CN119174818A

CN119174818A - OTUB2 inhibitor and application thereof in tumor immunity

Info

Publication number: CN119174818A
Application number: CN202410804288.1A
Authority: CN
Inventors: 黄承浩; 任文峰; 徐子龙; 常亚婷; 吴红宁; 徐晨航; 赵敏; 张军; 夏宁邵
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2023-06-21
Filing date: 2024-06-20
Publication date: 2024-12-24
Also published as: WO2024260410A1

Abstract

The present invention belongs to the field of biomedicine and relates to OTUB2 inhibitors and their applications in tumor immunity. Specifically, the present invention provides uses and methods of OTUB2 inhibitors for enhancing anti-tumor immune responses, reducing tumor immunosuppression, and/or treating tumors. In addition, the present invention also provides small molecule inhibitors targeting OTUB2.

Description

OTUB2 inhibitor and application thereof in tumor immunity

Technical Field

The invention belongs to the field of biological medicine, and relates to an OTUB2 inhibitor and application thereof in tumor immunity.

Background

The ability to enhance anti-tumor immunity in tumor microenvironments (Tumor microenvironment, TME) is critical to tumor control. Cancers often evolve different mechanisms that enable them to evade destruction of the immune system. One of the escape mechanisms involves interactions between anti-tumor cytotoxic T lymphocytes (Cytotoxic T lymphocytes, CTLs) and tumor cells. Tumor cells resist or attenuate CTLs attack by a variety of tumor cell intrinsic mechanisms, including mechanisms such as upregulation of inhibitory immune checkpoint receptors, impaired tumor antigen presentation, and altered secretion and metabolic pathways of inhibitory cytokines or ligands.

Otubin-2 (OTUB 2) is a deubiquitinase (Deubiquitinating enzyme, DUB), belongs to the Ovarian Tumor (OTU) protein superfamily (Nanao MH,Tcherniuk SO,Chroboczek J,Dideberg O,Dessen A,Balakirev MY.Crystal structure of human otubain 2.EMBO Rep 5,783-788(2004))., and although the molecular biology of OTUB2 protein has been studied, no report about the function of OTUB2 in regulating tumor immunity has been found yet.

Disclosure of Invention

It has been reported in the art that OTUB2 is expressed at a higher level in human malignant tumors and at a lower level in organs throughout the body, see e.g. https:// www.proteinatlas.org/ENSG00000089723-OTUB2/tissue, but there is no report that OTUB2 is related to tumor immunity. The invention discovers that OTUB2 has the function of immune regulation for the first time and plays a key role in tumor microenvironment. Targeting OTUB2 helps to improve tumor microenvironment and improve anti-tumor immunity, thereby exerting tumor treatment effect. Furthermore, it is notable that no OTUB 2-specific small molecule inhibitors have been developed. The invention provides a specific small molecule inhibitor targeting OTUB2 for the first time based on an OTUB2 binding pocket structure. The following invention is thereby provided.

Application of OTUB2 inhibitor in tumor immunity

In one aspect, the invention provides the use of an OTUB2 inhibitor in the manufacture of a medicament for use in anti-tumour immunotherapy. Also provided are methods for anti-tumor immunotherapy comprising administering to a subject in need thereof an effective amount of an OTUB2 inhibitor.

In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and/or reducing tumor immunosuppression in a subject.

In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell (e.g., cd8+ T cell) -mediated tumor killing in a subject.

In certain embodiments, the anti-tumor immunotherapy comprises promoting immune cell function in a subject. The immune cell function is measured, for example, by one or both of (i) activation of T cells (e.g., cd8+ T cells and/or cd4+ T cells), NK cells, or any combination thereof, and (ii) proliferation of T cells (e.g., cd8+ T cells and/or cd4+ T cells), NK cells, or any combination thereof. In certain embodiments, the activation is measured as an increased level of a cytokine (e.g., IFN-gamma, IL-2, TNF-alpha, etc.). In certain embodiments, the immune cell function is measured, for example, by activation and/or proliferation of cd8+ T cells.

In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor.

OTUB2 inhibitors

The OTUB2 inhibitors described herein may exert their inhibitory effect by any mechanism, for example by inhibiting the expression of a gene at the RNA or protein level (e.g., reducing or inhibiting transcription of a gene, and/or reducing or inhibiting translation of an mRNA product of the gene), or by inhibiting or blocking the biological activity of a protein product of the gene, or by degrading a protein product of the gene or disrupting its stability, for example.

