Ubiquitin high affinity cyclic peptides and methods of use thereof

Abstract

The present invention relates to polypeptides that bind ubiquitin (Ub), e.g., lysine 48 of Ub. Further provided are methods of using the polypeptides, e.g., for reducing deubiquitinating activity of a cell, and for treating cancer in a subject in need thereof.

Description

[0001] Reference to electronic sequence listing The electronic serial number (TECH-P-0296-PCT.xml; size: 5,410 bytes; creation date: December 22, 2024) is incorporated herein by reference in its entirety.

[0002] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 623,267, filed January 21, 2024, entitled “UBIQUITIN HIGH AFFINITY CYCLIC PEPTIDES AND METHODS OF USE THEREOF”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of peptide engineering and drug screening. Background Technology

[0004] The initial and mandatory step in ubiquitin (Ub) signaling is the ATP-dependent modification of protein substrates by Ub or poly-Ub chains. The formation of poly-Ub chains is achieved through the cooperation of three enzymes known as E1, E2, and E3. The most studied Ub chain is the K48 linker chain, known for its compact structure that targets modified proteins for proteasome degradation.

[0005] Ubiquitination is a major post-translational modification that affects many aspects of cell biology and is associated with a variety of diseases, including cancer. Several tumor inhibitors or oncogenes involved in Ub conjugation and deconjugation pathways are altered in cancer states, making the Ub system an excellent target for cancer therapy. In particular, the ubiquitin-proteasome system (UPS) has proven to be an effective target for the development of anticancer drugs, such as the approved small molecule bortezomib for the treatment of multiple myeloma and mantle cell lymphoma.

[0006] Another approach is to target the Ub chain itself. This offers significant potential for interfering with Ub signaling compared to targeting a single Ub-modifying enzyme. In this regard, the small molecule ubistatins emerged from chemogenetic screening and were found to bind to a single Ub and K48-linked chain, thereby impairing proteasome recognition and inhibiting protease degradation. However, ubistatins suffer from poor cell permeability, weak µM binding, and poor chain specificity.

[0007] Targeting Ub chains of specific lengths and connections can be advantageous and influence specific cellular processes. However, given the number of possible Ub chains and their subtle differences in structure and dynamics, this poses a significant challenge for molecular recognition.

[0008] Therefore, there is still a need for molecules or compounds that have specific binding affinity for single Ub, especially the K48 linker chain, and that can fully penetrate living cells. Summary of the Invention

[0009] This invention relates to polypeptides and methods of use thereof, for example, for reducing the deubiquitination activity of cells or for improving or treating cancer in subjects in need.

[0010] According to the first aspect, a polypeptide is provided comprising an amino acid sequence selected from FQYWOYATGVCR (SEQ ID NO:1); FQYWOYATGVCRR (SEQ ID NO:2); FQYWOYATGVCGR (SEQ ID NO:3); and FQYWOYATGVCGRR (SEQ ID NO:4).

[0011] According to another aspect, a pharmaceutical composition is provided comprising the polypeptide of the present invention and at least one acceptable carrier.

[0012] According to another aspect, a method for reducing cellular deubiquitination activity is provided, the method comprising contacting cells with an effective amount of a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1-4.

[0013] According to another aspect, a method for improving or treating cancer in a subject in need is provided, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising the amino acid sequences shown in SEQ ID NO:1-4.

[0014] In some implementations, the polypeptide is a cyclic polypeptide.

[0015] In some embodiments, at least one amino acid residue is methylated.

[0016] In some implementations, the polypeptide contains no more than 16 amino acid residues.

[0017] In some implementations, the amino acid at position 1 is a D amino acid.

[0018] In some embodiments, the amino acid at position 1 is conjugated with a cyclized molecule.

[0019] In some embodiments, the polypeptide is a cyclic polypeptide prepared using cyclized molecules containing halogens.

[0020] In some embodiments, the cyclized molecule is selected from: chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl (4-ClBz), or Cl2SAc.

[0021] In some embodiments, the polypeptide is characterized by having: cell penetration ability, ubiquitin (Ub) binding ability, or a combination thereof.

[0022] In some implementations, the peptide exhibits increased affinity for Ub compared to a control.

[0023] In some embodiments, the control is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5.

[0024] In some embodiments, the pharmaceutical composition is used to treat cancer in a subject in need.

[0025] In some implementations, the application is subcutaneous.

[0026] In some embodiments, the polypeptide: (i) has an affinity K of 0.05-100 nM. D (i) Combining with Ub; (ii) Containing no more than 16 amino acid residues; or both (i) and (ii).

[0027] In some implementations, the peptide exhibits increased affinity for Ub compared to a control.

[0028] In some implementations, Ub is aggregated Ub.

[0029] In some implementations, Ub includes a Ub monomer connected at its K48 position ( K48 Ub).

[0030] In some implementations, the peptide reduces the cell's deubiquitination activity by at least 30%.

[0031] In some implementations, the peptide reduces the proteasome degradation rate of ubiquitinated proteins by at least 40%.

[0032] In some implementations, the peptide exhibits increased pro-apoptotic activity compared to a control.

[0033] In some implementations, the increased pro-apoptotic activity resulted in at least a 20% increase in apoptosis compared to a control.

[0034] Unless otherwise defined, 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. While similar or equivalent methods and materials may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not necessarily limiting.

[0035] Further embodiments and the full scope of the invention will become apparent from the detailed description given below. However, it should be understood that while indicating preferred embodiments of the invention, the detailed description and specific examples are given only by way of illustration, as those skilled in the art will understand various changes and modifications within the spirit and scope of the invention from this detailed description. Attached Figure Description

[0036] Figure 1 A general, non-limiting schematic diagram of the cyclic polypeptide of the present invention.

[0037] Figure 2 The diagram includes vertical bar graphs showing comparative efficiency assays of the cyclic peptides of the present invention in MM1s cell lines, normalized to controls. The peptides are: Ub4a-CR (SEQ ID NO:1); Ub4a-CRR (SEQ ID NO:2); Ub4a-GR (SEQ ID NO:3); and Ub4a-GRR (SEQ ID NO:4).

[0038] Figure 3 The data includes vertical bar graphs showing comparative efficiency assays of the cyclic peptides of the present invention in BTZ900 (bortezomib-resistant cell line), normalized to control. The peptides are: Ub4a-CR (SEQ ID NO:1); Ub4a-CRR (SEQ ID NO:2); Ub4a-GR (SEQ ID NO:3); and Ub4a-GRR (SEQ ID NO:4).

[0039] Figure 4 The diagram includes a comparison of the time-dependent therapeutic effects in MM1s cell lines, normalized to a control, between Ub4a derivatives, the cyclic peptides of the present invention Ub4a-CR (SEQ ID NO:1), and Ub4a-GRR (SEQ ID ID:4), and is normalized to a control. Each cyclic peptide was applied at two concentrations: 2 µM and 5 µM.

[0040] Figure 5The diagram includes a comparison of the Ub4a derivatives, the cyclic peptides of the present invention: Ub4a-CR (SEQ ID NO:1) and Ub4a-GRR (SEQ ID ID:4), time-dependent efficacy in BTZ900 (bortezomib-resistant cell line), normalized to a control. Each cyclic peptide was applied at two concentrations: 2 µM and 5 µM.

[0041] Figure 6 Includes vertical bar graphs showing the efficacy of Ub4a-GRR (SEQ ID NO:4) in MM1S and BTZ900 (bortezomib-resistant cell lines), normalized to control. Ub4a-GRR (SEQ ID NO:4) was applied at two concentrations: 5 µM and 8 µM.

[0042] Figure 7 The chart includes vertical bar graphs showing a comparison of the efficacy of Ub4a (SEQ ID NO:5) and its derivatives, and the cyclic peptide Ub4a-GRR (SEQ ID NO:4) of the present invention, at two concentrations (2 µM and 5 µM) in the MM1S cell line, normalized to a control.

[0043] Figure 8 Includes a vertical bar graph showing the time-dependent stability of Ub4a-GRR (SEQ ID NO:4) in human plasma as determined by high performance liquid chromatography (HPLC).

[0044] Figure 9 Includes a vertical bar graph showing the concentration-related effects of the Ub K63 inhibitor CP-15 (as described by Vamisetti et al., Nature Communications (2022) 13:6174) on MM1S cell viability during 12 days of treatment.

[0045] Figure 10 Includes a graph showing the weight changes of 20 female NOD / SCID mice administered Ub4a-GRR (SEQ ID NO:4) via intravenous bolus (IV) or subcutaneous (SC). Data are plotted as a percentage change relative to initial body weight (day 1). Results are expressed as mean ± SEM for all mice in each group.

[0046] Figure 11 The graphs show the mean plasma concentrations of Ub4a-GRR (SEQ ID NO:4) after the following administration methods: IV bolus, SC, intraperitoneal (IP), and intramuscular (IM).

[0047] Figure 12 Includes a graph showing the plasma concentration of Ub4a-GRR (SEQ ID NO:4) after administration of 10.0 mg / kg SC.

[0048] Figure 13 Includes a graph showing the in vivo effects of UB4a (SEQ ID NO:5) on the mean and standard error of tumor volume over 40 days in 10 SCIDNOD mice implanted with MM tumors from the BTZ900 cell line. Three (3) different doses were administered daily: 2 mg / kg, 5 mg / kg, and 10 mg / kg. Changes in tumor volume were compared with those in vector-injected mice (daily) and bortezomib (weekly).

[0049] Figure 14 This includes a graph showing the in vivo effects of UB4a-GRR (SEQ ID NO:4) on the mean and standard error of tumor volume calculated over 38 days in three mice implanted with MM tumors of type SCIDNOD from the BTZ900 cell line. Tumor volume changes after treatment with 10 mg / ml were compared with those in untreated mice and mice with CP-15 Ub K63 inhibitors. Detailed Implementation

[0050] This invention relates to peptides and methods of use thereof, for example, for reducing deubiquitination activity of cells or for improving or treating cancer in subjects in need. The invention is partly based on the discovery that cyclic peptides possess an affinity K at nanomolar levels. D The invention is also based in part on the surprising discovery that cyclic peptides can penetrate cells and bind to polymeric ubiquitin in vivo.

[0051] polypeptide According to some embodiments, the present invention relates to polypeptides.

[0052] In some implementations, the polypeptide can penetrate cells (e.g., cancer cells), bind ubiquitin, or a combination thereof.

