In one embodiment, viral vectors that cannot spread laterally are produced. In one embodiment, this feature can be useful when the desired goal is to introduce a particular gene into only a local number of target cells. 【0152】 A variety of methods can be used to introduce an expression vector into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. Further, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992. 【0153】 In one embodiment, a plant expression vector is used. In one embodiment, the expression of the polypeptide coding sequence is driven by multiple promoters. In some embodiments, viral promoters such as the CaMV 35S RNA promoter and 19S RNA promoter [Brisson et al., Nature 310:511-514 (1984)], or the TMV coat protein promoter [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters such as the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)] are used. In one embodiment, constructs are introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those skilled in the art. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp421-463 (1988)]. Other expression systems such as insect and mammalian host cell lines well known in the art can also be used in the present invention. 【0154】 It will be understood that, in addition to containing elements necessary for transcription and translation of the inserted coding sequence (which encodes a polypeptide), the expression construct can also include sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed peptide. 【0155】 In some embodiments, the transformed cells are cultured under effective conditions that allow for the expression of large amounts of recombinant peptide. In some embodiments, effective culture conditions include, but are not limited to, an effective medium that allows for protein production, a bioreactor, temperature, pH, and oxygen conditions. In one embodiment, an effective medium refers to any medium in which cells are cultured to produce the recombinant polypeptide of the present invention. In some embodiments, the medium typically includes an assimilable carbon, nitrogen, and phosphate source, as well as an aqueous solution containing other nutrients such as appropriate salts, minerals, metals, and vitamins. In some embodiments, the cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates, and Petri dishes. In some embodiments, culturing is performed at a temperature, pH, and oxygen content suitable for the recombinant cells. In some embodiments, the culture conditions are within the expertise of those skilled in the art. 【0156】 In some embodiments, depending on the vector and host system used for production, the resulting peptide of the present invention remains within the recombinant cell, is secreted into the fermentation medium, is secreted into the space between two cell membranes such as the periplasmic space of E. coli, or is retained on the outer surface of the cell or virus membrane. In one embodiment, after a predetermined time during culturing, the recovery of the recombinant polypeptide is affected. 【0157】 In one embodiment, the phrase "recovering the recombinant peptide" as used herein refers to collecting the entire fermentation medium containing the peptide and does not necessarily mean additional steps of separation or purification. 【0158】 In one embodiment, the peptides of the present invention are purified using various standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and fractionation lysis. 【0159】 In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the invention and the fused cleavable moiety. In one embodiment, the fusion protein can be designed such that the polypeptide can be readily isolated by immobilization on a column specific for, for example, the cleavable moiety by affinity chromatography. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site (see, for example, Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)). 【0160】 In one embodiment, the peptide of the invention is recovered in a "substantially pure" form such that the protein can be effectively used in the applications described herein. 【0161】 As used herein, the term "substantially pure" refers to a peptide / polypeptide or other substance that has been separated from native contaminants. Typically, a monomeric peptide is substantially pure if at least about 60-75% of the sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence. A substantially pure peptide can comprise greater than about 85-90% of the peptide sample, and can be of a purity greater than 95%, greater than 97%, or greater than about 99%, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. Purity can be measured on a polyacrylamide gel and homogeneity can be determined by staining. Alternatively, for certain purposes, high resolution may be required and HPLC or similar purification means can be used. In most cases, a simple chromatography column or polyacrylamide gel can be used to determine purity. 【0162】 The term "purified" does not require that a substance be present in a form exhibiting absolute purity, excluding the presence of other compounds. Rather, it is a relative definition. A peptide is in a "purified" state after being purified at least one, two, or three orders of magnitude, or four or five orders of magnitude, from a starting material or natural material. 【0163】 In one embodiment, the peptides of the invention are substantially free of naturally associated host cell components. The term "substantially free of naturally associated host cell components" refers to a peptide or other substance that has been separated from the natural contaminants that accompany it in its native host cell state. Thus, a chemically synthesized peptide, or a peptide synthesized in a cell system different from the host cell from which it is derived, is free of naturally associated host cell components. 【0164】 In one embodiment, the peptide of the present invention can also be synthesized using an in vitro expression system. In one embodiment, the in vitro synthesis method is well-known in the art, and the components of the system are commercially available. Non-limiting examples of in vitro systems include, but are not limited to, in vitro translation. 【0165】 Pharmaceutical composition According to some embodiments, there is provided a composition comprising any one of (a) the peptide of the present invention, (b) the dimeric peptide of the present invention, and (c) a combination of (a) and (b), and an acceptable carrier. 【0166】 In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the carrier is a pharmaceutical carrier. 【0167】 In some embodiments, the pharmaceutical composition is for use in the treatment or prevention of K63Ub-related diseases in a subject in need thereof. In some embodiments, K63Ub-related diseases include diseases associated with dysregulation of Lys63Ub. 【0168】 The term "K63Ub-related disease" refers to any disease in which Lys63Ub is involved in the etiology, pathophysiology, or both of the disease, induces, enhances, propagates, or any combination thereof. 【0169】 In some embodiments, a subject suffering from a K63Ub-related disease or having an increased risk of developing a K63Ub-related disease is characterized by an increased or elevated level of Lys63Ub compared to a healthy subject. In some embodiments, a subject suffering from a K63Ub-related disease or having an increased risk of developing a K63Ub-related disease is characterized by an elevated level of ubiquitin E3 ligase compared to a healthy subject. In some embodiments, a subject suffering from a K63Ub-related disease or having an increased risk of developing a K63Ub-related disease is characterized by an increased level of ring finger protein 8 (RNF8) or RNF168 compared to a healthy subject. In some embodiments, a subject suffering from a K63Ub-related disease or having an increased risk of developing a K63Ub-related disease is characterized by an elevated level of mutant DNA compared to a healthy subject. In some embodiments, a subject suffering from a K63Ub-related disease or having an increased risk of developing a K63Ub-related disease is characterized by an increase in the level, abundance, phosphorylation level, or any combination thereof of histone H2A family member X (H2AX) compared to a healthy subject. 【0170】 In some embodiments, the K63-related disease includes a cell proliferation-related disease. In some embodiments, the cell proliferation-related disease includes cancer. In some embodiments, the cancer includes adenocarcinoma. In some embodiments, the cancer includes squamous cell carcinoma. In some embodiments, the cancer includes sarcoma. In some embodiments, the cancer includes osteosarcoma. 【0171】 The term "cell proliferation-related disease" refers to any disease in which dysregulation of cell proliferation is involved in at least one mechanism of disease development. 【0172】 In some embodiments, the pharmaceutical composition is characterized by having pro-apoptotic activity. In some embodiments, the pharmaceutical composition is characterized by having cell cycle arrest activity. 【0173】 As used herein, the term "pro-apoptotic activity" refers to the ability of a compound to induce, increase, enhance, propagate, facilitate, contribute to, be involved in, or any combination thereof, programmed cell death. 【0174】 As used herein, the term "cell cycle arrest" refers to delaying, stopping, inhibiting, blocking, or any combination thereof, the progression of a cell in the cell cycle. A cell can be induced to arrest at any point / phase / step of the cell cycle. In some embodiments, cell cycle arrest activity includes arresting, inhibiting, blocking, or any combination thereof, the progression of a cell through any one of the following: G0, G1, S, G2, or M. 【0175】 In some embodiments, the pharmaceutical composition is for use in the treatment of cancer in a subject in need thereof. 【0176】 In some embodiments, the pharmaceutical composition facilitates the administration of the compound to an organism. According to another embodiment, the present invention provides a pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the peptide of the present invention, the dimeric peptide of the present invention, or both. 【0177】 In another embodiment, the pharmaceutical composition of the present invention can be formulated in the form of a pharmaceutically acceptable salt of the peptide of the present invention or an analog or derivative thereof. In another embodiment, pharmaceutically acceptable salts include salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, iron(III) hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine. 【0178】 As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic compound is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, for example, of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Aqueous solutions of sodium chloride as well as dextrose and glycerol can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain, if desired, small amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, and tonicity modifiers such as sodium chloride or dextrose are also contemplated. The carrier can in total constitute from about 0.1 wt% to about 99.99999 wt% of the pharmaceutical composition presented herein. 【0179】 As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, such as a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory authority of the federal or state government or included in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, more specifically in humans. 【0180】 In another embodiment, the compositions of the present invention may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained release formulations, and the like. In another embodiment, the compositions of the present invention may be formulated as suppositories with conventional binders and carriers such as triglycerides, microcrystalline cellulose, tragacanth gum, or gelatin. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences (E.W. Martin). The content thereof is incorporated herein by reference. Such compositions preferably contain a therapeutically effective amount of the polypeptide of the present invention in a substantially purified form, together with a carrier in an amount suitable to provide a form for appropriate administration to a subject. 【0181】 According to one embodiment of the present invention, the pharmaceutical composition comprises from 0.1% to 95% of the peptide(s), functional analog(s), or derivative(s) of the present invention. According to another embodiment of the present invention, the pharmaceutical composition comprises from 1% to 70% of the peptide(s). According to another embodiment of the present invention, the composition or formulation to be administered may contain an amount of the peptide(s) effective to treat the condition or disease of the subject to be treated, in accordance with an embodiment of the present invention. 