In certain embodiments, the OTUB2 inhibitor is capable of inhibiting or down-regulating expression of an OTUB2 gene, inhibiting or blocking activity of an OTUB2 protein, or degrading an OTUB2 protein.

In certain embodiments, the inhibitor inhibits or blocks a biological function of the OTUB2 protein. In certain embodiments, the biological function of the OTUB2 protein comprises deubiquitinase activity. In certain embodiments, the biological function of the OTUB2 protein includes binding to the substrate protein and removing ubiquitination thereof (i.e., interacting with the substrate protein to stabilize the substrate protein from degradation by ubiquitination). Thus, by inhibiting or blocking the above-described functions of the OTUB2 protein, the degradation of the substrate protein can be caused to inhibit the expression of the substrate protein.

In certain embodiments, the inhibitor comprises a small molecule compound. Herein, the expression "small molecule compound" refers to an organic non-protein compound. In certain embodiments, the small molecule compound has a molecular weight of no greater than 1500Da. The small molecule compound can inhibit the functions of the above genes or expression products thereof. The small molecule compounds may be obtained by screening an existing library of small molecule compounds (e.g., chem Bridge, chem Div, inter Bio Screen, LIFE CHEMICALS, specs, or Vitas-m) and assaying the compounds for inhibitory activity on the expression level of OTUB2, binding activity to OTUB2 protein, or inhibitory ability to the biological function of OTUB2 protein.

In certain embodiments, the small molecule compound binds to or interacts with an OTUB2 protein, e.g., as determined by BLI techniques (e.g., octet apparatus) or SPR techniques (e.g., biacore apparatus). In certain embodiments, the small molecule compound binds to the catalytic pocket of an OTUB2 protein. In certain embodiments, the small molecule compound binds to an OTUB2 protein, blocking the interaction of OTUB2 with a substrate protein (e.g., PD-L1), thereby resulting in ubiquitinated degradation of the substrate protein (e.g., PD-L1).

In certain embodiments, the small molecule compound is selected from a compound represented by formula (I):

Wherein:

R ¹ is-C (O) X, X is hydroxy or C ₁-C₄ alkoxy, or is C3-7 monocyclic cycloalkyl, 4-6 membered monocyclic heterocyclyl or 5-6 membered monocyclic heteroaryl, wherein the 4-6 membered monocyclic heterocyclyl and 5-6 membered monocyclic heteroaryl each have 1-3 ring heteroatoms independently selected from N, O and S;

n is an integer selected from 1 to 12;

R ² is

Wherein R ^2a is selected from (C1-C4) alkyl, (C2-C4) alkenyl, or-C (O) Y, Y is hydroxy or (C1-C4) alkoxy;

Wherein R ^2b、R^2c、R^2d is each independently selected from halogen (e.g., -F, -Cl, -Br or-I), nitro, amino, hydroxy, mercapto, (C1-C4) alkyl or (C1-C4) alkoxy, preferably R ^2b、R^2c、R^2d are different from each other;

Wherein m is 1, 2,3 or 4.

In certain embodiments, R ¹ is-C (O) X, X is hydroxy or (C1-C4) alkoxy, and n is an integer selected from 1-12.

In certain embodiments, n is an integer selected from 1 to 11, for example 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.

In certain embodiments, R ¹ is-C (O) X, X is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, X is hydroxy, methoxy, or ethoxy. In certain embodiments, X is hydroxy.

In certain embodiments, R ¹ is C3-7 monocyclic cycloalkyl (e.g., C3-6 cycloalkyl, C3-5 cycloalkyl, C3-4 cycloalkyl, C4-6 cycloalkyl, or C4-5 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), 4-6 membered monocyclic heterocyclyl (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, or piperidinyl), or 5-6 membered monocyclic heteroaryl (imidazolyl, triazolyl, tetrazolyl, pyrazolyl, thiophenyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl), and n is 1, 2, 3, or 4. In certain embodiments, n is 1 or 2, preferably n is 1.