[0053] In some embodiments, the polypeptide of the present invention comprises or consists of an amino acid sequence selected from or composed of the following: FQYWOYATGVCR (SEQ ID NO: 1); FQYWOYATGVCRR (SEQ ID NO: 2); FQYWOYATGVCGR (SEQ ID NO: 3); or FQYWOYATGVCGRR (SEQ ID NO: 4), or analogues thereof, wherein the analogue has at least 80%, 90%, 95%, 97% or 99% homology or identity with the sequence, or any value and range thereof. In some embodiments, the polypeptide of the present invention comprises or consists of an amino acid sequence selected from or composed of the following: FQYWOYATGVCR (SEQ ID NO: 1); FQYWOYATGVCRR (SEQ ID NO: 2); FQYWOYATGVCGR (SEQ ID NO: 3); or FQYWOYATGVCGRR (SEQ ID NO: 4), or analogues thereof, said analogues having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with the sequence. Each possibility represents a separate embodiment of the invention.

[0054] In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCR (SEQ ID NO: 1), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polypeptide of the present invention consists of the amino acid sequence FQYWOYATGVCR (SEQ ID NO: 1), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention.

[0055] In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCRR (SEQ ID NO: 2), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCRR (SEQ ID NO: 2), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention.

[0056] In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCGR (SEQ ID NO: 3), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCGR (SEQ ID NO: 3), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention.

[0057] In some embodiments, the polypeptide of the present invention comprises the amino acid sequence FQYWOYATGVCGRR (SEQ ID NO: 4), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polypeptide of the present invention consists of the amino acid sequence FQYWOYATGVCGRR (SEQ ID NO: 4), or an analogue thereof, said analogue having 80-100%, 90-100%, 95-100%, 97-100%, or 99-100% homology or identity with said sequence. Each possibility represents a separate embodiment of the present invention.

[0058] The terms “homology” or “identity” used interchangeably in this article refer to the sequence identity between two amino acid sequences or two nucleic acid sequences; identity is a more stringent comparison. The phrases “identity or percentage of homology” and “identity or percentage of homology” refer to the percentage of sequence identity found when comparing two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be any value from 0 to 100% identical, or any value in between. Identity can be determined by comparing positions in each sequence that can be aligned with a reference sequence for comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, the molecules are identical at that position. The degree of identity of an amino acid sequence is a function of the number of identical amino acids at shared positions in the amino acid sequences. The degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at shared positions in the nucleic acid sequences. The degree of homology of an amino acid sequence is a function of the number of amino acids at shared positions in the polypeptide sequences.

[0059] The following is a non-limiting example of calculating homology or sequence identity between two sequences (these terms are used interchangeably throughout this document). Sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison). Using the GAP procedure in the GCG software package, with a Blossum 62 scoring matrix, a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5, the optimal alignment is determined as the best score. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences.

[0060] In some implementations, the basic local alignment search tool (BLAST) is used to calculate or determine the homology or identity percentage described herein. In some implementations, the Blossum 62 scoring matrix is ​​used to calculate or determine the homology or identity percentage described herein.

[0061] As used herein, “O” stands for “ornithine,” which refers to a non-protein amino acid known to participate in the urea cycle, as will be apparent to those skilled in the art.

[0062] In some embodiments, the polypeptides of the present invention are linear or cyclic. In some embodiments, the peptides or polypeptides are cyclic peptides or cyclic polypeptides.

[0063] As defined herein, the amino acid sequence (SEQ ID No: 1-4) of the polypeptides of the present invention is referenced from their N-terminus to their C-terminus. In some embodiments, the amino acid residue located at the N-terminus of the polypeptide is located at a first position of the polypeptide. In some embodiments, the reference position of a given amino acid residue in a cyclic polypeptide is determined based on the position of that amino acid residue in the linear form of the polypeptide.

[0064] In some embodiments, the cyclic polypeptide of the present invention is characterized by increased affinity for ubiquitin. In some embodiments, ubiquitin includes diubiquitin, tetraubiquitin, or both.

[0065] In some embodiments, the cyclic polypeptide of the present invention has increased affinity for diubiquitin, tetraubiquitin, or both, comprising or consisting of the amino acid sequences described in or composed of SEQ ID No: 1-4.

[0066] In some embodiments, the cyclic polypeptide of the present invention comprises no more than 16 amino acid residues. In some embodiments, no more than 16 amino acid residues comprises 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, or any value and range thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, no more than 16 amino acid residues comprises 7-9, 8-11, 7-12, 10-13, 12-15, 8-14, 9-12, or 10-15 amino acid residues. Each possibility represents a separate embodiment of the invention.

[0067] In some embodiments, the polypeptides of the present invention comprising or consisting of the amino acid sequences described in SEQ ID No:1-4 further comprise methylated amino acid residues.

[0068] In some embodiments, the polypeptides of the present invention comprising or composed of the amino acid sequences described in SEQ ID Nos. 1-4 are further functionalized, for example by conjugating the carbon chain to amino acid residues at positions 6 or 8, or both. In some embodiments, the carbon chain comprises one or more carbons. In some embodiments, the carbon chain comprising one or more carbons comprises at least 2, at least 3, at least 4, or at least 5 carbons, or any value and range thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the carbon chain comprising one or more carbons comprises 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 present invention as described above are functionalized by conjugation with methyl, ethyl, propyl, butyl, or any combination thereof.

[0069] In some embodiments, the polypeptides of the present invention are capable of binding ubiquitin (Ub). In some embodiments, the polypeptides of the present invention have a specific binding affinity for ubiquitin (Ub). As defined herein, the term "ubiquitin" refers to a regulatory protein added to other proteins through post-translational modification. In some embodiments, Ub is polymeric Ub. In some embodiments, polymeric Ub comprises at least 2, at least 3, at least 4, or at least 5 Ub monomers, or any value and range thereof. 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 polymer comprises Ub monomers linked together at a lysine (Lys) residue at position 48 (K48). In some embodiments, with polymeric... K11 Ub or cluster K63 Compared to Ub, the polypeptide of the present invention is effective against poly(peptides). K48 Ub has a greater binding affinity. In some embodiments, Ub is further conjugated to the protein. In some embodiments, the protein is post-translational modified by conjugation to a Ub monomer or polymer. In some embodiments, the Ub-conjugated protein is bound to the peptide of the present invention at the Ub site. In some embodiments, the peptide of the present invention reduces the deubiquitination of the Ub-conjugated protein. In some embodiments, the peptide of the present invention increases the proteasome degradation of the Ub-conjugated protein.

[0070] In some embodiments, the polypeptides of the present invention are capable of binding Ub in vitro, in vivo, ex vivo, or any combination thereof. In some embodiments, the polypeptides of the present invention are capable of penetrating cells. In some embodiments, the polypeptides do not require additional components to penetrate cells. In some embodiments, the polypeptides may be further prepared (formulated) with other components to enhance cell penetration. In some embodiments, the polypeptides may be used as a carrier or vehicle to carry other components into cells.

[0071] In some embodiments, the present invention relates to cyclic polypeptides of 12-16 amino acids, which are capable of penetrating cells and having an affinity K of 0.1-100 mM. D Combine with Ub.

[0072] In some embodiments, the peptides of the present invention exhibit increased affinity for Ub compared to a control. In some embodiments, the affinity is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or any value and range thereof, compared to a control. Each possibility represents a separate embodiment of the invention. In some embodiments, the increased affinity is at least 5%, 10%, 20%, 35%, 50%, 65%, 75%, 85%, 90%, 99%, or 100%, or any value and range thereof, compared to a control. Each possibility represents a separate embodiment of the invention. In some embodiments, the affinity is increased by 1-5%, 4-10%, 8-20%, 25-35%, 30-50%, 45-65%, 60-75%, 70-85%, 80-90%, 85-99%, or 95-100%, compared to a control. Each possibility represents a separate embodiment of the invention.

[0073] As used herein, “control” includes any baseline against which the binding affinity of the polypeptide of the invention to Ub is compared. In some embodiments, the control is a protein that has no Ub binding affinity. In some embodiments, the control is a protein with low Ub binding affinity. In some embodiments, the control is a protein known to have Ub binding capacity, such as ubiquitin receptors and proteins containing ubiquitin-binding domains, non-limiting examples of which include, but are not limited to: Rabex-5, STAM1, STAM2, IsoT, EAP45, S5a / Rpn10, Rpn13, or any other protein known in the art. In some embodiments, the control is the linear form of the polypeptide of the invention. In some embodiments, the control is the polypeptide of the invention that has been initially inactivated, such as by antibody neutralization, enzymatic digestion, denaturation, or other methods known in the art of protein inactivation, before incubation in vitro or in vivo in an environment containing Ub. In some embodiments, the control is a cyclic peptide. In some embodiments, the control is a peptide consisting of the amino acid sequence FQYWOYATGVCG (SEQ ID NO:5). In some embodiments, the control is a peptide containing the amino acid sequence FQYWOYATGVCG (SEQ ID NO:5). In some embodiments, the control is CP-15 (as described in Vamisetti et al., Nature Communications (2022) 13:6174).

[0074] In some embodiments, the polypeptide of the present invention has Ub binding affinity, wherein K DThe possible values ​​are 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, or 15-40 µM. Each possibility represents a separate embodiment of the invention. In some embodiments, the polypeptide of the invention has Ub binding affinity, wherein K D The values ​​and ranges are up to 0.1 nM, up to 0.5 nM, up to 1 nM, up to 5 nM, up to 10 nM, up to 20 nM, up to 30 nM, up to 40 nM, up to 50 nM, up to 60 nM, up to 70 nM, up to 80 nM, up to 90 nM, up to 100 nM, up to 110 nM, up to 150 nM, up to 250 nM, up to 500 nM, up to 750 nM, up to 1500 nM, up to 1 µM, up to 5 µM, up to 10 µM, up to 15 µM, up to 20 µM, or up to 30 µM, or any values ​​and ranges therebetween. Each possibility represents a separate embodiment of the invention.

[0075] This invention includes derivatives of the polypeptides of this invention. The term "derivative" or "chemical derivative" includes any chemical derivative of the polypeptide having one or more residues chemically derived via a side chain or functional group reaction. Such derivatized molecules include, for example, those in which the free amino group has been derivatized to form an amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl, or formyl. The free carboxyl group can be derivatized to form a salt, methyl ester, ethyl ester, or other types of ester or hydrazide. 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-m-benzylhistidine. Also included are those peptides as chemical derivatives containing one or more naturally occurring amino acid derivatives of twenty standard amino acid residues. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine (O) can replace lysine.