【0182】 Embodiments of the present invention relate to the peptides of the present invention, dimeric peptides, or both, provided in unit dosage forms and prepared by any of the methods well known in the pharmaceutical art. In one embodiment of the present invention, the unit dosage form is in the form of tablets, capsules, lozenges, wafers, patches, ampoules, vials, or prefilled syringes. Additionally, in vitro assays may optionally be used to assist in determining the optimal dosage range. The exact dosage used in the formulation also depends on the route of administration and the nature of the disease or disorder and needs to be determined according to the judgment of the physician and the circumstances of each patient. The effective dosage can be estimated from the dose-response curve obtained from biological assays or systems in in vitro or in vivo animal model tests. 【0183】 According to one embodiment, the composition of the present invention is administered in the form of a pharmaceutical composition comprising at least one of the active ingredients of the present invention (e.g., a peptide, such as, but not limited to, a cyclic peptide) together with a pharmaceutically acceptable carrier or diluent. In another embodiment, the composition of the present invention can be administered individually or together in any conventional oral, parenteral, or transdermal dosage form. In some embodiments, the pharmaceutical composition further comprises at least one anti-cancer agent, such as a chemotherapeutic agent. In some embodiments, the pharmaceutical composition is employed in combination with anti-cancer therapy, such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. 【0184】 As used herein, the terms "administer", "administration" and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that produces a therapeutic effect in sound medical practice. 【0185】 Depending on the location of the target tissue, the peptides of the present invention can be administered by any method suitable for supplying the peptides to cells within the target tissue. Thus, for example, a composition containing the peptides of the present invention can be introduced into the systemic circulation, for example, whereby the peptides will be distributed to the target tissue. Alternatively, the composition can also be applied locally to the target tissue (e.g., by injection, infusion as a continuous infusion, as a bolus within the tissue, application to the whole or part of the surface of the skin, etc.). 【0186】 In some embodiments, the pharmaceutical composition containing the peptide is administered via oral, rectal, vaginal, topical, nasal, ocular, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration. The route of administration of the pharmaceutical composition depends on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections such as intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection modalities known in the art. The bioavailability of the peptide administered by other routes may be lower than when administered by parenteral injection, but it is contemplated that by using appropriate formulations, the compositions of the present invention can be administered by transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular treatment modalities. Further, it may be desirable to introduce the pharmaceutical compositions of the present invention by any suitable route such as intracerebroventricular injection and intrathecal injection, and intracerebroventricular injection can be easily performed, for example, by an intracerebroventricular catheter attached to a reservoir. For example, pulmonary administration by the use of an inhaler or nebulizer can also be employed. 【0187】 In the case of topical application, the peptides of the present invention, their derivatives, functional analogs or fragments thereof can be combined with a pharmaceutically acceptable carrier so that an effective dosage is delivered based on the desired activity. The carrier can be in the form of, for example, but not limited to, ointments, creams, gels, pastes, foams, aerosols, suppositories, pads, or gel sticks. 【0188】 For oral use, the pharmaceutical composition can be in the form of tablets or capsules and can contain any of the following components or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum, gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, primogel, or corn starch; lubricants such as magnesium stearate; or glidants such as colloidal silicon dioxide. When the dosage unit form is a capsule, in addition to the substances of the above type, it can contain a liquid carrier such as fatty oil. Further, the dosage unit form can include various other materials that change the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The tablets of the present invention can be further film-coated. 【0189】 For parenteral administration, solutions of sesame oil or peanut oil, or solutions of aqueous propylene glycol, as well as sterile aqueous solutions of the corresponding water-soluble salts can be used. Such aqueous solutions can be suitably buffered if necessary, and the liquid diluent can first be made isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection purposes. 【0190】 According to some embodiments, the peptide, dimeric peptide, or both of the present invention can be delivered by a controlled release system. In another embodiment, an infusion pump can be used to administer, for example, a peptide used to deliver insulin or chemotherapy to a particular organ or tumor. In another embodiment, the peptide, dimeric peptide, or both of the present invention is administered in combination with a biodegradable and biocompatible polymer implant that releases the peptide over a controlled period at a selected site. Examples of preferred polymer materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers, and blends thereof (see Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, the controlled release system can be placed near the therapeutic target, which requires only a fraction of the systemic dose. 【0191】 The peptides described herein may also be included in artificially created structures such as liposomes, ISCOMs, sustained-release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Liposomes include emulsions, bubbles, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described in the present invention are formed from standard vesicle-forming lipids that generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is generally determined in consideration of factors such as the size of the liposome and its stability in blood. For example, as discussed in Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, various methods are available for the preparation of liposomes, and reference is made to U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. 【0192】 Compositions also include the incorporation of the active substance into particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, monolayers or multilayers of vesicles, erythrocyte ghosts, or spheroplasts. Such compositions affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. 【0193】 In one embodiment, it will be understood that providing the peptide, dimer peptide, or both of the present invention to an individual together with an additional active agent can achieve an improved therapeutic effect compared to treating each agent alone. In another embodiment, measures are taken against harmful side effects associated with combination therapy (e.g., administration and selection of complementary agents). 【0194】 In one embodiment, administration can be carried out as a single or multiple administrations depending on the severity and responsiveness of the condition being treated, and the course of treatment continues for several days to several weeks, or until healing is affected or symptom relief is achieved. 【0195】 In some embodiments, the peptide is administered in a therapeutically safe and effective amount. As used herein, the term "safe and effective amount" refers to an amount of the component that, when used in the methods described herein, results in the desired therapeutic response without undue harmful side effects (such as toxicity, irritation, or allergic response, etc.) commensurate with a reasonable benefit / risk ratio. In another embodiment, a therapeutically effective amount of the polypeptide is the amount of the polypeptide necessary for a measurable expected biological effect in vivo. The actual amount administered, as well as the rate and duration of administration, depend on the nature and severity of the condition being treated. The determination of the treatment regimen, such as dosage, timing, etc., is within the responsibility of the general practitioner or specialist and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, the preparation of the effective or dosage amount can first be estimated from in vitro assays. In one embodiment, the dosage can be formulated in an animal model and such information can be used to more accurately determine the useful dosage in humans. 【0196】 In one embodiment, the toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell culture, or in experimental animals by standard pharmaceutical procedures. In one embodiment, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating dosage ranges for use in humans. In one embodiment, the dosage will vary depending on the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by an individual physician in view of the patient's condition [see, e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1]. 【0197】 A pharmaceutical composition containing the peptide(s) described herein as an active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005). 【0198】 In one embodiment, a composition is prepared that contains a preparation of the invention formulated in a compatible pharmaceutical carrier, placed in a suitable container, and labeled for treatment in the indicated circumstances. 【0199】 In one embodiment, the composition of the present invention is provided in a pack or dispenser device, such as an FDA-approved kit, comprising one or more unit dosage forms containing the active ingredient. In one embodiment, the pack includes a metal foil or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser contains a notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceuticals, and this notice reflects the form of the composition or the approval by the agency for administration to humans or animals. In one such embodiment, for a prescription drug or an approved product insert, a label approved by the US Food and Drug Administration is affixed. 【0200】 Treatment method According to some embodiments, a method for ameliorating or treating a subject suffering from a K63Ub-related disease is provided, the method comprising administering to the subject a therapeutically effective amount of any one of the peptides of the present invention, the dimeric peptides of the present invention, the pharmaceutical compositions of the present invention, or any combination thereof, thereby ameliorating or treating a subject suffering from a K63Ub-related disease. 【0201】 According to some embodiments, a method for ameliorating or treating a subject suffering from a cell proliferation-related disease is provided, the method comprising administering to the subject a therapeutically effective amount of any one of the peptides of the present invention, the dimeric peptides of the present invention, the pharmaceutical compositions of the present invention, or any combination thereof, thereby ameliorating or treating a subject suffering from a cell proliferation-related disease. 【0202】 According to some embodiments, there is provided a method for ameliorating or treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of any one of the peptides of the present invention, the dimeric peptides of the present invention, the pharmaceutical compositions of the present invention, or any combination thereof, thereby ameliorating or treating the subject suffering from cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is osteosarcoma. 【0203】 In some embodiments, the present invention relates to a method for treating, ameliorating, reducing and / or preventing a condition associated with an increase in the accumulation of K63Ub in the cells of a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the present invention, the dimeric peptide of the present invention, or both. 