In certain embodiments, R ¹ is C3-7 monocyclic cycloalkyl, 4-6 membered monocyclic heterocyclyl, or 5-6 membered monocyclic heteroaryl, each having 1 ring heteroatom selected from N, O and S. In certain embodiments, R ¹ is C3-7 monocyclic cycloalkyl or 4-to 6-membered monocyclic heterocyclyl. In certain embodiments, R ¹ is C3-6 monocyclic cycloalkyl or 4-to 6-membered monocyclic heterocyclyl. In certain embodiments, R ¹ is cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, or a heterocyclyl selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, or piperidinyl, such as tetrahydrofuranyl or pyrrolidinyl. In certain embodiments, R ¹ is tetrahydrofuran.

In certain embodiments, R ² isWherein R ^2a is selected from methyl, ethyl, n-propyl, isopropyl, ethenyl, propenyl, or-C (O) Y, Y is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy. In certain embodiments, R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) Y, Y is hydroxy, methoxy, or ethoxy. In certain embodiments, R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) OH. In certain embodiments, m is 1,2, or 3.

In certain embodiments, R ² isWherein R ^2b、R^2c、R^2d is each independently selected from nitro, hydroxy, (C1-C4) alkyl or (C1-C4) alkoxy. In certain embodiments, each R ^2b、R^2c、R^2d is independently selected from nitro, hydroxy, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy. In certain embodiments, each R ^2b、R^2c、R^2d is independently selected from nitro, hydroxy, methyl, ethyl, methoxy, ethoxy. In certain embodiments, R ^2b、R^2c、R^2d are different from each other. In certain embodiments, R ^2b、R^2c、R^2d is nitro, hydroxy, methoxy, respectively. In certain embodiments, R ² isR ^2b、R^2c、R^2d is as defined above.

In certain embodiments, the compound has a structure represented by formula (Ia):

wherein R ¹、R^2a, m and n are as defined in formula (I).

In certain embodiments, in formula (Ia), R ¹ is-C (O) X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy), preferably, n is an integer selected from 1 to 11, for example, 1,2,3,4, 5, 6, 7, 8, 9 or 10.

In certain embodiments, in formula (Ia), R ¹ is C3-7 monocyclic cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) or 4-to 6-membered monocyclic heterocyclyl (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, or piperidinyl) having 1 ring heteroatom selected from N, O and S, preferably R ¹ is tetrahydrofuranyl or pyrrolidinyl, preferably n is 1 or 2, for example 1.

In certain embodiments, in formula (Ia), R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) Y, Y is hydroxy, methoxy, or ethoxy, preferably R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) OH.

In certain embodiments, in formula (Ia), m is 1, 2, or 3.

In certain embodiments, in formula (Ia):

r ¹ is-C (O) X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably X is hydroxy, methoxy or ethoxy), preferably n is an integer selected from 1 to 11, for example 1,2,3,4,5, 6, 7, 8, 9 or 10;

R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) Y, Y is hydroxy, methoxy, or ethoxy, preferably R ^2a is selected from methyl, ethyl, vinyl, propenyl, or-C (O) OH, and m is 1,2, 3, or 4, for example 1,2, or 3.

In certain embodiments, the compound is selected from the group consisting of:

6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid(CAS No.:300558-22-9);

2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid( Such as available from OTAVAchemicals company under the accession number 1663785);

2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid(CAS No.:313646-95-6);

11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid(CAS No.:374543-72-3);

11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid( For example, OTAVAchemicals company, cat No. 4349908) is available.

In certain embodiments, the small molecule compounds described herein may be chemically synthesized or purchased commercially.

In certain embodiments, the inhibitor inhibits its biological function by inhibiting the expression level of an OTUB2 gene. In such embodiments, the determination of the expression level may be performed at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, northern blotting, PCR, RT-PCR, or real-time (real) RT-PCR. Methods for determining expression at the protein level include, but are not limited to, western blotting or polyacrylamide gel electrophoresis in combination with protein staining techniques such as coomassie brilliant blue or silver staining, mass spectrometry, ELISA, and the like.

In certain embodiments, the inhibitor is selected from an antibody, an RNA interfering agent (e.g., a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a microrna (miRNA)) or an antisense oligonucleotide, a gene editing system (e.g., CRISPR-Cas system), or a small molecule compound.

In some embodiments, the inhibitor is an antibody. In certain embodiments, the antibody is a blocking antibody (blocking antibody) or a neutralizing antibody (neutralizing antibody). In certain embodiments, the antibody is capable of binding to OTUB2 protein and directly interfering with its biological function.