[0076] Furthermore, peptide derivatives can be chemically modified to differ from the natural sequence of the peptides of the present invention, including but not limited to terminal -NH2 acylation, acetylation, or mercaptoacetate amidation, as well as terminal carbamate, for example with ammonia, methylamine, etc. The peptides can be linear, cyclic, or branched, etc., having any conformation, which can be achieved using methods known in the art.

[0077] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a polymer of amino acid residues.

[0078] As used herein, the term "amino acid" refers to an organic compound containing both a basic amino group and an acidic carboxyl group. This term includes naturally occurring amino acids, modified, unusual, non-naturally occurring amino acids, and amino acids known to exist biologically in free or combinatorial forms but not normally present in proteins. The term includes modified and unusual amino acids, such as those disclosed in Roberts and Vellaccio (1983) The Peptides, 5: 342-429. Modified, unusual, or non-naturally occurring 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, ortholeucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, β-phenylproline, tertiary leucine, 4-aminocyclohexylalanine (Cha), N-methyl-ortholeucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidin-4-carboxylic acid, 6-aminohexanoic acid, and trans-4-(aminomethyl) -Cyclohexanecarboxylic acid, 2-, 3- and 4-(aminomethyl)benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminovaleric acid, n-valine (Nva), 4-O-methylthreonine (TMe), 5-O-methylhomoserine (hSM), tert-butylalanine (tBu), cyclopentylalanine (Cpa), 2-aminoisobutyric acid (Aib), N-methylglycine (MeG), N-methylalanine (MeA), N-methylphenylalanine (MeF), 2-thienylalanine (2Th), 3-thienylalanine (3Th), O-methyltyrosine (YMe), 3-benzothienylalanine (Bzt), and D-alanine (DAl).

[0079] The term "amino acid residue" used in this article refers to the amino acid portion present in a peptide.

[0080] The term "peptide bond" refers to a covalent amide bond formed by the loss of water molecules between the carboxyl group of one amino acid and the amino group of a second amino acid.

[0081] As used herein, the terms “peptide,” “polypeptide,” and “protein” include natural peptides, peptidomimetic peptides (typically including non-peptide bonds or other synthetic modifications), and peptide analogs such as peptides and hemipeptides, or any combination thereof. In another embodiment, the terms “peptide,” “polypeptide,” and “protein” apply to amino acid polymers wherein at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0082] Those skilled in the art will recognize that a single substitution, deletion, or addition of a peptide or protein sequence that alters, adds, or omits a single amino acid or a small subset of amino acids in the coding sequence is a variant of a conserved modification, wherein the alteration results in the substitution of an amino acid with similar charge, size, and / or hydrophobic characteristics, such as glutamic acid (E) being replaced by aspartic acid (D).

[0083] As used herein, the phrase “conservative substitution” also includes the use of chemically derived residues to replace non-derived residues, provided that the peptide exhibits the necessary function as defined herein.

[0084] Peptide derivatives according to the principles of the present invention may further include side chain 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-, as well as skeletal modifications, such as modified peptide bonds. The peptide bonds (-CO-NH-) within a peptide can be substituted, for example, by the following bonds: N-methylation (-N(CH3)-CO-); ester bond (-C(R)HCOOC(R)HN); ketomethylene bond (-CO-CH2-); α-aza bond (-NH-N(R)-CO-), where R is any alkyl group, such as methyl; carba bond (-CH2-NH-); hydroxyethylene bond (-CH(OH)-CH2-); thioamide bond (-CS-NH); olefin double bond (-CH=CH-); and peptide derivative (-N(R)-CH2-CO-), where R is a "normal" side chain naturally present on a carbon atom. These modifications can occur on one or more bonds on the peptide chain, or even simultaneously on several (e.g., 2-3) bonds.

[0085] This invention also includes peptides and their derivatives, which may contain D-isomers of one or more amino acids. The production of retro-inverso D-amino acid peptides, in which at least one amino acid and possibly all amino acids are D-amino acids, is well known in the art. When all amino acids in a peptide are D-amino acids and the N- and C-termini of the molecule are reversed, the result is a molecule that has the same structural groups at the same positions as in the L-amino acid form of the molecule. However, this molecule is more stable to proteolytic degradation and can therefore be used in many of the applications described herein. Due to their higher water solubility, lower immunogenicity, and lower sensitivity to proteolytic degradation, diastereomeric peptides may be more advantageous than all-L- or all-D-amino acid peptides having the same amino acid sequence. The term "diasteremeric peptide" as used herein refers to a peptide containing both L-amino acid residues and D-amino acid residues. The number and position of D-amino acid residues in the diastereomeric peptides of this invention can be variable, as long as the peptide exhibits the function of the chimeric peptides disclosed herein.

[0086] According to one embodiment, the polypeptides of the present invention can be synthesized or prepared by any method and / or technique known in the art for peptide synthesis. According to another embodiment, the polypeptides can be synthesized using Merrifield's solid-phase peptide synthesis method (see J. Am. Chem. Soc., 85:2149, 1964). According to yet another embodiment, the polypeptides of the present invention can be synthesized using standard solution methods well known in the art (e.g., see Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, 1984).

[0087] Generally, the synthetic method involves sequentially adding one or more amino acids, or appropriately protected amino acids, to a growing peptide chain bound to a suitable resin. Typically, the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. Then, under conditions favorable for amide bond formation, the protected or derived amino acid can be attached to an inert solid support (resin), or used in solution by adding the next amino acid in the sequence with a complementary (amino or carboxyl) group that has appropriate protection. The protecting group is then removed from this newly added amino acid residue, and the next amino acid (appropriately protected) is added, and so on. After all the desired amino acids are linked in the correct order, any remaining protecting groups are removed sequentially or simultaneously, and if synthesized by a solid-phase method, the peptide chain is cleaved from the solid support to obtain the final peptide.

[0088] In solid-phase peptide synthesis, the α-amino group of an amino acid is protected by an acid- or base-sensitive group. This protecting group should be stable to peptide bond formation conditions and easily removed without disrupting the growing peptide chain. Suitable protecting groups include tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, (α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylthio, 2-cyano-tert-butoxycarbonyl, and 9-fluorenemethoxycarbonyl (Fmoc). In solid-phase peptide synthesis, the C-terminal amino acid is linked to a suitable solid support. Suitable solid supports for the above synthesis are those that are inert to the reagents and reaction conditions of the stepwise condensation deprotection reaction and insoluble in the solvent medium used. Suitable solid supports include chloromethyl polystyrene-divinylbenzene polymers and hydroxymethyl polystyrene-divinylbenzene polymers. The conjugation reaction is carried out in solvents such as ethanol, acetonitrile, and N,N-dimethylformamide (DMF). The conjugation of sequentially protected amino acids can be performed in automated peptide synthesizers well known in the art.

[0089] In another embodiment, the polypeptide of the present invention can be synthesized such that one or more bonds of the amino acid residues linking the peptide are non-peptide bonds. In another embodiment, non-peptide bonds include, but are not limited to, imino, ester, acylhydrazine, aminourea, and azo bonds, which can be formed by reactions well known to those skilled in the art.

[0090] The term "linker" refers to a molecule or macromolecule used to connect different parts of a peptide or polypeptide. In one embodiment, the linker may also facilitate other functions, including but not limited to maintaining biological activity and maintaining subunit and domain interactions.

[0091] In some embodiments, the polypeptides of the present invention can be attached to or linked to another molecule via chemical linkers. Chemical linkers are well known in the art and include, but are not limited to, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), maleimide-benzoyl-N-hydroxysuccinimide ester (MBS), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), and N-isobutoxy-carbonyl-2-isobutoxy-1,2-dihydroquinoline (IIDQ). In another embodiment, the linker can also be a monomeric entity, such as a single amino acid. In another embodiment, amino acids having small side chains, or small polypeptide chains, or polymeric entities of several amino acids are particularly preferred. In another embodiment, the length of the polypeptide linker is 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. In one embodiment, the linker can be a nucleic acid encoding a small polypeptide chain. In another embodiment, the linker encodes a polypeptide linker with a length of 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0092] Recombinant technology can be used to express the peptides of the present invention, as is well known in the art. In another embodiment, the linker may be a cleavable linker that, once delivered to a selected tissue or cell, results in the cleavage of the peptides of the present invention. In this embodiment, the cell or tissue will have an endogenous (naturally occurring enzyme or recombinantly engineered to express an enzyme) or an exogenous (e.g., by injection, absorption, etc.) enzyme capable of cleaving the cleavable linker.

[0093] In another embodiment, the linker may be biodegradable, allowing the peptides of the present invention to be further processed intracellularly via hydrolysis and / or enzymatic cleavage. In one embodiment, a tumor-specific expressed protease may be used as a prodrug for delivering a cytotoxic agent, wherein the linker is selective for site-specific protein hydrolysis. In some embodiments, cleavable groups include acetyl, trimethylacetyl, butyryl, methylsuccinoyl, tert-butylsuccinoyl, ethoxycarbonyl, methoxycarbonyl, benzoyl, 3-aminocyclohexylene, etc.

[0094] According to another aspect, a polynucleotide encoding the polypeptide of the present invention is provided.

[0095] In another embodiment, the nucleic acid sequence encoding the polypeptide has at least 70%, or optionally at least 80%, or optionally at least 90%, or optionally at least 95%, or optionally at least 99% homology, or any value and range therebetween, to the nucleic acid sequence of the polypeptide of the present invention or a derivative thereof. Each possibility represents a separate embodiment of the invention.

[0096] In some embodiments, the polynucleotide molecules of the present invention encode polypeptides containing non-canonical amino acids.

[0097] In some embodiments, the polynucleotide of the present invention is ligated into an expression vector containing transcriptional control of a cis-regulatory sequence (e.g., a promoter sequence). In some embodiments, the cis-regulatory sequence is adapted to guide constitutive expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is adapted to guide tissue-specific expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is adapted to guide inducible expression of the polypeptide of the present invention.

[0098] The term "polynucleotide" refers to a nucleic acid (such as DNA or RNA) sequence containing the coding sequence required for the production of a polypeptide. In one embodiment, a polynucleotide refers to a single-stranded or double-stranded nucleic acid sequence isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a complex polynucleotide sequence (e.g., a combination of the above).

[0099] In one embodiment, a "complementary polynucleotide sequence" refers to a sequence obtained by reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. In one embodiment, the sequence can then be amplified in vivo or in vitro using a DNA polymerase.