【0204】 In some embodiments, the present invention relates to a method for treating, ameliorating, reducing and / or preventing a condition associated with an increase in the proliferative activity of cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the present invention, the dimeric peptide of the present invention, or both. 【0205】 In some embodiments, the present invention relates to a method for treating, ameliorating, reducing and / or preventing a condition or disease associated with an increase in the apoptosis resistance activity of cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. 【0206】 In some embodiments, any one of the K63Ub-related diseases is characterized by an increase in the accumulation of K63Ub in the cells of the subject. 【0207】 As used herein, the terms "cancer" or "pre-malignant tumor" refer to diseases associated with cell proliferation. Types of cancer include, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, blastomas, and germ cell tumors. In one embodiment, carcinoma refers to a tumor derived from epithelial cells, including but not limited to breast cancer, prostate cancer, lung cancer, pancreatic cancer, and colon cancer. In one embodiment, sarcoma refers to a tumor derived from mesenchymal cells, including but not limited to sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma. In one embodiment, lymphoma refers to a tumor derived from hematopoietic cells that tend to mature in lymph nodes after emerging from the bone marrow, including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and immunoproliferative diseases. In one embodiment, leukemia refers to a tumor derived from hematopoietic cells that tend to mature in the blood after emerging from the bone marrow, including but not limited to acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, and adult T-cell leukemia. In one embodiment, blastoma refers to a tumor derived from immature progenitor cells or embryonic tissue, including but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma multiforme. In one embodiment, germ cell tumor refers to a tumor derived from germ cells, including but not limited to germ cell tumors of the gonadal or nongonadal type (GGCT, SGCT) and nongerm cell tumors or nongonadal tumors (NGGCT, NSGCT). In one embodiment, tumors of the gonadal or nongonadal type include, but are not limited to, germinoma, undifferentiated germinoma, and gonadoblastoma. In one embodiment, nongerm cell tumors or nongonadal tumors refer to pure and mixed germ cell tumors, including but not limited to fetal carcinoma, endodermal sinus tumor, choriocarcinoma, tearoom, polyembryoma, gonadoblastoma, and teratocarcinoma. 【0208】 As used herein, "cancer or pre - malignant cell proliferation" is a molecular process that requires an increase in deubiquitination activity in addition to an increase in cell proliferation rate. 【0209】 According to some embodiments, a method for increasing the amount of fragmented DNA in a cell of a subject is provided, the method comprising contacting the cell with an effective amount of any one of the peptide of the invention, the dimeric peptide of the invention, or both. 【0210】 According to some embodiments, a method for decreasing the amount, level, or both of Lys63Ub in a cell is provided, the method comprising contacting the cell with an effective amount of any one of the peptide of the invention, the dimeric peptide of the invention, and a composition comprising any one of these. 【0211】 According to some embodiments, a method for inducing or enhancing the apoptosis rate of a cell is provided, the method comprising contacting the cell with an effective amount of any one of the peptide of the invention, the dimeric peptide of the invention, and a composition comprising any one of these. 【0212】 According to some embodiments, a method for inducing cell cycle arrest in a cell is provided, the method comprising contacting the cell with an effective amount of any one of the peptide of the invention, the dimeric peptide of the invention, and a composition comprising any one of them. 【0213】 In some embodiments, the cell is a cancer cell or a cancerous cell. In some embodiments, the cell is a cancer cell or a cancerous cell of a subject. In some embodiments, the cell is obtained from or derived from a subject. 【0214】 In some embodiments, the method comprises reducing the deubiquitination activity or rate, the abundance or level of deubiquitinated protein(s) within the cell, reducing the proteasome activity or proteasome proteolytic rate, or any combination thereof. In some embodiments, the cell is a cancer cell or a cancerous cell. In some embodiments, the cell is a cell of a subject. In some embodiments, the cell is obtained from or derived from a subject. 【0215】 In some embodiments, treating or ameliorating comprises increasing the rate of DNA fragmentation in cancer cells or cancerous cells in a subject, such as an increase in the number of mutations (including, but not limited to, deleterious mutations), an increase in the rate of accumulation, level, or both of fragmented DNA, or a combination thereof. In some embodiments, treating comprises reducing the drug resistance of cancer cells or cancerous cells in a subject. In some embodiments, cancer cells or cancerous cells are characterized by an increased deubiquitination activity as compared to non-cancerous cells or benign cells. In another embodiment, treating comprises reducing the deubiquitination activity in cancer cells or cancerous cells in a subject. In some embodiments, treating comprises reducing the survival rate or reducing the survival of cancer cells or cancerous cells in a subject. In some embodiments, reducing the deubiquitination activity comprises increasing the apoptosis rate in cancer cells or cancerous cells in a subject. 【0216】 In some embodiments, the terms "decreasing" or "decreased" include a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, decreasing is a decrease of 1-5%, 4-10%, 8-20%, 15-30%, 25-40%, 35-55%, 50-70%, 60-80%, 75-90%, 90-99%, or 95-100%. Each possibility represents a separate embodiment of the invention. In some embodiments, decreasing is a decrease of at least one-half, at least one-third, at least one-fifth, at least one-tenth, at least one-fifteenth, at least one-twentieth, at least one-fortieth, at least one-seventy-fifth, at least one-hundredth, at least one-hundred-fiftieth, at least one-two-hundredth, at least one-five-hundredth, or at least one-thousandth, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. 【0217】 The terms "inhibiting", "decreasing", and "reducing" are interchangeable. 【0218】 In some embodiments, the term "increase" or "increasing" as used in the above embodiments (e.g., pro-apoptotic activity, cell apoptosis rate, etc.) means at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, or any value and range therebetween, compared to a control. Each possibility represents a separate embodiment of the present invention. In some embodiments, increasing means 1-5%, 4-10%, 8-20%, 15-30%, 25-40%, 35-55%, 50-70%, 60-80%, 75-90%, 90-99%, or 95-100% compared to a control. Each possibility represents a separate embodiment of the present invention. In some embodiments, increasing means at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 40-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 500-fold, or at least 1,000-fold, or any value and range therebetween, compared to a control. Each possibility represents a separate embodiment of the present invention. 【0219】 In some embodiments, the dynamics or dynamics of ubiquitination / deubiquitination are detected by any assay known to those skilled in the art, such as immunoassays, Western blots, immunohistochemistry, etc., and those for detecting Lys63Ub. In some embodiments, proteolysis and proteasome activity are detected by any acceptable method, such as immunoassays, Western blots, immunohistochemistry, pulse chase assays, etc. All of these are well known to those skilled in the art. 【0220】 As used herein, the term "subject" refers to animals, more specifically non-human mammals and human organisms. Subjects that are non-human animals can include animals in a prenatal form such as embryos or fetuses. Non-limiting examples of non-human animals include horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, pigs, and the like. In one embodiment, the subject is human. Human subjects can include fetuses. In one embodiment, the subject in need thereof is a subject suffering from and / or at risk of suffering from a condition associated with cell proliferation, increased deubiquitination activity, or a combination thereof. 【0221】 As used herein, the term "treating" or "treatment" of a disease, disorder, or condition encompasses alleviating at least one symptom thereof, reducing the severity thereof, or inhibiting its progression. Treatment need not mean that the disease, disorder, or condition is completely cured. For a treatment to be effective, a useful composition herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement to the quality of life of the patient or subject. 【0222】 As used herein, the term "prevention" of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of the disease, disorder, or condition. When used in accordance with the subject matter described herein, the term "prevention" relates to the process of prevention in which a subject is exposed to a peptide described herein before the disease / disorder process is induced or has occurred. This can be done if the individual has a genetic lineage indicating a predisposition to the occurrence of the disease / disorder to be prevented. For example, this may be applicable in the case of an individual whose ancestors show a predisposition to a particular type of disease such as an inflammatory disorder. The term "suppression" is used to describe a state in which the disease / disorder process has already started but the obvious symptoms of the condition have not yet manifested. Thus, the cells of an individual may have a disease / disorder, but the external signs of the disease / disorder are not clinically recognized. In either case, the term prevention may be applied to include both prevention and suppression. Conversely, the term "treatment" refers to the clinical use of an active agent to combat existing symptoms that are already manifesting clinically in a patient. 【0223】 As used herein, the term "condition" includes an anatomical and physiological deviation from a normal condition. This includes the impairment of the normal condition of a living animal or one of its parts and the interference with or alteration of the performance of body functions. 【0224】 In some embodiments, the method further comprises administering to the subject any one of a peptide of the invention, a dimeric peptide of the invention, and a pharmaceutical composition of the invention, in combination with a therapeutically effective amount of an anti-cancer therapy such as, but not limited to, surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, cytokine therapy, or any combination thereof. 【0225】 In some embodiments, a single pharmaceutical composition comprising a peptide of the invention, a dimeric peptide of the invention, and a pharmaceutical composition of the invention is combined with an anti-cancer therapy. In some embodiments, the combination comprises separate pharmaceutical compositions, wherein the first composition comprises a peptide of the invention, a dimeric peptide of the invention, and a pharmaceutical composition of the invention, and the second composition comprises an anti-cancer therapy. 【0226】 In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition described herein. These are provided simultaneously or separately. 【0227】 As used herein, the term "high binding affinity" refers to binding with a dissociation constant in the nanomolar scale. In some embodiments, the high binding affinity has a KD in the range of 0.1 nM to 10 nM, 1 nM to 100 nM, 50 nM to 250 nM, 15 nM to 1,500 nM, or 20 nM to 300 nM. Each possibility represents a separate embodiment of the invention. 【0228】 General The concentration ranges, percentage ranges, or ratio ranges described herein are understood to include concentrations, percentages, or ratios of any integer and fractions thereof (such as one-tenth and one-hundredth of an integer) within the range, unless otherwise specified. 