[0100] In one implementation, a “genomic polynucleotide sequence” refers to a sequence derived (or isolated) from a chromosome, and therefore represents a continuous portion of the chromosome.

[0101] In one embodiment, a "complex polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, the complex sequence may include some exon sequences required to encode the polypeptide of the present invention, and some intron sequences interposed therebetween. In one embodiment, the intron sequences may be from any source, including other genes, and may generally include conserved splicing signal sequences. In one embodiment, the intron sequences include cis-acting expression regulatory elements.

[0102] In some embodiments, the polynucleotides of the present invention are prepared using PCR technology or any other method or procedure known to those skilled in the art.

[0103] In one embodiment, the polynucleotide of the invention is inserted into an expression vector (e.g., a nucleic acid construct) to enable the recombinant polypeptide to be expressed. In one embodiment, the expression vector includes additional sequences that adapt the vector for replication and integration in prokaryotes. In one embodiment, the expression vector includes additional sequences that adapt the vector for replication and integration in eukaryotes. In one embodiment, the expression vector includes a shuttle vector that adapts the vector for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector contains transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[0104] In one embodiment, various prokaryotic or eukaryotic cells can be used as host expression systems to express the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing polypeptide coding sequences; yeast transformed with recombinant yeast expression vectors containing polypeptide coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV); or plant cell systems transformed with recombinant plasmid expression vectors containing polypeptide coding sequences (e.g., Ti plasmid).

[0105] In some embodiments, non-bacterial expression systems (e.g., mammalian expression systems) are used to express the polypeptides of the present invention. In one embodiment, an expression vector is used to express the polynucleotides of the present invention in mammalian cells.

[0106] In some embodiments, a variety of expression vectors can be advantageously selected in the bacterial system depending on the intended use of the expressed polypeptide. In one embodiment, a large quantity of polypeptide is required. In another embodiment, a vector is needed to guide high levels of protein product expression, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the bacterial periplasm or a culture medium in which the protein product can be easily purified. In one embodiment, certain fusion proteins are engineered with specific cleavage sites to aid in polypeptide recovery. In one embodiment, vectors suitable for this operation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0107] In one embodiment, a yeast expression system is used. In one embodiment, many vectors containing constitutive or inducible promoters, such as those disclosed in U.S. Patent No. 5,932,447, can be used in yeast. In another embodiment, a vector that promotes the integration of exogenous DNA sequences into the yeast chromosome is used.

[0108] In one implementation, the expression vector may also include additional polynucleotide sequences, which, for example, allow the translation of several proteins from a single mRNA, such as internal ribosome entry sites (IRES).

[0109] In some embodiments, the 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.

[0110] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses may be used. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Ebolavirus contain pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the guidance of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrosis protein promoter, or other promoters that are effectively expressed in eukaryotic cells.

[0111] 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, a process in which a single infected cell produces numerous progeny viral particles that bud and infect neighboring cells. In one embodiment, the result is a large-scale, rapid infection, with most of the virus initially uninfected by the original viral particles. In one embodiment, the resulting viral vector cannot spread laterally. In one embodiment, this feature may be useful if the desired objective is to introduce a specific gene into only a localized number of target cells.

[0112] Various methods can be used to introduce expression vectors 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 al. [Biotechniques 4 (6):504-512, 1986], and include, for example, stable or transient transfection, liposome transfection, electroporation, and infection with recombinant viral vectors. In addition, please see the positive and negative selection methods in U.S. Patent Nos. 5,464,764 and 5,487,992.

[0113] In one embodiment, a plant expression vector is used. In another embodiment, expression of the polypeptide-coding sequence is driven by multiple promoters. In some embodiments, viral promoters are used, such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the capsid protein promoter of TMV [Takamatsu et al., EMBO J.6:307-311 (1987)]. In another embodiment, plant promoters are used, 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)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those skilled in the art. See, for example, Weissbach and Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463 (1988)]. The invention may also utilize other expression systems well known in the art, such as insect and mammalian host cell systems.

[0114] It should be understood that, in addition to the elements required for transcription and translation of the inserted coding sequence (coding polypeptide), the expression construct may also include sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0115] In some embodiments, transformed cells are cultured under efficient conditions that allow for high-level expression of the recombinant peptide. In some embodiments, efficient culture conditions include, but are not limited to, efficient culture media, bioreactors, temperature, pH, and oxygen conditions that allow for protein production. In one embodiment, efficient culture media refers to any culture medium used to culture cells to produce the recombinant peptide of the present invention. In some embodiments, the culture medium typically comprises an aqueous solution having assimilated carbon, nitrogen, and phosphate sources, as well as appropriate salts, minerals, metals, and other nutrients (such as vitamins). In some embodiments, cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and culture dishes. In some embodiments, culture is carried out at temperatures, pH, and oxygen levels suitable for the recombinant cells. In some embodiments, the culture conditions are within the scope of the expertise of those skilled in the art.

[0116] In some embodiments, depending on the vector and host system used for production, the resulting peptides of the present invention may be retained within recombinant cells, secreted into a fermentation medium, secreted into the space between two cell membranes, such as the periplasmic space of *E. coli*, or retained on the outer surface of a cell or viral membrane. In one embodiment, the recovery of the recombinant peptides is affected after a predetermined culture time.

[0117] In one implementation, the phrase “recover recombinant peptide” as used herein refers to collecting the entire fermentation medium containing the peptide, without implying additional separation or purification steps.

[0118] In one embodiment, the polypeptides 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, chromatographic focusing, and differential solubilization.

[0119] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and the cleavable fusion moiety. In one embodiment, the fusion protein can be designed such that the polypeptide can be easily separated by affinity chromatography; for example, by immobilization on a column specific to the cleavable moiety. In one embodiment, a cleavage site is designed between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or reagent that specifically cleaves the fusion protein at that site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0120] In one embodiment, the polypeptide of the present invention is recovered in a "basically pure" form, which allows for the effective use of the protein in the applications described herein.

[0121] As used herein, the term "substantially pure" describes peptides / polypeptides or other substances isolated from their natural contaminants. Generally, a monomeric peptide is essentially pure when at least about 60% to 75% of the sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence. Substantially pure peptides can comprise more than about 85% to 90% of the peptide sample and can be more than 95% pure, more than 97% pure, or more than about 99% pure, or any value and range in between. Each possibility represents a separate embodiment of the invention. Purity can be measured on a polyacrylamide gel, and homogeneity determined by staining. Alternatively, for certain purposes, high resolution may be required, and HPLC or similar methods may be used for purification. In most cases, purity can be determined using a simple chromatography column or polyacrylamide gel.

[0122] The term "purified" does not require the material to exist in a form that demonstrates absolute purity, excluding the presence of other compounds. Rather, it is a relative definition. A peptide is considered "purified" after purification of the starting material or natural material by at least one, two, three, four, or five orders of magnitude.

[0123] In one embodiment, the polypeptides of the present invention are substantially free of naturally bound host cell components. The term "substantially free of naturally bound host cell components" describes a peptide or other substance that is isolated from its natural contaminants in its natural host cell state. Therefore, peptides synthesized chemically or in cellular systems with host cells different from their natural origin will not contain their naturally associated host cell components.

[0124] In one embodiment, the polypeptide 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, as described below.

[0125] In some embodiments, the polypeptide of the present invention is cyclized. In some embodiments, the first amino acid residue at the N-terminus of the polypeptide of the present invention is conjugated to a cyclizing molecule. In some embodiments, the first amino acid residue (located at the N-terminus) of the polypeptide and another amino acid residue (located at the C-terminus) of the polypeptide bind to each other, thereby forming a cyclic polypeptide. In some embodiments, the cyclizing molecule binds to the first amino acid residue and the other amino acid residue located at the C-terminus. In some embodiments, the cyclizing molecule promotes the binding of the first amino acid residue (at the N-terminus) and the C-terminal amino acid residue. In some embodiments, the cyclizing molecule is released after binding to the first and C-terminal amino acid residues. In some embodiments, the cyclizing molecule conjugates to the first and C-terminal amino acid residues upon binding. In some embodiments, the first amino acid residue (located at the N-terminus) of the polypeptide and the cysteine ​​residue located at the C-terminus of the polypeptide bind to each other, thereby forming a cyclic polypeptide. In some embodiments, the cyclizing molecule binds to the first amino acid residue and the cysteine ​​amino acid residue located at the C-terminus. In some embodiments, the cyclizing molecule promotes the binding of the first amino acid residue (at the N-terminus) and the C-terminal cysteine ​​amino acid residue. In some embodiments, the cyclizing molecule is released after binding to the first and C-terminal amino acid residues. In some embodiments, the cyclized molecule conjugates to the first amino acid residue and the C-terminal cysteine ​​residue of the polypeptide upon binding. In some embodiments, the cyclic polypeptide comprises a cyclized molecule conjugated to the first amino acid residue at the N-terminus of the cyclic polypeptide and the cysteine ​​residue at the C-terminus of the cyclic polypeptide.

[0126] As defined herein, a “C-terminal amino acid residue” refers to an amino acid residue located closer to the C-terminus of a linear polypeptide than to its N-terminus. In some embodiments, the C-terminal amino acid residue is located at the 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st position from the end, or the last amino acid residue in the linear polypeptide. Each possibility represents a separate embodiment of the invention. According to a non-limiting example, the 9th amino acid residue of a polypeptide comprising 16 amino acid residues is considered the C-terminal amino acid residue. Various methods can be used to cyclize the polypeptide (e.g., macrocyclization), as reviewed by White and Yudin (2011). In some embodiments, the C-terminal amino acid residue of the cyclic peptide of the present invention is cysteine.

[0127] In some embodiments, the cyclized molecule comprises one or more halogen atoms selected from fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At), or any combination thereof. Non-limiting examples of halogen-containing cyclized molecules include, but are not limited to, chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl (4-ClBz), or Cl2SAc. In one embodiment, the cyclized molecule containing the 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 at the C-terminus of the polypeptide, thereby producing a cyclic polypeptide.

[0128] In some embodiments, the polypeptide of the present invention is characterized by increased solubility compared to a control. In some embodiments, solubility is in vitro solubility, in vivo solubility, ex vivo solubility, or any combination thereof. In some embodiments, the polypeptide of the present invention is characterized by increased solubility in serum, plasma, blood, whole blood, or any combination thereof compared to a control. In some embodiments, the polypeptide is characterized by reduced toxicity to the subject administering the polypeptide compared to a control. In some embodiments, administration is intravenous. In some embodiments, the polypeptide of the present invention is characterized by increased solubility, increased apoptotic activity, and reduced toxicity compared to a control. In some embodiments, the polypeptide of the present invention is SEQ ID NO:5, and the control includes SEQ ID NO:5, CP-15, or both.