【0229】 Any numerical range described herein in relation to any physical characteristics such as polymer subunits, size, or thickness is understood to include any integer within the recited range, unless otherwise indicated. 【0230】 As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" refer to any subject for which treatment is desired, particularly a mammalian subject, such as a human. 【0231】 In this discussion, unless otherwise specified, adjectives such as "substantially" and "about" that modify the conditions or relative characteristics of the features (if any) of the embodiments of the present invention are understood to mean that the condition or characteristic is defined within an acceptable range tolerated by the operation of the embodiment for the intended use. Unless otherwise expressly stated, the word "or" in the specification and claims is to be construed as an inclusive "or" rather than an exclusive "or", indicating at least one of the items being combined, or any combination thereof. 【0232】 It should be understood that the terms "a" and "an" used above and elsewhere in this specification refer to "one or more" of the recited components. It will be apparent to those skilled in the art that, unless otherwise stated, the use of the singular includes the plural. Thus, in this application, the terms "a", "an", and "at least one" are used interchangeably. 【0233】 For a better understanding of the present teachings and not in any way limiting the scope of the teachings, unless otherwise expressly stated, all numbers representing quantities, percentages, or ratios, as well as other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Thus, unless otherwise noted, the numerical parameters set forth in the following specification and the appended claims are approximations and may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques. 【0234】 In the specification and claims of this application, each of the verbs "comprise", "include", and "have" and their conjugations are used to indicate that the object (if any) of the verb is not necessarily an exhaustive listing of the components, elements, or parts of the subject (if any) of the verb. 【0235】 Other terms used in this specification shall be defined by their well-known meanings in the relevant technical field. 【0236】 Further objects, advantages, and novel features of the present invention will become apparent to those skilled in the art by considering the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention described above and claimed in the following claims sections will find experimental support in the following examples. 【0237】 For clarity, it is understood that specific features of the present invention described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, the various features of the present invention may, for brevity, be described in the context of a single embodiment and also provided separately, or in any suitable sub-combination, or as appropriate in any other described embodiment of the present invention. Specific features described in the context of various embodiments are not considered essential features of those embodiments unless the embodiments would not operate without those elements. 【0238】 Examples Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well explained in the literature. For example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series" Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods described below, U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al.(Eds.), see 「Basic and Clinical Immunology」 (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), 「Strategies for Protein Purification and Characterization - A Laboratory Course Manual」 CSHL Press (1996); all of which are incorporated by reference. Other general references are provided herein. 【0239】 Materials and Methods Peptides are synthesized by a solid-phase peptide synthesis (SPPS) approach using an automated peptide synthesizer (CS336X, CSBIO) or manually with a syringe equipped with a Teflon filter purchased from Torviq. Unless otherwise specified, all chemicals used are of analytical grade. Palladium(II) chloride (PdCl2), 4-bromomethyl-7-methoxycoumarin, dimethyl sulfoxide (DMSO), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), iodoacetamide, decafluorobiphenyl (reagent grade), hexafluorobenzene (reagent grade), benzyl bromide (reagent grade) were purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA), dichloromethane (DCM), diisopropylethylamine (DIEA), and N,N-dimethylformamide (DMF) were purchased from Biolab. Tetramethylrhodamine-5-maleimide (TAMRA) and fluorescein-5-maleimide (FITC) were purchased from ThermoFisher Scientific. 3-(Chloromethyl)benzoic acid, 2-chloroacetic acid were purchased from Acros Organics. Tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids were purchased from GL Biochem. Resin was purchased from CreoSalus. Dithiothreitol (DTT) and triisopropylsilane (TIPS) were purchased from Alfa Aesar. All coupling reagents [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylidene]-dimethylazanium hexafluorophosphate (HCTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU)] and hydroxybenzotriazole (HOBt) were purchased from GL Biochem and Luxembourg Bio Technologies.For high-performance liquid chromatography (HPLC) analysis, a Thermo instrument (Dionex Ultimate 3000) using an Xbridge (4.6×150 mm, 3.5 μm, BEH300C4, waters) column was used with a flow rate of 1.2 ml / min. For the Thermo Scientific instrument (Dionex Ultimate 3000), Jupiter C4 (250×10 mm, 10 μm, 300 Å, Phenomenex) was used for semi-preparative HPLC at a flow rate of 4.0 ml / min. For the Thermo Scientific instrument (Dionex Ultimate 3000), Jupiter C4 (250×22.4 mm, 10 μm, 300 Å, Phenomenex) was used for preparative HPLC at a flow rate of 15.0 ml / min. All peptides were purified by HPLC and characterized by mass spectrometry. 【0240】 Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine, antibiotics (penicillin / streptomycin), trypsin / EDTA, and phosphate-buffered saline (PBS) were purchased from the biological industry. The Trans-Blot Turbo (0.2um PVDF) membrane for blotting and electrophoresis setups was purchased from Bio-Rad. Streptavidin agarose resin, Hoechst 33342 solution (20 mM), Imperial Blue stock, cell culture plates, and high-capacity streptavidin agarose resin were purchased from Thermo-fisher. The non-protein instant block buffer for Western blotting applications was purchased from Gene Bio-Application L.T.D. Immobilon Crescendo Western HRP substrate was purchased from Millipore. The Comet assay kit was purchased from Abcam. The MEBCYTO apoptosis kit or Annexin V-FITC kit was purchased from medical and biological laboratories co., LTD., the μ-Slide 8 well for live cell confocal microscopy was purchased from ibidi, and polylysine hydrobromide was purchased from Sigma. The recombinant monoclonal gamma H2A.X (phosphorylated S139) antibody, rabbit monoclonal histone H2A.X [EPR895], recombinant monoclonal ubiquitin (linkage-specific Lys63) antibody, ubiquitin (linkage-specific Lys48) antibody, secondary goat anti-rabbit or anti-mouse IgG (HRP) antibody were purchased from Abcam, the recombinant monoclonal FLAG (R) M2 antibody was purchased from Sigma, and the recombinant mouse monoclonal ubiquitin (P4D1) antibody was purchased from Santa Cruz Biotechnology. 【0241】 Synthesis of Biotinylated Lys63-linked Di-Ub For the synthesis of biotinylated Lys63-linked Di-Ub chains, Ub building blocks 1 and 2 (biotin-Ub-MMP) were prepared using standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS) with the aforementioned modifications (Kumar KSA et al., 2010). Building blocks 1 and 2 were ligated to obtain 3. Next, 3 was subjected to radical-mediated desulfurization and then purified by HPLC (using a C4 column and a gradient flow of 0-60% B) and FPLC purification steps to obtain the desired native Lys63-linked Di-Ub chain 4 in high purity. Other Ub chains such as Lys11-linked Di-Ub, Lys29-linked Di-Ub, Lys48-linked Di-Ub, linear Di-Ub, and Lys48-linked Tetra-Ub were prepared as previously described (Kumar KSA et al., 2011). 【0242】 RaPID method for the identification of cyclic peptide 1 In aminoacylation of tRNA, mClBz was added to initiator tRNA using enhanced flexible ribosomes (eFX), which is designated as tRNAfMetCAU. The reaction conditions for preparing the tRNA solution were used as described in (Suga H et al., 2020). Next, microhelix RNA addition experiments were performed as previously explained (Suga H et al., 2020). For flexible in vitro translation (FIT), the components of the translation system used in (Jongkees SAK et al., 2017) were used. 【0243】 RaPID selection was performed using buffer selection = 1×TBS-T (50 mM Tris, 150 mM NaCl, 0.1% Tween20, pH 7.60) and 33 μM mClBz-tRNAfMetCAU as described in (Nawatha M et al., 2019). Positive and negative readings were collected by clone assay, and clones with P / N > 20 were selected as candidate peptides. 【0244】 After extracting the obtained sequences, PCR amplification was performed and classification was carried out based on a Python script. Thereby, a motif corresponding to the NNK library region having the correct length was obtained. 【0245】 Screening of peptides using a fluorescence-based competitive assay Microplates (96-well plates) coated with streptavidin were washed with HEPES buffer (50 mM HEPES, 150 mM NaCl, pH 7.30), and in each well, 100 μl of the same buffer containing 1 μg of biotinylated Lys63-linked Di-Ub was incubated at room temperature for 30 minutes. Additional wells were kept without biotinylated Lys63-Di-Ub for blank subtraction. After the washing step, a peptide standard (CP1) and an unlabeled peptide (5.0 molar equivalents relative to biotin-Lys63-linked Di-Ub) were incubated at room temperature for 30 minutes to achieve saturation binding to the target. Next, a 1 molar equivalent of FITC-labeled peptide standard (CP1-FITC) relative to biotinylated Lys63-linked Di-Ub was incubated at room temperature for 30 minutes to compete with the unlabeled peptide. After releasing the bound peptide by treatment with 6 M guanidinium chloride (Gnd·HCl), the fluorescence values were measured (λex = 480 nm and λem = 525 nm). These values were normalized, and the relative change in binding was calculated using the following formula: Y = [1 - (a / b)] × 100 Where Y = the change in signal (%) relative to the standard (CP1). a = the measured signal value (relative fluorescence units) of each peptide candidate. b = the measured signal value of the standard peptide (CP1). The dissociation constant (KD) of the fluorescein-labeled peptide (CP1-FITC) was calculated according to the procedure in the literature (Vamisetti GB et al., 2021). 【0246】 Synthesis of cyclic peptide 1 As shown in Fig. 3A, for the synthesis of Peptide 1, Fmoc-SPPS was carried out on Rink amide resin (0.26 mmol / g, 0.1 mmol scale). The peptide was synthesized at room temperature using amino acids (4.0 eq), HCTU (4.0 eq), and DIEA (8.0 eq). The Fmoc protecting group was removed by treating the resin with 20% piperidine in DMF containing 0.1 mmol HOBt (3:5:3 min). 3-(Chloromethyl)benzoic acid was coupled to the N-terminus of the sequence according to the literature procedure. The tetrapeptide was cleaved from the resin using a cocktail of TFA / H2O / TIS (95:2.5:2.5), then precipitated with cold diethyl ether and lyophilized. Cyclization was carried out by dissolving the crude peptide in 6 M Gnd·HCl, adjusting the pH to 8.0 with NaOH, and then incubating at 42 °C for 4 h. Next, the crude peptide was purified by HPLC using a C4 column with a gradient flow of 0 - 60% B for 60 min. 【0247】 Synthesis of Cyclic Peptide 2 - 14 Fmoc-SPPS was applied to the synthesis of cysteine mutant cyclic peptides. To synthesize different cyclic peptides with Cys mutations, orthogonally protected Cys(Acm) was incorporated at various positions of 1 (CP1). After cleavage from the resin and cyclization, purification was performed. In the case of Acm removal, the peptide CP1-S8C-Acm (10.0 mg, 5.04X10-3 mmol, 1.0 eq) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2524.8 μl, 2 mM). Next, PdCl2 (8.94 mg, 10.0 eq) was dissolved in 100 μl of 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50) at 37 °C for 10 min and added to the peptide solution. The reaction mixture was incubated at 37 °C for 1 h. Next, the reaction mixture was treated with dithiothreitol (DTT) (40.0 eq, 2.05×10 -1It was quenched with (mmol). After centrifugation, the supernatant was injected into HPLC with a gradient flow of 0 - 60% B for 60 minutes using a semi-preparative C4 column, and the free thiol-containing cyclic peptide 9, CP1-L1C (3.85 mg, yield 40%) was obtained. The same procedure was applied to the synthesis of other cyclic peptides 2 - 13 and cyclic peptide 14. 