[0129] Composition In some embodiments, the present invention relates to a composition comprising a polypeptide comprising or consisting of the following: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any combination thereof.

[0130] According to another embodiment, the present invention provides a pharmaceutical composition comprising the polypeptide of the present invention as an active ingredient, and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, the pharmaceutical composition facilitates the administration of the compound to a living organism. According to another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of the present invention as an active ingredient.

[0131] In another embodiment, the pharmaceutical composition of the present invention may be formulated as a pharmaceutically acceptable salt of the polypeptide of the present invention or an analogue or derivative thereof. In another embodiment, the pharmaceutically acceptable salt includes those salts that form with a free amino group, such as salts derived from non-toxic inorganic or organic acids, such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those salts that form with a free carboxyl group, such as salts derived from non-toxic inorganic or organic bases, such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0132] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic compound. Such a drug carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous glucose and glycerin solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerin, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; and agents for regulating tension, such as sodium chloride or glucose, may also be envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical composition described herein.

[0133] As used herein, the term "pharmaceuticalally acceptable" means suitable for administration to subjects, such as humans. For example, the term "pharmaceuticalally acceptable" can mean approved by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeias, for use in animals, especially humans.

[0134] In another embodiment, the compositions of the present invention are in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations, etc. In another embodiment, the compositions of the present invention can be formulated as suppositories having 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, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's Remington's Pharmaceutical Sciences, the contents of which are incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the polypeptide of the present invention, preferably in a substantially purified form, and an appropriate amount of carrier to provide a suitable form of administration to the subject.

[0135] According to one embodiment of the present invention, the pharmaceutical composition contains 0.1-95% of the polypeptide, its derivative, or analogue of the present invention. According to another embodiment of the present invention, the pharmaceutical composition contains 1-70% of the polypeptide (one or more). According to another embodiment of the present invention, the composition or formulation to be administered may contain a certain amount of the polypeptide (one or more), which, according to embodiments of the present invention, is effective in treating the condition or disease of the treated subject.

[0136] One embodiment of the invention relates to the polypeptide of the invention, which is present in a unit dosage form and prepared by any method well known in the pharmaceutical field. In one embodiment of the invention, the unit dosage form is in the form of tablets, capsules, lozenges, wafers, patches, ampoules, vials, or pre-filled syringes. Furthermore, in vitro assays may optionally be used to help determine 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 should be determined based on the judgment of a professional and the individual patient's situation. The effective dosage can be deduced from dose-response curves obtained from in vitro or in vivo animal model bioassays or systems.

[0137] According to one embodiment, the compositions of the present invention are administered as pharmaceutical compositions comprising at least one active ingredient (cyclic polypeptide) of the present invention and a pharmaceutically acceptable carrier or diluent. In another embodiment, the compositions of the present invention may be administered alone or in combination with any conventional oral, parenteral, or transdermal dosage form. In some embodiments, the pharmaceutical compositions further comprise at least one anticancer agent, such as a chemotherapeutic agent. In some embodiments, the pharmaceutical compositions are used in combination with anticancer therapies such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery.

[0138] As used herein, the terms “administering,” “administration,” and similar terms refer to any method, in sound medical practice, of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect.

[0139] Depending on the location of the tissue of interest, the polypeptides of the present invention can be administered in any manner suitable for delivering the polypeptide to cells within the tissue of interest. Thus, for example, a composition containing the polypeptides of the present invention can be introduced into, for example, systemic circulation, which will distribute the peptide to the tissue of interest. Alternatively, the composition can be applied locally to the tissue of interest (e.g., as a continuous infusion injection or pump, or as a pill within the tissue, applied to all or part of the skin surface, etc.).

[0140] In some embodiments, pharmaceutical compositions comprising peptides are administered via oral, rectal, vaginal, topical, nasal, ocular, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous routes. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, such as intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection method known in the art. While the bioavailability of peptides administered via other routes may be lower than that administered via parenteral injection, it is conceivable, through the use of appropriate formulations, that the compositions of the invention can be administered via transdermal, oral, rectal, vaginal, topical, nasal, inhalation, and ocular therapeutic routes. Furthermore, it may be desirable to introduce the pharmaceutical compositions of the invention via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter attached to a reservoir. Lung administration may also be employed, for example, through the use of an inhaler or nebulizer.

[0141] For local application, the polypeptides, derivatives, analogs, or fragments thereof of the present invention can be bound to a pharmaceutically acceptable carrier to deliver an effective dose based on desired activity. The carrier may be in the form of, for example, but not limited to, ointments, creams, gels, pastes, foams, aerosols, suppositories, pads, or gel sticks.

[0142] For oral administration, the pharmaceutical composition may be in the form of tablets or capsules and may contain any of the following components or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate; or gliding agents, such as colloidal silica. When the dosage unit is in capsule form, it may also contain a liquid carrier, such as fatty oil, in addition to the materials of the types described above. Furthermore, the dosage unit form may contain a variety of other materials that can alter the physical form of the dosage unit, such as coatings with sugar, shellac, or other enteric solvents. The tablets of the present invention may also be coated with a film.

[0143] For parenteral administration, sterile aqueous solutions of sesame or peanut oil or aqueous propylene glycol, along with the corresponding water-soluble salts, can be used. If necessary, these solutions can be appropriately buffered, and the liquid diluent should first be isotonic with sufficient saline or glucose. These solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injections.

[0144] According to some embodiments, the peptides of the present invention can be delivered in a controlled-release system. In another embodiment, an infusion pump can be used to administer the peptide, such as a peptide for delivering insulin or chemotherapy to a specific organ or tumor. In yet another embodiment, the peptides of the present invention are administered in conjunction with a biodegradable, biocompatible polymer implant that releases the peptide at a selected site within a controlled time. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyvinyl acetate, copolymers thereto, and blends thereof (see Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the entire contents of which are incorporated herein by reference). In yet another embodiment, the controlled-release system can be placed near the therapeutic target, thus requiring only a fraction of the system dose.

[0145] The peptides described herein can also be contained in artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other carriers that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes used with the peptides described herein are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is usually determined by factors such as liposome size and blood stability. Various methods can be used to prepare liposomes, as reviewed, for example, by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and also see US Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0146] The composition also includes incorporating active materials into or onto particulate formulations of polymeric compounds, such as polylactic acid, polyethylene glycol, hydrogels, etc., or incorporating liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte shadows, or spheroplasts. Such compositions will affect physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0147] In one implementation, depending on the severity and responsiveness of the condition to be treated, administration may be a single or multiple doses, and the treatment process may last from several days to several weeks, or until it affects a cure or alleviates the disease state.

[0148] 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 manner described herein, is sufficient to produce the desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic reactions), and is proportionate to a reasonable benefit / risk ratio. In another embodiment, a therapeutically effective amount of peptide is the amount of peptide required for a measurable, expected biological effect in vivo. The actual dosage, rate of administration, and timing will depend on the nature and severity of the condition being treated. The determination of treatment prescriptions, such as dosage, timing, etc., is the responsibility of a general practitioner or specialist, typically taking into account the barrier to be treated, the individual patient's condition, 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 edition, Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, the effective amount or dose may initially be estimated from in vitro studies. In one embodiment, the dose may be formulated in an animal model, and this information may be used to more accurately determine the useful dose for humans.

[0149] In one embodiment, the toxicity and efficacy of the active ingredient described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture, or in laboratory animals. In one embodiment, data obtained from these in vitro and cell culture analyses, as well as animal studies, can be used to formulate dosage ranges for human use. In one embodiment, the dosage varies depending on the dosage form and route of administration used. In one embodiment, the exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition. [See, for example, Fingl et al. (1975), "The Pharmacological Basis of Therapeutics," Chapter 1, page 1].

[0150] Pharmaceutical compositions containing the currently described peptides as active ingredients can be prepared according to conventional pharmaceutical formulation techniques. See, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa. (1990). See also, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0151] In one embodiment, a composition comprising the formulation of the present invention in a compatible pharmaceutical carrier is prepared, placed in a suitable container, and labeled for treating a specified condition.

[0152] In one embodiment, the composition of the invention is contained in a packaging or dispenser device, such as an FDA-approved kit containing one or more unit dose forms of an active ingredient. In one embodiment, the packaging includes, for example, metal or plastic foil, such as blister packs. In one embodiment, the packaging or dispenser device includes instructions for use. In one embodiment, the packaging or dispenser is equipped with instructions relating to the container in a form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that agency's approval of the composition form or method of administration for human or veterinary use. In one embodiment, the instructions are a prescription drug label approved by the U.S. Food and Drug Administration (FDA) or an approved product information leaflet.

[0153] How to use According to another aspect, a method for reducing cellular deubiquitination activity is provided, comprising contacting cells with a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No:1-4.

[0154] According to another aspect, a method for inducing or increasing the rate of apoptosis is provided, comprising contacting cells with a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No:1-4.

[0155] According to another aspect, a method is provided for improving or treating cancer or pre-malignancy in a subject in need, comprising administering to the subject a therapeutically effective amount of a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No:1-4.

[0156] In some embodiments, the present invention relates to a method for treating, improving, reducing, and / or preventing conditions associated with increased cellular deubiquitination activity in a subject of need, the method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No: 1-4.

[0157] In some embodiments, the present invention relates to a method for treating, improving, reducing, and / or preventing a condition associated with increased cell proliferation activity in a subject of need, the method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No: 1-4.

[0158] In some embodiments, the present invention relates to a method for treating, improving, reducing, and / or preventing conditions associated with increased anti-apoptotic activity in a subject of need, the method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a cyclic polypeptide comprising the amino acid sequence described in SEQ ID No: 1-4.

[0159] In some implementations, conditions associated with increased cell deubiquitination activity, enhanced cell proliferation activity, increased cell anti-apoptotic activity, or any combination thereof, include cancer.

[0160] In some embodiments, the method of the present invention for treating or improving cancer in a subject in need includes administering to the subject any of: (i) the polypeptide of the present invention; or (ii) the pharmaceutical composition of the present invention.