【0248】 【Table 1】 【0249】 Synthesis of cysteine-modified derivatives of cyclic peptide 2 CP1-L1C, which is cyclic peptide 2, was further modified with alkylating and arylating reagents to prepare different Cys-modified cyclic peptides as shown below: 【0250】 (a) Synthesis of cysteine alkylation derivatives 15 - 19 Various cysteine alkylation derivatives from CP1-L1C-Acm, which is cyclic peptide 14, were prepared in one pot as previously described (Vamisetti GB et al., 2021). The synthesis of each derivative was carried out as follows. 【0251】 (i) Synthesis of CP1-L1C-CH3, which is cyclic peptide 15 The Acm of CP1-L1C-Acm (10.0 mg, 5.11×10 -3 mmol, 1.0 equivalent), which is peptide 14, was removed. After the quenching and centrifugation steps of the reaction mixture, the supernatant was separated and the pH was adjusted to 8.0 with NaOH. 500 equivalents of iodomethane (CH3I) was dissolved in 500 μl of DMF and added to the reaction mixture at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 3 hours. Next, the reaction mixture was purified using a semi-preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain 15, CP1-L1C-CH3 (4.27 mg, yield 44%). 【0252】 (ii) Synthesis of CP1-L1C-CH2C6H5, which is cyclic peptide 16 After removing peptide 14 (10.0 mg, 5.11×10 -3 mmol, 1.0 equivalent) with Acm, the aqueous portion was separated and the pH was adjusted to 8.0 with NaOH. Then, 500 equivalents of benzyl bromide (C6H5CH2Br) dissolved in 500 μl of DMF was added to the reaction mixture at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 6 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-CH2C6H5 (4.04 mg, 40% yield), which is 16. 【0253】 (iii) Synthesis of CP1-L1C-CH2CONH2, which is cyclic peptide 17 After removing peptide 14 (10.0 mg, 5.11×10 -3 mmol, 1.0 equivalent) with Acm, the aqueous portion was separated and the pH was adjusted to 8.0 with NaOH. Next, 500 equivalents of 2-iodoacetamide (ICH2CONH2) dissolved in 500 μl of DMF was added to the reaction mixture at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 5 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-CH2CONH2 (4.76 mg, 49% yield), which is 17. 【0254】 (iv) Synthesis of cyclic peptide 18, CP1-L1C-CH2C10H7 Peptide 14 (10.0 mg, 5.11×10 -3After removing Acm (1.0 equivalent, mmol), the aqueous portion was separated and the pH was adjusted to 8.0 with NaOH. Next, 500 equivalents of 2-(bromomethyl)naphthalene (C10H7CH2Br) dissolved in 500 μl of DMF was added to the reaction mixture at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 7 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-CH2C10H7 (4.14 mg, 40% yield) with a retention time of 18. 【0255】 (v) Synthesis of cyclic peptide 19, CP1-L1C-CH2C10H7O3 14 (10.0 mg, 5.11×10 -3 After removing Acm (1.0 equivalent, mmol), the aqueous portion was separated and the pH was adjusted to 8.0 with NaOH. After the quenching and centrifugation steps of the reaction mixture, the supernatant was separated and the pH was adjusted to 8.0 with NaOH. Then, 500 equivalents of 4-bromomethyl-7-methoxycoumarin (BrCH2C10H7O3) dissolved in 500 μl of DMF was added to the reaction mixture at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 8 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-CH2C10H7O3 (4.23 mg, 40% yield) with a retention time of 19. 【0256】 (b) Synthesis of cysteine arylation derivatives 20 - 21 (vi) Synthesis of cyclic peptide 20, CP1-L1C-C6F5 Peptide 2, CP1-L1C (10.0 mg, 5.31×10 -3(1.0 equivalent) was dissolved in 2.7 mL of DMF containing 50 mM Tris base. Next, a solution of 100 μL of DMF containing hexafluorobenzene (6.15 μL, 10.0 equivalents) was added to the peptide solution. Then, the reaction mixture was vigorously mixed for 30 seconds and stored at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 4 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-C6F5 (4.03 mg, 37% yield) with a retention time of 20 minutes. 【0257】 (vii) Synthesis of cyclic peptide 21, CP1-L1C-C10F9 Peptide 2, CP1-L1C (10.0 mg, 5.31×10 -3 (1.0 equivalent) was dissolved in 2.7 mL of DMF containing 50 mM Tris base. Next, a solution of 100 μL of DMF containing decafluorobiphenyl (5.32 mg, 3.0 equivalents) was added to the peptide solution. Then, the reaction mixture was vigorously mixed for 30 seconds and stored at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed in 5 hours and purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain CP1-L1C-C10F9 (4.08 mg, 35% yield) with a retention time of 21 minutes. 【0258】 Synthesis of CP1-FITC and CP1-TAMRA To synthesize the FITC-labeled cyclic peptide (CP1-FITC), Fmoc-Cys(Acm) was coupled as the first amino acid for the late-stage modification. After the cyclization and Acm removal steps, the peptide with a free thiol functional group was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2 mM). Next, fluorescein-5-maleimide (2.0 equiv) / tetramethylrhodamine-5-maleimide (TAMRA, 1.50 equiv) dissolved in DMF was added to the peptide solution and stored in the dark at room temperature. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 min. The reaction was completed in 2 h and filtered and purified by injecting it into HPLC using a C4 semi-preparative column with a gradient flow of 0 - 60% for 60 min. 【0259】 Synthesis of Cyclic Peptide 23 The Fmoc-SPPS method was used for the synthesis of peptide 22. All amino acids were coupled using an automatic peptide synthesizer. When synthesizing the TAMRA-labeled peptide, cysteines at positions 1 and 18 were orthogonally protected with Acm and -StBu, respectively, for modification in later steps. After coupling 3-(chloromethyl)benzoic acid to the N-terminus, the resin was washed with DMF, MeOH, and DCM and dried under vacuum. The peptide was cleaved from the resin using a TFA / H2O / TIS (95:2.5:2.5) cocktail, then precipitated in cold diethyl ether and lyophilized to obtain 22. Cyclization was performed by dissolving the crude peptide 22 (4.0 mM) in 6 M Gnd·HCl / 200 mM phosphate buffer, adjusting the pH to 8.0 with NaOH, and incubating at 42 °C. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 min. The reaction was shown to be completed within 4 h and purified by HPLC using a preparative column C4 with a gradient flow of 0 - 60% for 60 min, and peptide 23 was obtained in a 43% yield. 【0260】 Synthesis of Cyclic Peptide 24 Peptide 23 (10.0 mg, 4.52×10 -3 mmol, 1.0 equivalent) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2,258 μl, 2 mM). To this, a solution of TCEP (50.0 equivalents) dissolved in H2O was added, the pH was adjusted to 2.50, and the mixture was incubated at 37 °C. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 4 hours and purified by HPLC using a preparative column C4 with a gradient flow of 0 - 60% for 60 minutes to obtain Peptide 24 (3.84 mg, yield 40%). 【0261】 Synthesis of Cyclic Peptide 25 Peptide 24 (10.0 mg, 4.70×10 -3 mmol, 1.0 equivalent) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2,351 μl, 2 mM). To this, tetramethylrhodamine-5-maleimide (3.40 mg, 1.50 equivalents) dissolved in 50 μl of DMF was added under dark conditions at room temperature. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 2 hours and purified by HPLC using a preparative column C4 with a gradient flow of 0 - 60% for 60 minutes to obtain Peptide 25 (5.15 mg, yield 42%). 【0262】 Synthesis of Cyclic Peptide 2-TAMRA 26 Peptide 25 (10.0 mg, 3.84×10 -3(1.0 equivalent) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, approximately 1,918 μl, 2 mM). Next, PdCl2 (approximately 1.36 mg, 2.0 equivalents) was dissolved in 50 μl of 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50) at 37 °C for 10 minutes. After adjusting the pH to 1.50, this was added to the peptide solution and the reaction was maintained at room temperature. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes, and it was shown that the reaction was completed in 30 minutes. Next, dithiothreitol, DTT (8.0 equivalents, 3.07×10 -2 mmol) was added to quench the reaction mixture, which was then centrifuged. The supernatant was injected into HPLC using a semi-preparative C4 column with a gradient flow of 0 - 60% B for 60 minutes to obtain peptide 26 (1.85 mg, yield 19%) containing free thiol. 【0263】 Synthesis of cyclic peptide 27 Peptide 26 (10.0 mg, 3.94×10 -3 mmol, 1.0 equivalent) was dissolved in approximately 2 ml of DMF containing 50 mM tris base. Next, a solution of 100 μl of DMF containing hexafluorobenzene (6.15 μl, 10.0 equivalents) was added to the peptide solution. The reaction mixture was vigorously mixed for 30 seconds and stored at room temperature. The progress of the reaction mixture was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes, and it was shown that the reaction was completed within 4 hours. Next, the reaction mixture was purified using a preparative C4 column with a gradient flow of 0 - 60% for 60 minutes to obtain 27 (3.30 mg, yield 31%). 【0264】 Selectivity of peptides 1 and 2 for Ub chains with different linkages Using surface plasmon resonance (SPR), the binding of cyclic peptide 1 to Lys11- and Lys29-linked Di-Ub was examined (Fig. 24, i and ii). The SPR analysis was performed according to the reported protocol. The relative binding affinities of cyclic peptides 1 and 2 to linear Di-Ub, Lys48-linked Di-Ub, and Lys48-linked Tetra-Ub were tested by applying our fluorescence-based assay as shown in (iii), (iv), and (v), respectively (Fig. 24). 【0265】 Synthesis of FITC-labeled CP1-L1C-Acm cyclic peptide 28 The same procedure described in Section 10 was applied to peptide 24 (10.0 mg, 4.70×10 -3 mmol, 1.0 equivalent). Peptide 24 was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2,351 μl, 2 mM), and then fluorescein-5-maleimide (6.02 mg, 3.0 equivalents) dissolved in 100 μl of DMF was added. The reaction was kept at room temperature in the dark. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 2 hours and purified by HPLC using a semi-preparative column C4 with a gradient flow of 0 - 60% for 60 minutes, and peptide 28 was obtained in a 44% isolated yield (5.28 mg). 【0266】 Synthesis of cyclic peptide 2-FITC29 The same procedure as the synthesis of cyclic peptide 25 was applied to peptide 28 (10.0 mg, 3.92×10 -3It was applied to (1.0 equivalent in mmol). 28 was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 1,958 μl, 2 mM). Next, PdCl2 (1.39 mg, 2.0 equivalents) was dissolved in 50 μl of 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50) at 37 °C for 10 minutes, adjusted to pH 1.50, and then added to the peptide solution, and the reaction was maintained at room temperature. After completion of the reaction (30 minutes), the reaction mixture was centrifuged and quenched with DTT, and the supernatant was injected into HPLC. Using a semi-preparative C4 column, a peptide 29 (2.04 mg, yield 21%) containing free thiol was obtained with a gradient flow of 0 - 60% B for 60 minutes. 【0267】 Determination of KD (i) Determination of the KD of 1 using SPR The binding of cyclic peptide 1 (CP1) to the target Lys63-linked Di-Ub was determined using a BIACORE T100 instrument (GE Healthcare) equipped with a biotin Capture kit series S chip. 50 mM HEPES (pH = 7.30, 150 mM NaCl, 0.05% Tween 20, 2.0 mM DTT, 0.2% DMSO) was used as the buffer. Biotinylated Lys63-linked Di-Ub was loaded onto the SPR chip. By using various concentrations of the peptide. 【0268】 (ii) Determination of the KD value of CP1-FITC As previously described (Vamisetti GB et al., 2021), the KD value of FITC-labeled cyclic peptide 1 (CP1-FITC) was determined. The dissociation constant KD was calculated to be 95.8 ± 2.3 nM. 【0269】 (iii) Determination of the KD value of CP1-TAMRA The fluorescence value was measured in the same manner as CP1-FITC (λex = 510 nm, λem = 565 nm). The dissociation constant (KD) value of TAMRA-labeled CP1 (CP1-TAMRA) was calculated to be 101.9 ± 3.6 nM. All measurements were repeated 3 times. 【0270】 (iv) Determination of the KD value of 2-FITC29 The dissociation constant (KD) value of the FITC-labeled cyclic peptide 29 was calculated in the same manner as CP1-FITC and was found to be 43.2 ± 4 nM. 【0271】 (v) Determination of the KD value of 2-TAMRA26 The fluorescence values were measured in the same manner as described for CP1-FOTC (λex = 510 nm, λem = 565 nm). The dissociation constant (KD) value of the TAMRA-labeled 26 (2-TAMRA) was calculated to be 47.4 ± 5.9 nM. All measurements were performed in triplicate. 【0272】 Synthesis of biotin-labeled cyclic peptide 31 Peptide 24 (10.0 mg, 4.70×10 -3 mmol, 1.0 equivalent) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, approximately 2,351 μl, 2 mM). Next, biotinylated PEG6-maleimide (6.60 mg, 9.40×10 -3 mmol, 2.0 equivalents) was dissolved in 50 μl of DMF and added to the peptide solution at room temperature. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 45 minutes and purified by HPLC using a semi-preparative C4 column with a gradient flow of 0 - 60% B for 60 minutes to obtain peptide 30 (9.04 mg, yield 68%). Next, the Cys(Acm) deprotection step was carried out according to the procedure described for the synthesis of the cyclic peptide 2-TAMRA26. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B for 30 minutes. The reaction was completed within 30 minutes and quenched with DTT. After centrifugation, the mixture was injected into the HPLC using a semi-preparative C4 column with a gradient flow of 0 - 60% B for 60 minutes to obtain peptide 31 with an isolated yield of 22% (1.94 mg). 【0273】 Synthesis and selectivity of scrambled cyclic peptide 33 Similar to that described in Section 6, Fmoc-SPPS was applied for the synthesis of the scrambled cyclic peptide 33 of cyclic peptide 2. After cleavage from the resin and cyclization, purification was carried out. For Acm removal, 5 peptide 32 (10.0 mg, 5.12×10 -3 mmol, 1.0 equiv) was dissolved in 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50, 2561.5 μl, 2 mM). Next, PdCl2 (9.07 mg, 10.0 equiv) was dissolved in 100 μl of 6 M Gnd·HCl / 200 mM phosphate buffer (pH 7.50) at 37 °C for 10 min, and this was added to the peptide solution. The reaction mixture was incubated at 37 °C for 1 h. Next, the reaction mixture was quenched with dithiothreitol (DTT) (40.0 equiv, 2.05×10 -1 mmol). After centrifugation, the supernatant was injected into HPLC using a semi-preparative C4 column with a gradient flow of 0 - 60% B for 60 min to obtain the free thiol-containing cyclic peptide 33 (4.05 mg, yield 42%). Next, the relative binding affinity of cyclic peptide 33 to Lys63-linked Di-Ub was compared with that of the specific Lys48-linked Di-Ub binder mJ08-L8W in the same process as described for cyclic peptides 1 and 2. 【0274】 Synthesis of biotinylated labeled scrambled cyclic peptide 38 The same Fmoc-SPPS procedure described in Section 4 was applied to the synthesis of Peptide 34. When synthesizing the biotinylated peptide, the cysteines at positions 1 and 18 were orthogonally protected with Acm and -StBu, respectively, for modification at a later stage. After coupling 3-(chloromethyl)benzoic acid to the N-terminus, the resin was washed with DMF, MeOH, and DCM and dried under vacuum. The peptide was cleaved from the resin using a TFA / H2O / TIS (95:2.5:2.5) cocktail, then precipitated in cold diethyl ether and lyophilized to obtain 34. Cyclization was carried out by dissolving the crude peptide 34 (4.0 mM) in 6 M Gnd·HCl / 200 mM phosphate buffer, adjusting the pH to 8.0 with NaOH, and then incubating at 42 °C. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B over 30 minutes. The reaction was shown to be complete within 4 hours and was purified by HPLC using a preparative C4 column with a gradient flow of 0 - 60% over 60 minutes, yielding Peptide 35 in 40% yield. The same procedure as described for the synthesis of cyclic peptide 24 was applied to the preparation of Peptide 36. Next, biotinylated cyclic peptide 37 was prepared according to the procedure described for the synthesis of biotinylated cyclic peptide 31. Next, the Cys(Acm) deprotection step was carried out according to the procedure described in Section 12. The progress of the reaction was monitored by HPLC using a C4 analytical column with a gradient flow of 0 - 60% B over 30 minutes. The reaction was complete within 30 minutes and was quenched with DTT. After centrifugation, the mixture was injected into an HPLC using a semi-preparative C4 column with a gradient flow of 0 - 60% B over 60 minutes, yielding Peptide 38 in 20% isolated yield. 【0275】 Cell culture procedures HeLa (CCL-2™) cells and 293T (CCL-2™) cells were cultured in DMEM (high glucose) medium supplemented with 10% FBS, 0.2 mM L-Gln, and antibiotics (penicillin / streptomycin) in a humidified incubator at 37 °C and 5% CO2. U2OS (HTB-96TM) cells were cultured in DMEM (low glucose) medium supplemented with 10% FBS, 0.2 mM L-Gln, and antibiotics (penicillin / streptomycin) in a humidified incubator at 37 °C and 5% CO2. To detach cells from the culture flask, the medium was aspirated, the flask was washed with PBS without calcium and magnesium, and the cells were treated with a 0.02% EDTA solution containing 0.25% trypsin and returned to the incubation chamber for 4 - 5 minutes. Trypsin was quenched by adding supplemented medium. The cell suspension was collected and pelleted (2 minutes at 1,000 x g). Next, the medium was aspirated, and the cell pellet was resuspended in fresh medium. Cell density was measured using an automated cell counter (Countess II, Invitrogen) and seeded accordingly. 【0276】 Cell uptake assay Cells were seeded on ibidi 8-well μ-slides treated with poly-L-lysine for 24 hours and reached approximately 90% confluence. Next, the cells were washed three times with warmed PBS and then incubated for 1 hour with warmed serum-free medium (DMEM) containing the peptide. Thereafter, the cells were washed twice with warmed PBS. Before imaging, the cells were washed with optical culture medium and stained with Hoechst (2 μg / ml). Live cell CLSM images were taken using a confocal Zeiss LSM710 equipped with a 40x NA 1.2 water immersion objective lens using a 1 AU pinhole setting. Different lasers were used for different tags (Hoechst, TAMRA, FITC), and during analysis, the μ-slide was maintained at 37 °C in a humidified chamber. 【0277】 Induction of histone H2AX phosphorylation The accumulation of histone H2AX phosphorylated at serine 139 (γ-H2AX) was examined by Western blot using a standard lysis protocol. Briefly, U2OS cells or HeLa cells (3×10 6 ) treated with the sample (2 μM cyclic peptide or DMSO) were harvested and lysed on ice for 1 hour with 100 μl of hot lysis buffer (50 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride). The samples were then incubated at 95 °C for 15 minutes, and after the solution was returned to room temperature, the samples were incubated again with benzonase nuclease at 37 °C for 20 minutes. The solution was centrifuged at 4 °C, 15,000 rpm for 20 minutes. Cell lysates containing 20 μg of total cell protein were separated on a 12% SDS-PAGE gel and blotted onto a polyvinylidene fluoride (PVDF) transfer membrane. After blocking with non-protein InstaBlock buffer, the membrane was incubated overnight at 4 °C with the primary antibody anti-γ-H2A.X (phosphorylated S139), which is an antibody against phosphorylated H2AX (1:1500). After washing three times with Tris-buffered saline Tween20 (TBST), the secondary anti-rabbit IgG (HRP) antibody was added at room temperature for 2 hours. Finally, the crescendo Western HRP solution was added to the membrane and the blot was imaged using a Fusion-400 ECL detection system. The γ-H2AX signal at 15 kDa in the blot was quantified using Fiji software. Anti-H2A.X was used as a loading control. 【0278】 DNA damage test by comet assay Preparation of samples and slides The comet assay was performed with some modifications according to the protocol provided by the Abcam Comet Assay Kit (ab238544). Briefly, U2OS cells were cultured and treated with the same peptide as described in the previous conditions for the investigation of histone H2AX phosphorylation. After treating the samples with serum-free medium for 8 hours, the cells were scraped from the 60 mm dish. Then, to obtain cell pellets, the cell suspension was transferred to a conical tube, centrifuged (at 2,600 rpm for 3 minutes), and washed twice with ice-cold phosphate-buffered saline (PBS, without Mg2+ and Ca2+). After counting the cells, the cells were resuspended in ice-cold PBS without Mg2+ and Ca2+ to a concentration of 1×10 5 cells / ml. For slide preparation, low melting point comet agarose was pipetted onto the attached 3-well comet slide to obtain the base layer and incubated at 4°C for 20 minutes. Next, the cell sample was thoroughly mixed with the comet agarose at 37°C at a ratio of 1:10 (v / v), and 75 μL of the suspension was gently transferred immediately on top of the base layer without disturbing the base layer. The slide was incubated again at 4°C for 20 minutes. To avoid damage to the cell samples by ultraviolet light, all procedures were carried out under minimal light conditions. 【0279】 Alkaline electrophoresis Cells fixed on the slide were lysed with an alkaline lysis solution (Triton X-100 (1:100), DMSO (1:10), 2.5 M NaCl, 100 mM Na3EDTA, 10 mM Tris Base, pH 10). The slide with cells embedded was transferred to a container containing pre-cooled alkaline lysis solution and incubated overnight at 4°C in the dark. Then, the solution was replaced with pre-cooled alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na3EDTA, pH > 13) and left in the dark at 4°C for 30 minutes. The slide was transferred directly into the electrophoresis chamber horizontally, and the chamber was filled with pre-cooled alkaline electrophoresis solution. Next, electrophoresis was performed at a constant current setting of 300 mA at 1 V / cm for 30 minutes. After electrophoresis was completed, the slide was transferred horizontally to a container containing pre-cooled DI H2O for 2 minutes. The slide was washed two more times in the same manner. Finally, the slide was transferred horizontally to a container containing cold 70% ethanol and incubated for 5 minutes. Then, the slide was removed from the 70% ethanol horizontally and air-dried at room temperature for an additional 2 hours. DNA was stained with the provided bis-tag green DNA dye for 15 minutes at room temperature in the dark. Then, the slide was prepared for microscopic analysis. 【0280】 Comet assay and quantification Images of cells with comets were taken with a fluorescence microscope (Axio Observer Z1 LSM 700, Zeiss) equipped with a 63x Plan-APOCHROMAT 63X / 1.4 oil DTC objective lens (Zeiss) and a camera (AxioCam MRm, Zeiss). "Tail moment" is suggested to be an appropriate indicator of DNA damage induced when considering the movement of genetic material. Tail moment intensity profiles were analyzed using the "OpenComet" software plugged into Fiji. For the estimation, fluorescence signals of at least 100 cells were considered for each data point. 【0281】 Transfection of cells with flag-tagged wt or mutant RNF168 gene and immunoprecipitation (IP) against flag antibody pcDNA3-Flag-RNF168 and pcDNA3-Flag-RNF168 deletion MIU1 / MIU2 (purchased from Addgene) were overexpressed in human embryonic kidney 293T cells. The treatment was carried out for 12 hours using polyethyleneimine (PEI) reagent. After transfection, the cells were treated with 1 μM of cyclic peptide or DMSO for 36 hours, and then exposed to 10 Gy of ionizing radiation (IR) using an X-ray device (CellRad). After a 6-hour recovery, the cells were harvested and lysed using IP buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% NP-40, 10 mM EDTA, 20 mM β-glycerophosphate) containing protease inhibitor. Finally, the protein was immunoprecipitated using a flag antibody, and Western blot analysis was performed. In this experiment, protein A / G PLUS-agarose beads (purchased from Santa Cruz) were washed and blocked with IP buffer containing 5% BSA at 4°C for 2 hours, and whole cell extracts were prepared using NP-40 lysis buffer and pre-cleared. 【0282】 Apoptosis assay using Annexin V-FITC / PI double staining Sample preparation Apoptotic cell death was estimated using a standard MEBCYTO® Apoptosis Kit (MBL) protocol. Briefly, seeded HeLa cells were treated with samples (peptide 2 or 1 μM DMSO) at 37°C and 5% CO2 for 96 hours. After sample treatment, the cells were harvested from the 60 mm dish by trypsinization and centrifuged at 2,600 rpm for 4 minutes. The cells were washed with phosphate-buffered saline (PBS, Mg 2+ and Ca 2+Washed once (without any additives), resuspended in the supplied binding buffer, and then stained with annexin V-FITC and propidium iodide (PI). Annexin V-FITC-positive cells were regarded as apoptotic cells, and furthermore, early apoptotic cells and late apoptotic cells were distinguished by negative PI signals and positive PI signals, respectively. Due to the solubility problem of the cyclic peptide in the buffer medium, the inventors were unable to proceed with increasing the concentration of the cyclic peptide. 