[0161] In some embodiments, the treatment includes reducing the volume, size, weight, or any combination thereof of a tumor in the subject. In some embodiments, the treatment includes reducing the tumor volume in the subject. In some embodiments, the reduction is compared to a control. In some embodiments, the reduction is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or any value and range therebetween compared to a control. In some embodiments, the reduction is at least 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, or 99-100% compared to a control. Each possibility represents a separate embodiment of the invention.

[0162] In some embodiments, the control group includes untreated subjects with cancer. In some embodiments, the control group includes subjects who have received CP-15 or a composition containing CP-15. In some embodiments, the control group includes subjects who have received Ub4a (SEQ ID NO:5) or a composition containing it.

[0163] In another embodiment, the polypeptide of the present invention, or a composition comprising thereof, is used to treat, improve, reduce, and / or prevent cancer or premalignant conditions in a subject of need. In some embodiments, the composition comprises an effective amount of the polypeptide for treating or preventing cancer or premalignant conditions in a subject of need. In some embodiments, the composition comprises a therapeutically effective amount of the polypeptide, which comprises or is composed of an amino acid sequence selected from or consisting of SEQ ID No: 1-4, for treating or preventing cancer or premalignant conditions in a subject of need. In some embodiments, the composition further comprises at least one anticancer agent, such as a chemotherapeutic agent. In some embodiments, the composition is used in combination with anticancer therapies such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery.

[0164] In some embodiments, the compositions of the present invention comprise an effective amount of a polypeptide for preparing a medicament to treat, improve, reduce, or prevent diseases in subjects of need associated with increased cell proliferation, deubiquitination activity, anti-apoptotic activity, or a combination thereof. In some embodiments, the present invention relates to the use of compositions comprising an effective amount of a polypeptide in the preparation of a medicament for treating diseases in subjects of need associated with increased cell proliferation, deubiquitination activity, or a combination thereof, wherein the polypeptide comprises or is composed of an amino acid sequence selected from or consisting of SEQ ID No. 1-4.

[0165] In one embodiment, the polypeptide of the present invention is provided to a subject in its own form. In one embodiment, one or more (e.g., "multiple") polypeptides of the present invention are provided to a subject in their own form. In one embodiment, the polypeptide of the present invention is provided to a subject as part of a pharmaceutical composition, wherein the polypeptide is mixed with a pharmaceutically acceptable carrier. In one embodiment, one or more polypeptides of the present invention are provided to a subject as part of a pharmaceutical composition, wherein they are mixed with a pharmaceutically acceptable carrier.

[0166] In some implementations, the disease associated with increased cell deubiquitination activity, increased cell proliferation activity, increased anti-apoptotic activity, or a combination thereof, is cancer.

[0167] As used herein, the term "cancer" or "pre-malignancy" refers to a disease associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma, and germ cell tumor. In one embodiment, carcinoma refers to a tumor originating 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 originating from mesenchymal cells, including but not limited to botryoid sarcoma, chondrosarcoma, Ewing sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma. In one embodiment, lymphoma refers to a tumor originating from hematopoietic cells that leaves the bone marrow and tends to mature in lymph nodes, including but not limited to Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and immunoproliferative disorders. In one embodiment, leukemia refers to a tumor originating from hematopoietic cells that leaves the bone marrow and tends to mature in the blood, 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 originating from immature precursor cells or embryonic tissue, including but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuroblastoma, retinoblastoma, and glioblastoma multiforme. In one embodiment, germ cell tumor refers to a tumor originating from germ cells, including but not limited to germ cell tumors or seminomatomas (GGCT, SGCT) and non-germ cell tumors or non-seminoblastomas (NGGCT, NSGCT). In one embodiment, germ cell tumors or seminomatomas include but are not limited to germ cell tumors, dysgerminomas, and seminomas. In one implementation, non-germ cell tumors or non-seminomatous tumors refer to simple and mixed germ cell tumors, including but not limited to embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, teratoma, polyembryoma, gonadal blastoma, and teratocarcinoma.

[0168] As used herein, “cancer or precancerous cell proliferation” is a molecular process, and further increasing cell proliferation requires increased deubiquitination activity. In some embodiments, the method of the present invention involves reducing deubiquitination activity. In some embodiments, reducing deubiquitination activity leads to increased proteasome activity. In some embodiments, reducing deubiquitination activity leads to increased protein degradation. In some embodiments, reducing deubiquitination activity further reduces drug resistance. In some embodiments, cancer cells have increased deubiquitination activity compared to non-cancer or benign cells. In another embodiment, reducing deubiquitination activity reduces cancer cell viability. In some embodiments, reducing deubiquitination activity increases the apoptosis rate in or around cancer cells. In some embodiments, increased apoptosis leads to decreased cell viability.

[0169] In some embodiments, the terms "reduce" or "reducing" (e.g., for deubiquitination activity of cells, proteasomal degradation of ubiquitinated proteins, cell viability, or others) used in the above embodiments, compared to a control, are 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. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction is 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 invention. In some embodiments, the reduction compared to a control is at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 40 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 500 times, or at least 1000 times, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0170] The terms “inhibiting,” “reducing,” and “decreasing” are interchangeable.

[0171] In some embodiments, the term "increase" or "increasing" (e.g., for pro-apoptotic activity, apoptosis rate, or others) used in the above embodiments, compared to a control, is 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. Each possibility represents a separate embodiment of the invention. In some embodiments, the increase compared to a control is 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, the increase compared to the control is at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 40 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 500 times, or at least 1000 times, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0172] In some embodiments, the peptides of the present invention are used at half-maximum inhibitory concentrations (IC50) of 0.01-1 nM, 0.05-2 nM, 1-5 nM, 4-10 nM, 5-50 nM, 10-100 nM, 50-250 nM, 200-750 nM, 0.5-1.5 µM, 1-3 µM, 2-4 µM, 3-6 µM, 4-7 µM, 5-8 µM, 6-9 µM, 8-12 µM, 10-13 µM, 12-14 µM, 11-15 µM, 13-17 µM, 16-19 µM, 15-20 µM, 14-22 µM, 20-25 µM, or 26-35 µM. 50 This reduces cell viability. Each possibility represents a separate implementation of the invention.

[0173] In some embodiments, ubiquitination / deubiquitination kinetics or dynamics are detected by any assay known in the art, including immunoassays, Western blotting, immunohistochemistry, etc., for example, for detection K48 Ub. In some embodiments, protein degradation and proteasome activity are detected by any acceptable method, including immunoassay, Western blotting, immunohistochemistry, pulse tracking assay, etc., all of which are well known to those skilled in the art.

[0174] As used herein, the term "subject" refers to an animal, and more specifically, a non-human mammal and a human organism. Non-human animal subjects may also include animals in prenatal forms, such as embryos or fetuses. Non-limiting examples of non-human animals include: horses, cattle, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In one embodiment, the subject is a human. Human subjects may also include fetuses. In one embodiment, the desired subject is a subject who has and / or is at risk of having a condition associated with increased cell proliferation, deubiquitination activity, or a combination thereof.

[0175] As used herein, the term "treatment" or "treating" a disease, disorder, or condition includes relieving at least one of its symptoms, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a treatment to be effective, the useful compositions described herein need only reduce the severity of the disease, disorder, or condition, decrease the severity of associated symptoms, or improve the quality of life of the patient or subject.

[0176] As used herein, the term “prevention” for a disease, disorder, or condition includes delaying, preventing, suppressing, or inhibiting the onset of a disease, disorder, or condition. As used in light of the subject matter currently described, the term “prevention” refers to a preventative process in which a subject is exposed to the peptide currently described prior to the induction or onset of a disease / disorder process. This can be done in individuals with a genetic lineage indicating susceptibility to the occurrence of the disease / disorder to be prevented. For example, this can be done in individuals whose ancestors showed susceptibility to certain types, such as inflammatory disorders. The term “suppression” is used to describe a situation where a disease / disorder process has begun but no obvious symptoms have yet appeared. Thus, an individual’s cells may have a disease / disorder, but no external signs of the disease / disorder have yet been clinically detected. In either case, the term prevention can be used to include both prevention and suppression. Conversely, the term “treatment” refers to the clinical application of an active agent to combat an existing condition whose clinical manifestations have already been achieved in a patient.

[0177] As used herein, the term “condition” includes anatomical and physiological deviations from the normal state that constitute impairment of the normal state of a living animal or a part thereof, thereby disrupting or altering the performance of bodily functions.

[0178] Unless otherwise stated, any concentration range, percentage range or ratio range described herein shall be understood to include any integer and fractional concentration, percentage or ratio within that range, such as one-tenth and one-hundredth of an integer.

[0179] Unless otherwise stated, any numerical ranges relating to any physical characteristic (such as polymer subunits, size, or thickness) described herein should be understood to include any integers within the range.

[0180] As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal” refer to any subject in need of treatment, particularly a mammalian subject, such as a human.

[0181] In this discussion, unless otherwise stated, adjectives such as “substantially” and “about” modifying conditional or relational features of one or more features of an embodiment of the invention are understood to mean that the condition or feature is defined within an operationally acceptable tolerance range for the intended application of that embodiment. Unless otherwise stated, the word “or” in the specification and claims is considered inclusive, not exclusive, and indicates at least one or any combination of the items it connects.

[0182] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the listed components. Those skilled in the art will appreciate that, unless otherwise specified, the use of the singular includes the plural. Therefore, the terms “a”, “an”, and “at least one” are used interchangeably in this application.

[0183] To better understand this teaching, and without limiting its scope in any way, all figures and other numerical values ​​representing quantities, percentages, or proportions used in the specification and claims, unless otherwise stated, should in all cases be understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters described in the following specification and appended claims are approximate values ​​and may vary depending on the desired characteristics sought. At a minimum, each numerical parameter should be understood based on the number of significant figures reported and using conventional rounding techniques.

[0184] In the specification and claims of this application, the verbs “comprise,” “include,” and “have,” and each of their various inflectional forms, are used to indicate that one or more objects of the verb are not necessarily an exhaustive enumeration of the components, elements, or parts of one or more subjects of the verb.

[0185] Other terms used herein are meant to be defined by their well-known meaning in the field.

[0186] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon review of the following embodiments, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the invention, as described above and claimed in the following claims, has been experimentally supported in the following embodiments.

[0187] It should be understood that, for clarity, certain features of the invention described in the context of a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be provided individually, or in any suitable sub-combination, or appropriately provided in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of those embodiments unless the embodiment does not function without these elements.

[0188] Example Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. These techniques are well explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, Ausubel, RM, 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 in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "CellBiology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed.(1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996); all of these are incorporated herein by reference. Other general references are provided in this document.

[0189] Materials and Methods Surface plasmon resonance (SPR) Cyclic peptides and, for example, Ub1 were measured using a BIACORE T100 instrument (GE Healthcare) equipped with a Biotin-CAPture kit S-series chip. K48 Ub2 and K48 Interactions between Ub4+. All buffer solutions used in the experiments contained 50 mM HEPES, pH 7.3, 150 mM NaCl, 0.05% Tween 20, 2 mM DTT, and 0.2% DMSO. K48 Ub2 and K48 Ub4 was loaded onto the SPR chip in approximately equal molar amounts, allowing different concentrations of cyclic peptides to flow through at 100 µL / s for 120 seconds, followed by initiating dissociation by flowing buffer through at 100 µL / s for another 700 seconds. The curves were fitted using the simplest two-state binding model. Assuming pseudo-first-order conditions, binding was fitted with a single exponential function; the dissociation curves were fitted with a single exponential function plus a drift term.

[0190] In vitro deubiquitination assay In vitro deubiquitination reaction in a solution containing 2 µM K48 Ub 2 / 4The reaction was performed in Tris buffer (50 mM Tris, 1 mM TCEP, pH 7.7) followed by the addition of a specific DUB (50 mM Tris, 0.5 mM EDTA, 1 mM TCEP, and 0.5 mg / ml ovalbumin, pH 7.5). The reaction mixture was incubated at 37°C, and the reaction was stopped at specified time points by taking aliquots of the sample, mixing them with 3× sample buffer, and boiling. Equal volumes of sample were loaded onto 14% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and detected with an anti-ubiquitin (anti-Ub) antibody. Bands were quantified using an Image Quant LAS 4000 (GE Healthcare).

[0191] In vitro proteasome degradation assay HA-α-globin-K48 linked to tetra-Ub and 150 nM proteasome (Enzo) was synthesized in 12.5 µL: 5.3 µM and incubated at 37 °C for 50 min in the presence of 2 mM ATP, 40 mM Tris, 2 mM DTT, and 5 mM MgCl2. The reaction was terminated by adding 3× sample buffer. The reaction product was loaded onto 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and detected with rabbit anti-HA antibody. Quantitative analysis was performed using ECL imaging software (Fuji).

[0192] Radioactive pulse tracking To analyze the inhibition of protein synthesis, cells were incubated with DMSO, the ribosome inhibitor actinone (CHX), or the polypeptides of this invention (SEQ ID No: 1-4). After 4 hours, the culture medium was replaced with a solution containing DMSO, ribosome inhibitor actinone (CHX), or the polypeptides of this invention (SEQ ID No: 1-4). 35 Cells were pulse-labeled for 4 hours in a medium containing S-methionine and cysteine ​​(20 µCi). The culture plates were thoroughly washed with ice-cold PBS, and proteins were extracted from the cells using trichloroacetic acid (TCA) precipitation. Radiometric readings were determined using a scintillation counter.

[0193] For the degradation inhibition assay, cells were used... 35 S-methionine and cysteine ​​(20 µCi) were labeled for 16 hours, followed by tracking in cold medium for 4 hours in the presence of DMSO, the proteasome inhibitor MG132, or the polypeptide of this invention (SEQ ID No: 1-4). The medium was then collected, and amino acids were extracted using TCA precipitation. Radioactive readings were determined using a scintillation counter.

[0194] Cellular uptake of the peptides of this invention HeLa cells were seeded in 96-well glass-bottomed (#1.5) plates and incubated for a specified time in the presence of DMSO or fluorescein-5-maleimide-peptide of the present invention (SEQ ID No: 1-4). The culture medium was then aspirated, and fresh culture medium was added to the wells. Live-cell imaging was performed using a Zeiss LSM-700 confocal microscope equipped with an environmental control module.

[0195] Cell viability assay and kinetic experiments To assess the effect of the cyclic peptide on HeLa cells, toxicity was determined using the MTT assay. Equal volumes of cells (8,000 cells / well) were seeded in triplicate in 96-well plates. After 24 hours, adherent cells were exposed to increasing concentrations of the cyclic peptide and incubated for another 24 hours. Subsequently, 10 µL of 12 mM MTT (Vybrant® MTT Cell Proliferation Assay Kit, ThermoFisher) was added to each well according to the manufacturer's instructions, and the cells were incubated at 37 °C for 4 hours. Then, 100 µL of SDS-HCl solution was added, mixed thoroughly, and incubated at 37 °C for 4 hours. Absorbance was measured at 590 nm, with 630 nm as the reference wavelength. The half-maximal inhibitory concentration (IC50) was determined. 50 The concentration is defined as the concentration at which cell growth is reduced by 50% compared to the value of untreated control cells, and is expressed as the mean ± standard deviation of relative activity.

[0196] Fluorescence-activated cell sorting (FACS) analysis Using the annexin V-FITC apoptosis assay kit (BD Biosciences), following the manufacturer's instructions, the induction of apoptosis in HeLa cells by cyclic peptide treatment was determined after 24 and 48 hours of dose-dependent incubation. 2 × 10⁶ cells were used. 5 Cells / well were seeded in 6-well plates and treated with an inhibitor in a dose-dependent manner for 24 / 48 hours. Increase in fluorescence intensity (indicating apoptosis levels in treated cells) was monitored using flow cytometry and compared with untreated cells containing DMSO (as a negative control) and MG132 (as a positive control).

[0197] Example 1 The cyclic peptide, starting from scratch, is tightly bound to the K48-linked Ub chain. To test the binding and specificity of the peptides of this invention, each peptide was prepared chemically for surface plasmon resonance (SPR) analysis. Each peptide was flowed through a Ub1 matrix immobilized on an SPR chip. K48 Ub2 and K48 Ub2, and calculate K D Value. It is expected that no peptide will bind to Ub1.

[0198] Example 2 Cyclic peptides protect the Ub chain from in vitro DUB cleavage. The inventors further investigated the effects of cyclic peptides on two types of DUBs: OTUB1, a K48-linked chain-specific DUB, and USP2, which uses K48-linked di- and tetra-Ub chains as substrates and non-specifically cleaves most Ub chains. The inhibitory effect on OTUB1 or USP2 cleavage was quantified.

[0199] Example 3 Cyclic peptides prevent ubiquitinated proteins from being degraded by the proteasome. The inventors investigated whether the binding of the cyclic peptide to the K48-Ub chain prevented the proteasome from recognizing and inhibiting the degradation of proteins labeled with these Ub chains. To test this, the inventors incubated α-globulin-K48-linked tetra-Ub and 26S proteasomes with or without the peptides of the present invention and monitored the degradation of the proteasomes.

[0200] Example 4 Cyclic peptides enter cancer cells To assess the feasibility of using the peptides of the present invention in cultured cells, the inventors tested whether the peptides could cross the cell membrane of human cells. The inventors synthesized fluorescein-labeled peptides of the present invention and used live-cell imaging to monitor and determine their entry into HeLa cells, USOS osteosarcoma cells, and U87 primary glioblastoma cells.

[0201] Example 5 The polypeptide of this invention promotes the accumulation of Ub conjugates. Under basal conditions, the level of Ub conjugates reflects the dynamic homeostasis between the enzymatic binding of Ub to proteins and the removal of the conjugated Ub moiety by DUB, followed by proteasome degradation. The inventors investigated in vitro whether the cyclic peptide of the present invention protects the Ub chain from DUB cleavage and whether it also prevents proteasome recognition. Both effects are expected to prevent degradation and lead to the accumulation of proteins labeled with the K48-Ub chain in cells. The inventors monitored the cellular levels of Ub conjugates in cells treated with the peptide of the present invention and determined whether treatment with the cyclic peptide of the present invention resulted in an increase or decrease in Ub conjugate levels. Similar effects are expected to be observed when cells are treated with the direct proteasome inhibitor MG132.

[0202] To directly measure the effect of the present invention's peptides on proteolysis, the inventors employed radioactive labeling of cellular proteins and then conducted follow-up experiments in the presence of either the present invention's peptides or the proteasome inhibitor MG132. Compared to untreated cells or cells treated with DMSO, protein degradation in cells treated with the present invention's peptides was expected to be significantly reduced, with degradation rates similar to those observed in the presence of MG132. A dose-dependent effect on cellular protein degradation was also proposed. The inventors verified the inhibitory effects of known proteasome substrates, proteins p53 and p27 (the true substrates of UPS), on protein degradation. In the presence of the present invention's peptides, p53 and p27 were predicted to accumulate over time to levels similar to those observed in the presence of MG132.

[0203] Example 6 The cyclic polypeptide of the present invention induces apoptosis The inventors further investigated whether the peptides of the present invention inhibited cell growth and induced apoptosis in cancer cells. To test this, HeLa cells were treated with each cyclic peptide, and cell viability was assessed using the MTT assay. The inventors predicted that the peptides of the present invention would effectively inhibit cell growth. The effect of the cyclic peptides of the present invention on apoptosis was assessed using FACS analysis. Similar to the direct proteasome inhibitor MG132, the peptides of the present invention were predicted to increase apoptosis after 24 and 48 hours of treatment.

[0204] Example 7 Synthesis and activity determination of Ub4a derivatives To enhance the efficacy of peptides, novel Ub4a structures and derivatives were created to address their challenging solubility issue, which may be a factor in their cellular permeability and efficacy. To this end, novel structures were designed that function under the same mechanism of action as Ub4a (SEQ ID NO:5) but with enhanced solubility and / or activity.

[0205] To this end, Ub4a (SEQ ID NO:5) variants with added arginine (R) and glycine (G) were designed and synthesized. Four (4) Ub4a derivatives were synthesized: Ub4a-CR: FQYWOYATGVCR (SEQ ID NO: 1); Ub4a-CRR: FQYWOYATGVCRR (SEQ ID NO: 2); Ub4a-GR: FQYWOYATGVCGR (SEQ ID NO: 3) and Ub4a-GRR: FQYWOYATGVCGRR (SEQ ID NO: 4). Figure 1 ).

[0206] After incubation with 8 µM and 5 µM peptides for 14 days, the efficacy of four different Ub4a derivatives was compared on two cell lines, MM1S and BTZ900, using the Annexin PI apoptosis assay.