【0283】 Flow cytometry measurement Various populations were analyzed using a CYTEK Aurora flow cytometer. The final fluorescence of annexin V-FITC was obtained for sample analysis in independent replicates in 20,000 cells each. Non-distorted cells and single-distorted cells were analyzed as reference controls. None of the treated samples were mixed after measurement. Finally, the unmixed data files were analyzed using FCS Express6 software. 【0284】 Cell cycle analysis Cell cycle analysis was performed using the FxCycle™ PI / RNase staining solution. HeLa cells were treated as before (Section 23). After detachment, the cells were washed twice with cold PBS and then fixed overnight at -20 °C with 70% ice-cold ethanol. The fixed cell pellet was washed twice with cold PBS and then incubated with the PI / RNase staining solution for 30 minutes on ice in the dark. Finally, flow cytometry (CYTEK Aurora) analysis of the cell cycle was performed to confirm the cell arrest events considering 15,000 cell counts for all samples. Cells at various cell cycle stages were quantified using standard heat plot analysis using FCS Express6 software. 【0285】 Pull-down and proteomics analysis The cell lysate suspension pre-incubated with biotinylated peptide 31 was pulled down from streptavidin beads using a standard protocol with some modifications. Briefly, U2OS cells (3×10 6 ) were collected as cell pellets after trypsinization. Immediately, lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM HEPES pH 7.5, 1 μM NMM, and 1 μM IAA) was added to the pellet, left on ice for 30 minutes, and centrifuged at 4 °C for 15 minutes. The cell lysate suspension was aliquoted into two parts and incubated overnight at 4 °C on a rotating wheel with biotinylated peptide 31 or 38. Streptavidin agarose beads (high-capacity streptavidin agarose resin, Thermo Scientific) were equilibrated three times with lysis buffer under shaking conditions. The washed beads were added to each treated suspension and incubated at 4 °C for 1 hour. The beads were washed five times with wash buffer containing PBS pH 7.5, and the protein complex was eluted by heating at 95 °C for 5 minutes with reducing buffer containing DTT. The eluted mixture was examined by Western blot using anti-Ub (Lys63 specific) or anti-Ub (Lys48 specific). As a positive control, 0.5% v / v input was included. 【0286】 For proteomic analysis, elution solutions of both samples (peptide 31 and peptide 38) with the same lysate concentration were used. The protein components were cleaved with trypsin and analyzed by LC / MS using a QEplus (Thermo) mass spectrometer. The data were analyzed using Proteome Discoverer 2.4 and the human Uniprot database, and 1% FDR and at least two peptides were identified. The abundance ratio of eluted proteins between peptide samples was calculated, considering specific proteins (at least twice that of the control). 【Table 2】 【0287】 Example 1 Discovery of Cyclic Peptide 1 of K63Ub Chain Using the RaPID Method To screen for selective Lys63-linked Di-Ub binders, the inventors utilized chemical protein synthesis to prepare Lys63-linked Di-Ub with a biotin tag at the N-terminus of the distal Ub (Figs. 6A - 6B). Next, the inventors used this chain as bait for RaPID display and screened a library of 1 trillion mRNA·cDNA-tagged cyclic peptides against Lys63-linked Di-Ub (Figs. 1A - 1B), performing the first selection round of cyclic peptides. Thereafter, by introducing additional selection rounds, the monoubiquitin (Ub1) binders were removed. After repeating 2 - 5 rounds, as a result of deep sequencing of the cDNA library, a very specific and strongly binding de novo cyclic peptide 1 (CP1) (Fig. 7) with a low nanomolar dissociation constant (KD) value of 16 nM as measured by surface plasmon resonance (SPR) was discovered (Fig. 27). 【0288】 To further screen and determine the relative binding affinities of additional chemically modified cyclic peptides based on the lead compound, the inventors used a recently developed fluorescence-based assay (Figs. 2A and 8). For this purpose, cyclic peptide 1 tagged with fluorescein (CP1-FITC; Fig. 18) was prepared, and the KD value was determined to be 95.8 ± 2.3 nM (Fig. 2E). 【0289】 Example 2 Preparation and Characterization of Cyclic Peptide 1 Derivatives In the RaPID system, cyclization of the peptide occurs by selective nucleophilic attack of the thiol side chain of Cys located in the C-terminal region on non-amino acid initiators such as chloroacetyl (ClAc), 3-(chloromethyl)benzoic acid (mClBz), or 4-(chloromethyl)benzoic acid (pClBz). In particular, when using this approach for subsequent functionalization, no chemoselective cyclization occurs, so no additional Cys can be added within the sequence. 【0290】 To expand the library by chemical mutagenesis using chemical Cys modification without interfering with the cyclization step, the inventors prepared derivatives of cyclic peptide 1 having Cys orthogonally protected with acetamidomethyl (Acm) at various positions using Fmoc solid-phase peptide synthesis (Fmoc-SPPS) (Figure 2B, Figures 10A - 10B, and Table 1). Upon overall deprotection and cleavage, a free thiol group remains on the trityl-protected Cys residue, which cyclizes with the mClBz moiety. In the next step, as shown in a representative example of the synthesis of cyclic peptide 9 (CP1-S8C, Figure 2C), Acm was removed via PdCl2 to obtain a free thiol. Using the same strategy, additional derivatives of cyclic peptide 1 having a second Cys residue at different positions were prepared (Figures 10A - 10B, and Table 1). 【0291】 Having obtained cyclic peptides 2 - 13 and using a fluorescence-based assay, the inventors then examined the affinity of the peptides for Lys63-linked Di-Ub. The relative binding of each cyclic peptide was normalized to cyclic peptide 1 as a reference. The binding affinity of each cyclic peptide is inversely proportional to the FITC fluorescence of CP1-FITC. Using this assay, the inventors identified that cyclic peptide 2 is a more effective binder of Lys63-linked Di-Ub compared to 1 (Figure 2D). 【0292】 To further attempt to improve the binding affinity of cyclic peptide 2, the inventors utilized the advantage of the free thiol in cyclic peptide 2 for chemical modification with different groups. In particular, alkylation was performed in one pot using removal of the Acm protecting group and treatment with various alkyl halides. This in situ reaction enabled rapid preparation of five different alkylated (15 - 19) and arylated (20 - 21) derivatives of 2 (Figure 3A and 11 - 17). All modified peptides showed a decrease in binding compared to cyclic peptides 1 and 2 (Figure 3B). 【0293】 Next, the inventors investigated the selectivity of cyclic peptide 2 for Lys63-linked Di-Ub compared to other Ub chain types. Since no binding to Lys11- and Lys29-linked Di-Ub chains was detected for lead cyclic peptide 1 (Figure 24), the binding affinity of cyclic peptide 2 to other Ub chains was further investigated. For this purpose, the binding affinity of 2 to 1 was compared to the reported Lys48-linked Di-Ub and Tetra-Ub binders, mJ08-L8W and Ub4_ix, respectively (Figure 24). From the results, it was revealed that 2 had significantly lower binding affinity for linear Di-Ub, Lys48-linked Di-Ub, and Lys48-linked Tetra-Ub. In all these experiments, cyclic peptide 2 exhibited high selectivity for Lys63-linked Di-Ub chains compared to cyclic peptide 1. 【0294】 Example 3 Investigation of the Cellular Effects of Cyclic Peptides Targeting K63Ub To confirm the cellular effects of the cyclic peptides, the inventors first aimed to examine the cell permeability of cyclic peptide 2. For this purpose, to facilitate labeling with maleimide dyes, FITC- or TAMRA-labeled peptide 2-FITC(29) and 2-TAMRA(26) incorporating Cys(StBu) were prepared, and then Cys1 was exposed by removing the orthogonal palladium-promoted Acm. To examine the effect of the fluorophore on the binding of cyclic peptide 2, fluorescence-based assays were used to determine the KD values of cyclic peptide 2-FITC(29) and 2-TAMRA(26) to be 43.2 ± 4.0 and 47.4 ± 5.9 nM, respectively (Figures 30 and 31). These results indicated that the choice of fluorophore (i.e., FITC or TAMRA) did not affect the binding affinity of the cyclic peptide to Lys63-linked Di-Ub. Next, the delivery of these cyclic peptides into U2OS cells was measured by laser scanning confocal microscopy (LSCM), and it was confirmed that 2-TAMRA(26) had efficient cell permeability (Figures 4A-4B and 35). Notably, efficient delivery of 2-FITC(29) was not observed (Figure 35). This suggests that the choice of fluorescent dye greatly changes the cell permeability behavior of the labeled cyclic peptide. 【0295】 After demonstrating the cell permeability of 2-TAMRA, the inventors aimed to examine whether unlabeled cyclic peptide 2 could modulate cellular pathways in which Lys63-linked chains are known to be involved. Lys63-linked Ub chains have been reported to regulate various cellular pathways such as DNA damage repair (DDR), signal transduction, protein transport, and immune response. Misregulation of these pathways can lead to stress, accumulation of mutations, apoptosis, and cell cycle arrest, which can cause various pathological conditions. 【0296】 γ-H2AX, formed by phosphorylation of the Ser-139 residue of histone variant H2AX, is the oldest known marker of double-strand breaks (DSBs). After the formation of γ-H2AX, DNA repair proteins are recruited to the DSB site. Among these proteins, the RNF8 and RNF168 ubiquitin E3 ligases generate Lys63-linked Ub conjugates on histones and non-histone proteins (e.g., 53BP1 and BRCA1) surrounding the DSB site, thereby promoting repair. The availability of Lys63-linked Ub chains in these processes is assumed to be affected by the presence of external modulators. Therefore, the inventors predicted that the interaction between cyclic peptide 2 and Lys63-linked Di-Ub chains would inhibit the interaction between these chains and their endogenous partners, thereby inhibiting DSB repair and, as a result, potentially accumulating fragmented DNA. 【0297】 To investigate the effect of the Lys63-linked Di-Ub binders disclosed herein on DSB repair, U2OS cells and HeLa cells were treated with cyclic peptide 2, and Western blot was used to compare γ-H2AX levels with those of control cells. As a result, it was clearly shown that when treated with cyclic peptide 2, the level of γ-H2AX increased 3-fold in U2OS cells and 3.5-fold in HeLa cells. Importantly, in 33 (scrambled sequence of cyclic peptide 2) that has no traceable binding affinity for K63-linked Di-Ub (Figure 33C), the level of γ-H2AX (Figures 4C - 4D) did not change. Furthermore, by including cyclic peptide 20, which has a low binding affinity for the chain compared to 2, an increase in γ-H2AX levels of 1.8-fold in U2OS cells and 2.4-fold in HeLa cells was presented (Figures 4E - 4F and Figure 36). An increase in γ-H2AX levels was observed, indicating that cyclic peptides 2 and 20 inhibit the repair of DSBs, which is dependent on their affinity for Lys63-linked chains. 【0298】 To further validate the discovery, the inventors sought to directly measure the integrity of DSB repair at the single-cell level using the comet assay. The results disclosed herein show that cells treated with cyclic peptide 2 exhibit a significant increase in the amount of fragmented DNA, as evident by the “comet-like” bis-tag green signal from the DNA of individual cells (Figure 4G). Furthermore, the relative tail moment, which directly measures the extent of DNA damage, shows a significant improvement (8-fold) in cells treated with cyclic peptide 2 compared to the control group. Similarly, cyclic peptide 20 also induced DNA damage, but to a lesser extent compared to cyclic peptide 2 as in the previous experiment (Figure 4H). 【0299】 At the DSB site, RNF168 accumulates downstream of RNF8 by itself, which interacts with ubiquitinated H2A and catalyzes the formation of Lys63-linked Ub-conjugates. The MIU motifs of RNF168 (MIU1 and MIU2) mediate the binding to Lys63-linked Ub chains on histones, which is required to promote the DNA damage response. Therefore, to examine the direct binding activity of the developed cyclic peptide 2 to intracellular Lys63-linked Ub chains, the inventors transfected 293T cells with the flag-tagged RNF168 (wt) gene and the MIU1 / MIU2-deleted RNF168 (mutant) gene. The transfected cells were treated with cyclic peptide 2 (1 μM) or DMSO for 36 hours, exposed to ionizing radiation (IR) (10 Gy), and stimulated the accumulation of DSBs with associated Lys63-linked Ub chains. Immunoprecipitation using an anti-flag antibody and Western blot analysis showed several additional signals in RNF168 (wt)-transfected cells, which were found to be RNF168 and its ubiquitinated forms, further verified by Ub signals. On the other hand, treatment with cyclic peptide 2 removed these additional signals of RNF168 and conjugated Ub, strongly supporting the regulatory activity of 2 on intracellular Lys63-linked Ub chains. Importantly, the effect of peptide 2 was not seen in cells transfected with mutant RNF168 (Figure 4I). 