[0207] Figures 2-3 The results showed that the apoptotic response in MM1S was better after incubation with Ub4a-CR (SEQ ID NO:2) and Ub4a-GRR (SEQ ID NO:4), while lower viability was observed in BTZ900 after incubation with Ub4a-CR (SEQ ID NO:2) and Ub4a-GRR (SEQ ID NO:4). Based on these results, further experiments were conducted using Ub4a-CR (SEQ ID NO:2) and Ub4a-GRR (SEQ ID NO:4).

[0208] Time-dependent responses were investigated in multiple myeloma (MM) cell lines MM1S and BTZ900, and the effects of Ub4a-CR (SEQ ID NO: 2) and Ub4a-GRR (SEQ ID NO: 4) on cell viability after incubation with 2 µM and 5 µM peptides for 5 and 10 days were examined.

[0209] Figures 4-6 All results showed that Ub4a-GRR (SEQ ID NO:4) had a significantly higher negative impact on cell viability.

[0210] To ensure that these modifications enhanced peptide efficacy, the effects of Ub4a (SEQ ID NO:5) and Ub4a GRR (SEQ ID NO:4) on cell viability were compared after 8 days of incubation with MM1S and BTZ900 cell lines at 5 µM or 2 µM. The results surprisingly showed that Ub4a-GRR (SEQ ID NO:4) exhibited significantly improved efficacy compared to Ub4a. Figure 7 ).

[0211] Furthermore, the stability of Ub4a GRR (SEQ ID NO:4) in human plasma was investigated by incubating Ub4a-GRR (SEQ ID NO:4) in human plasma at a concentration of 0.5 mg / ml at 37°C for 1 hour. Peptide extraction was then performed by precipitating plasma proteins in cold acetone at -20°C for 1 hour. The samples were then centrifuged at 14000 RPM for 10 minutes. The supernatant was collected and analyzed by HPLC. The recovery and stability of Ub4a-GRR in plasma were assessed by comparing the Ub4a-GRR (SEQ ID NO:4) peak with a control Ub4a-GLR diluted in 5% DMSO water using the same incubation and extraction method. Stability was observed for at least 8 hours. Figure 8 ).

[0212] To calculate the half-maximum effective concentration (EC) 50 The study investigated concentration-related responses by detecting cell viability after 12 days of treatment with different concentrations. 50 The calculation is based on Figure 9 The results shown were obtained with a value of approximately 0.44. Therefore, the EC of the "mother peptide" Ub4a (SEQ ID NO:5) was discovered. 50 Greater than 3µM (data not shown). Therefore, it can be concluded that Ub4a-GRR is likely an improved derivative of Ub4a, at least in terms of inhibitory activity.

[0213] Given the promising in vitro efficacy data described above, and the enhanced efficacy / activity compared to the "parent peptide" Ub4a (SEQ ID NO:5), it is crucial to assess the safety profile of Ub4a-GRR (SEQ ID NO:4) to ensure its suitability for further development. This experiment provides key insights into the potential toxicities associated with Ub4a-GRR and helps identify any dose-limiting toxicities.

[0214] Therefore, a maximum tolerated dose (MTD) study was conducted. The primary objective of the MTD study was to determine the highest dose of Ub4a-GRR (SEQ ID NO:4) that could be safely administered without causing serious / adverse reactions. Establishing the MTD is a critical step in the drug development process because it helps define the dose range for subsequent preclinical and clinical studies.

[0215] In this study, 20 female NOD / SCID mice were used and divided into six groups, with one, three, or six animals per group. The treatment group was treated with Ub4a-GRR for 5 days, after which morbidity and mortality were observed, and body weight and food consumption were assessed. Then, on day 6, the animals were euthanized and gross autopsies were performed to detect abnormalities.

[0216] Weight changes such as Figure 10 As shown. Based on the results, the MTD was not reached because Ub4a-GRR did not cause any significant damage at any of the tested doses (5-45 mg / kg SC and 0.5-2 mg / kg IV).

[0217] according to Figure 11 The results shown evaluated the pharmacokinetic characteristics of Ub4a-GRR after intravenous (IV) bolus, subcutaneous (SC), intraperitoneal (IP), and intramuscular (IM) administration.

[0218] Notably, the SC administration route showed a significant advantage because it was the only administration route where the compound remained detectable after 8 hours, indicating prolonged systemic exposure and a prolonged half-life. Figures 10-11 Given this prolonged duration and advantageous characteristics, SC administration appears to be the preferred route for further in vivo studies. Furthermore, due to its rapid absorption, twice-daily SC administration of Ub4a-GRR (SEQ ID NO:4) may be the optimal option for maintaining adequate plasma levels over time.

[0219] Furthermore, the anticancer effects (one or more) of the peptides of the present invention were detected in vivo. Briefly, UB4a (SEQ ID NO:5) was administered to mice with MM tumors resistant to bortezomib. SCID NOD mice were implanted with MM tumors derived from the BTZ900 cell line, with 10 mice in each group receiving different treatments. The UB4a treatment group received three different doses of UB4a (2, 5, and 10 mg / kg) intramuscularly daily for 40 days. Tumor volume changes were monitored throughout the study to assess the effect of treatment on tumor growth. The results were compared with the vector control group and the bortezomib treatment group. The results showed that tumor growth was significantly inhibited after 22 days, with the UB4a peptide (SEQ ID NO:5) administered... Figure 13 After 40 days of treatment, a reduction in tumor volume of up to 44% was observed.

[0220] In addition, a study was conducted using comparable negative controls as described above. Figure 13 The in vivo efficacy of UB4a-GRR (SEQ ID NO:4) in a KMS-12-BM-BPR subcutaneous multiple myeloma tumor model in female NSG mice (bortezomib-resistant MM tumors) was examined. Three mice in a group received a subcutaneous (SC) dose of 2 mg / ml daily. Tumor volume changes were monitored throughout the study to assess the effect of treatment on tumor growth. Results were compared with the untreated group, showing that tumor growth was significantly inhibited 10 days after initial administration, and tumor volume was reduced by up to 63% after 24 days of treatment. Figure 14In addition, a second control group was used, which included CP-15, a known inhibitor that binds to Ub K63 (as described by Vamisetti et al., Nature Communications (2022) 13:6174). Results showed that CP-15 activity was comparable to the untreated control group, and both were found to be significantly worse than Ub4a-GRR. Figure 14 Furthermore, CP-15 was found to have rather low plasma solubility (data not shown). In addition, in vivo (mouse) pharmacokinetic studies showed that intravenous injection of CP-15 was lethal, with no detectable levels in plasma (data not shown). Subsequent in vitro studies indicated that CP-15 precipitates in plasma, potentially contributing to its unfavorable pharmacokinetic profile.

[0221] These results highlight that the Ub4a-GRR peptide is significantly improved in vivo compared to its precursor Ub4a and other suitable controls (such as CP-15).

[0222] While certain features of the invention have been described and illustrated herein, many modifications, substitutions, alterations, and equivalents will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations that conform to the true spirit of the invention.

Claims

1. A polypeptide comprising an amino acid sequence selected from FQYWOYATGVCR (SEQ ID NO:1); FQYWOYATGVCRR (SEQ ID NO:2); FQYWOYATGVCGR (SEQ ID NO:3); and FQYWOYATGVCGRR (SEQ ID NO:4).

2. The polypeptide according to claim 1, wherein at least one amino acid residue of the polypeptide is methylated.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises no more than 16 amino acid residues.

4. The polypeptide according to any one of claims 1 to 3, wherein the amino acid at position 1 is a D amino acid.

5. The polypeptide according to any one of claims 1 to 4, wherein the amino acid at position 1 is conjugated to a cyclized molecule.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide is a cyclic polypeptide prepared using a cyclized molecule containing a halogen.

7. The polypeptide according to claim 6, wherein the cyclized molecule is selected from chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl or Cl2SAc.

8. The polypeptide according to any one of claims 1 to 7, characterized in that... It possesses: cell penetration ability, ubiquitin (Ub) binding ability, or a combination thereof.

9. The polypeptide according to claim 8, compared with the control, has increased affinity for Ub.

10. The polypeptide of claim 9, wherein the control is a polypeptide consisting of the amino acid sequence described in SEQ ID NO:

5.

11. The polypeptide according to any one of claims 1 to 10, wherein it is a cyclic polypeptide.

12. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 11 and at least one acceptable carrier.

13. The pharmaceutical composition according to claim 12, for treating cancer in a subject in need.

14. A method for reducing the deubiquitination activity of cells, the method comprising contacting the cells with an effective amount of a polypeptide comprising the amino acid sequence described in SEQ ID NO:1-4.

15. A method for improving or treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, the polypeptide comprising the amino acid sequences described in SEQ ID NO:1-4.

16. The method of claim 15, wherein the application is subcutaneous.

17. The method of claim 15 or 16, wherein the application comprises multiple applications.

18. The method of claim 17, wherein the multiple applications comprise at least two applications, spaced 4 to 12 hours apart.

19. The method of claim 17 or 18, wherein the multiple applications comprise at least two applications, spaced 4 to 10 hours apart.

20. The method according to any one of claims 14 to 19, wherein the polypeptide: (i) has an affinity K of 0.05-100 nM. D (i) Combining with Ub; (ii) Containing no more than 16 amino acid residues; or both (i) and (ii).

21. The method according to any one of claims 14 to 20, wherein the amino acid at position 1 of the polypeptide is a D amino acid.

22. The method according to any one of claims 14 to 21, wherein the amino acid at position 1 of the polypeptide is conjugated to a cyclized molecule.

23. The method according to any one of claims 14 to 22, wherein the polypeptide is a cyclic polypeptide prepared using a cyclized molecule containing a halogen.

24. The method according to claim 23, wherein the cyclized molecule is selected from chloroacetyl chloride, 3-chlorobenzoyl (3-ClBz), 4-chlorobenzoyl (4-ClBz), or Cl2SAc.

25. The method according to any one of claims 14 to 24, wherein the polypeptide has: cell penetration ability, Ub binding ability, or a combination thereof.

26. The method according to any one of claims 14 to 25, wherein the polypeptide has increased affinity for Ub compared to a control.

27. The method according to any one of claims 20 to 26, wherein the Ub is polymeric Ub.

28. The method of claim 26, wherein the polymerized Ub comprises a Ub monomer (K) linked at its K48 position. 48 Ub).

29. The method of claim 28, wherein the polypeptide has increased pro-apoptotic activity compared to a control.

30. The method according to any one of claims 14 to 29, wherein the polypeptide is a cyclic polypeptide.

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