【0300】 When unrepaired DNA damage persists, in most cases, cell cycle arrest and apoptosis occur. Therefore, the inventors investigated the effect of cyclic peptide 2 on the cell cycle progression of HeLa cells treated with 1 μM of cyclic peptide 2 for 72 hours and 96 hours and subjected to flow cytometry analysis. It was observed that treatment with cyclic peptide 2 resulted in an increase in the cell population in the G2 / M phase after 72 hours (about 5.2% and about 13% after 72 hours and 96 hours, respectively) (Figures 4J and 38). Subsequently, the apoptosis rate of HeLa cells treated with cyclic peptide 2 was examined using an annexin V-FITC / PI double staining kit. The inventors' results showed that treatment of HeLa cells with 1 μM of cyclic peptide 2 for 96 hours increased the overall annexin V-positive cells (about 14.5%) (Figures 4K and 37). These results suggest that apoptosis is increased by cyclic peptide 2 treatment. Collectively, the inventors have shown evidence that cyclic peptide 2 inhibits DSB repair by binding to Lys63-linked Ub chains, thereby causing G2 / M cell cycle arrest and apoptosis. 【0301】 Example 4 Identification of potential ligands of cyclic peptides targeting K63Ub To examine those ubiquitinated proteins modified via Lys63-linked Ub chains that can bind to cyclic peptide 2, the inventors modified its structure with an S-biotinylation reagent for a pull-down experiment to generate 31. Using derivative 31 and streptavidin beads, Lys63-linked Ub chains and their binders were enriched from U2OS lysates containing biotinylated scrambled peptide 38 as a non-specific control. These binders were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then Western blot analysis was performed using antibodies against Lys63-linked and Lys48-linked Ub chains (Figure 5B). Cyclic peptide 31 specifically enriched proteins modified with Lys63-linked Ub chains without containing Lys48-linked chains, which supports the specificity of the binding of Lys63-linked Ub chains by cyclic peptide 2. 【0302】 To identify proteins rich in 31, the inventors performed label-free proteomics after digestion on beads (Figure 5A). The inventors concentrated a substantial amount of proteins (about 1,100), of which a significant number (about 450) are involved in various cellular processes (such as DNA repair, transport, cell cycle, and histone modification) mainly involving Lys63-linked Ub chains, and these are shown by colored dots in the volcano plot (Figure 5C). The inventors identified 68 improved terms for 31 DNA repair proteins and presented them in a cluster format with a string network (Figure 39 and Table 2). Importantly, the inventors concentrated several proteins with specific affinity for K63-Ub chains, such as UBR5, PCNA, BABAM1, and PSMD14. Gene ontology analysis showed enrichment of GO terms such as DSB repair, regulation of the mitotic cell cycle, condensation of mitotic chromosomes, transport, and response to stress (Figure 5D), suggesting that only 31 pull-down proteins are adhered to K63-linked ubiquitin. Combining all these results, it is suggested that cyclic peptide 2 specifically binds to Lys63-linked Ub chains, thereby regulating cellular processes such as DDR and cell cycle mainly involving the Lys63 chain type. 【0303】 Conclusion The therapeutic approach of the present invention targeting components of the ubiquitination machinery focuses on inhibiting the activity of specific enzymes. Modulating DNA damage repair by targeting Lys63-linked Ub chains is a novel approach that has not been tested hitherto. Furthermore, the absence of pharmacological inhibitors of Lys63-linked Ub chain E2-E3 enzymes emphasizes the urgency to establish orthogonal approaches for targeting Lys63-linked Ub chains. Since most human cancers have defects in repair pathways, targeting the remaining functional repair pathways by modulating Lys63-linked Ub chains has great therapeutic potential. Many therapeutic approaches experience acquired drug resistance, but targeting conserved Lys63-linked Ub chains may potentially avoid standard drug resistance mechanisms. Additionally, developing cyclic peptides that target Lys63-linked Ub chains increases the repertoire of drug therapy targets. 【0304】 The inventors discovered macrocyclic modulators of Lys63-linked Di-Ub by combining chemical protein synthesis, RaPID selection, and late-stage modifications. Leveraging the power of chemical synthesis, additional libraries of Cys mutants and their modified analogs were created. This multi-disciplinary screening approach differentiated between different Ub chain types and generated efficient cyclic peptide binders that bind tightly to Lys63 chains. Considering the flexible open structure of Lys63 chains in solution, this discovery is notable. Importantly, the effective cyclic peptides do not bind to linear Di-Ub chains that have similar structural features to Lys63-linked Di-Ub. The molecular basis for such selectivity and the mode of interaction between the cyclic peptides disclosed herein and Lys63-linked chains remain unclear, but these will enable further modifications to improve functional properties. Slight structural modifications in cyclic peptide-based Ub binders were observed to have a wide-ranging impact on binding efficiency. This emphasizes the strength of the approach of the present invention in selecting specific Ub chain binders despite minor differences at the molecular level for potential drug development. 【0305】 It has been found that the cyclic peptide is a cell-permeable modulator of the DDR pathway that leads to the accumulation of DNA damage, cell cycle arrest in the G2 / M phase, and apoptosis. Furthermore, proteome analysis of proteins rich in cyclic peptides has revealed important elements of the DDR pathway involving Lys63-linked Ub chains. 【0306】 The inventors' approach provides new opportunities for basic research related to the Ub system. These are considered to be valuable means for this macrocyclic peptide to regulate Ub signaling and DNA damage. Selectively inhibiting Lys63-linked polyubiquitin chains with cyclic peptides could be a promising strategy for cancer therapy. 【0307】 Example 5 Optimization of Solubilization and Cell Penetration Ability of Cyclic Peptides The cyclic peptide modulator 2 (CP2) of the present invention for Lys63-linked Di-Ub, which combines chemical protein synthesis, RaPID selection, and late-stage modification, has low water solubility, thus limiting its application to advanced biological tests and other uses. To overcome this problem, Arg (R) was incorporated at intra-ring and extra-ring positions to generate 39 - 42 (Figure 40A), thereby improving the overall solubility of the cyclic peptide in a buffer (aqueous solution) medium (more than 5 - 10 times that of 2). Interestingly, disruption of the DNA damage repair ability of the cyclic peptide (40) with an "RR" modification at the extra-ring position was relatively more compared to the parent cyclic peptide 2, but its binding affinity for Lys63-linked Di-Ub was shown to be relatively lower than that of 2 (Figures 40B - 40C). This is thought to be because the modification with RR improved the cell permeability of the cyclic peptide. Therefore, it was concluded that the intracellular penetration of the cyclic peptide could be further improved by adjusting the structure of the cyclic peptide at other position(s). 【0308】 The inventors hypothesized that the linker for cyclization is an important factor that imparts unique conformational characteristics to cyclic peptides. To investigate this, the inventors prepared eight different linkages on the RR peptide and created libraries 43-50 (Figure 41A). The results of the in vitro binding affinity and intracellular DNA damage induction screening of the present invention showed that 43 had the highest binding affinity among all cyclic peptides and a significantly improved DNA damage induction ability (Figures 41B-41C). This suggests that the "cyclization linker" plays an important role in improving the cellular uptake of cyclic peptides. 【0309】 Non-limiting exemplary synthetic schemes for peptides 39-50 are shown in Figures 42-43. 【0310】 Certain features of the present invention are illustrated and described herein, but many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Therefore, it is to be understood that the appended claims are intended to cover all modifications and changes that fall within the true spirit of the present invention.

Claims

[Claim 1] A cyclic peptide containing the amino acid sequence LLIWIGSSKNPYILCG (SEQ ID NO: 1) or a functional analog thereof having at least 80% homology. [Claim 2] The cyclic peptide according to claim 1, comprising at least one cysteine ​​residue in which the amino acid residue of SEQ ID NO: 1 is substituted. [Claim 3] CLIWIGSSKNPYILCG (SEQ ID NO: 2); LCIWIGSSKNPYILCG (SEQ ID NO: 3); LLCWIGSSKNPYILCG (SEQ ID NO: 4) LLICIGGSSKNPYILCG (SEQ ID NO: 5); LLIWCGSSKNPYILCG (SEQ ID NO: 6); LLIWIICSSKNPYILCG (SEQ ID NO: 7) LLIWIGCSKNPYILCG (SEQ ID NO: 8); A cyclic peptide according to claim 1 or 2, comprising an amino acid sequence selected from the group consisting of LLIWIGSSKNPYILCG (SEQ ID NO: 9); LLIWIGSSKCPYILCG (SEQ ID NO: 10); LLIWIGSSKNCYILCG (SEQ ID NO: 11); LLIWIGSSKNPICILCG (SEQ ID NO: 12); and LLIWIGSSKNPYCLCG (SEQ ID NO: 13). [Claim 4] A cyclic peptide according to claim 1, comprising 14 to 20 amino acid residues. [Claim 5] The cyclic peptide according to claim 1, wherein the amino acid at position 1 of the N-terminus is conjugated to a cyclization molecule. [Claim 6] The cyclized molecule, 【Chemistry 1】 The cyclic peptide according to claim 5, comprising any one of the following, wherein each wavy bond represents an attachment site to a first amino acid residue or a C-terminal amino acid. [Claim 7] The cyclized molecule is -CH ２ - The cyclic peptide according to claim 1. [Claim 8] The cyclic peptide according to claim 1, wherein the cyclic peptide is chemically modified. [Claim 9] The cyclic peptide according to claim 8, wherein the chemical modification is selected from the group consisting of alkylation, arylation, addition of a thiol protecting group, and any combination thereof. [Claim 10] The cyclic peptide according to claim 1, characterized by having cell penetration ability, ubiquitin (Ub) binding ability, or any combination thereof. [Claim 11] The cyclic peptide according to claim 10, wherein the Ub is polymer Ub. [Claim 12] The cyclic peptide according to claim 11, wherein the polymer Ub comprises a Ub monomer (K63Ub) linked at the 63rd position. [Claim 13] The cyclic peptide according to claim 1, wherein the affinity for Lys63-linked Ub chains is increased compared to a control Ub chain. [Claim 14] The increased affinity is the dissociation constant (K Ｄ The cyclic peptide according to claim 13, wherein the binding affinity is 0.05 to 150 nM. [Claim 15] The cyclic peptide according to claim 1, further comprising at least one arginine residue. [Claim 16] The cyclic peptide according to claim 15, wherein the at least one arginine residue is located at the C-terminus of the cyclic peptide. [Claim 17] A cyclic peptide according to claim 15 or 16, comprising an amino acid sequence selected from the group consisting of CLIWIGSSKNPYILCGRR (SEQ ID NO: 15); CLIWIGSSKNPYILCRR (SEQ ID NO: 16); and CLIWIGSSKNPYILCR (SEQ ID NO: 17). [Claim 18] A dimeric cyclic peptide comprising the cyclic peptide described in claim 1. [Claim 19] A pharmaceutical composition comprising a cyclic peptide according to claim 1, or a dimeric cyclic peptide according to claim 18, and an acceptable carrier. [Claim 20] The pharmaceutical composition according to claim 19 for use in the treatment of K63Ub-related diseases. [Claim 21] The pharmaceutical composition for use according to claim 20, wherein the K63Ub-related disease is a cell proliferation-related disease. [Claim 22] The pharmaceutical composition for use according to claim 21, wherein the cell proliferation-related disease includes cancer.

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