In other embodiments, the inhibitor is a nucleic acid inhibitor, including an RNA interfering agent or an antisense oligonucleotide. The RNA interference agent inhibits the expression of the gene. The antisense oligonucleotide specifically binds to the gene or its mRNA product to inhibit expression.

As used herein, the expression "RNA interference agent" refers to any agent that inhibits the expression of a target gene by an RNA interference (RNAi) mechanism. "RNA interference (RNAi)" is an evolutionarily conserved process in which the expression or introduction of RNA of the same or highly similar sequence as a target gene results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from the target gene, thereby inhibiting the expression of the target gene. Thus, one skilled in the art will recognize that small interfering RNA (siRNA) or small RNA (microRNA, miRNA) molecules may be designed based on the selection of target sequences for a particular gene or mRNA transcribed from that gene. The siRNA or miRNA molecule can interfere with transcription, translation or post-transcriptional and post-translational modification of genes, thereby affecting the expression of proteins.

In addition, to prolong the inhibition of siRNA on target gene expression, a specific pair of oligonucleotide sequences can be designed, annealed and cloned into a vector, and the transcription product of the recombinant vector, namely short hairpin RNA (short HAIRPIN RNA, SHRNA), can be folded and paired to form a stem-loop (stem-loop) structure with the stem length of 19-21 bases, wherein the 19-21 bases are a specific sequence derived from target gene mRNA, and precursors of the stem-loop structure are rapidly cut in cells to form functional siRNA. The siRNA formed by shearing shRNA expressed by the vector has the characteristics of stable expression quantity and long lasting time, thereby being capable of causing long-acting inhibition of target gene expression.

In this context, shRNA refers to short hairpin RNAs (short HAIRPIN RNA) comprising two short inverted repeats, separated by a stem-loop (loop) sequence in the middle, constituting a hairpin structure, controlled by the polIII promoter. Then, 5-6T's were ligated as transcription terminators for RNA polymerase III. Cloning of the siRNA sequence into a plasmid vector allows for the delivery of "small interfering RNAs" (siRNAs) in vivo. When delivered into an animal, the hairpin sequence is expressed to form a stem-loop structure that is cleaved into functional siRNAs that exert gene silencing effects.

Herein, siRNA refers to SMALL INTERFERING RNA, a small RNA molecule consisting of about 21-25 nucleotides, processed by Dicer (an enzyme specific for double-stranded RNA in RNAASEIII family), which is the main member of siRISC, triggering silencing of the complementary target mRNA.

In this context, micrornas (mirnas) refer to a naturally occurring class of non-coding RNA molecules, approximately 21-25 nucleotides in length, which, based on complementarity to the sequence of the target mRNA, are capable of post-transcriptional expression regulation of the gene by causing degradation or inhibiting translation of the target mRNA by base pairing specific for the target mRNA. mirnas can be referenced to the miRBase database.

In certain embodiments, the RNA interfering agent is selected from small interfering RNAs (siRNA), small hairpin RNAs (shRNA), or micrornas (miRNA).

In this context, the expression "antisense oligonucleotide" refers to a molecule complementary to a sense nucleic acid, for example complementary to the coding strand of an OTUB2 gene or complementary to the mRNA sequence of an OTUB2 gene. Thus, the antisense oligonucleotide can form hydrogen bonds with (i.e., anneal to) the sense nucleic acid.

The antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biostability of the molecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, antisense nucleic acids can be produced biologically using expression vectors that contain a target nucleic acid subcloned into them, and RNA transcribed from the inserted nucleic acid will have an antisense orientation with the target nucleic acid.

In certain embodiments, the antisense oligonucleotide is an antisense RNA or an antisense DNA.

After the RNA interfering agent or antisense oligonucleotide is obtained, the inhibitory activity of the RNA interfering agent or antisense oligonucleotide on OTUB2 expression levels can be further determined using the methods described above.

In certain exemplary embodiments, the inhibitor is an shRNA, e.g., comprising a sequence set forth in SEQ ID NO. 3 or 4.

In other embodiments, the inhibitor is a gene editing system. Exemplary gene editing systems include ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas, and other site-specific nuclease technologies. The use of gene editing systems to knock down or knock out a target gene (e.g., OTUB 2) is well known to those skilled in the art. In certain embodiments, the inhibitor is a CRISPR/Cas, such as CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas12b, CRISPR/Cas13a, or CRISPR/Cas14a.

In other embodiments, the inhibitor is PROTAC (protein degradation targeting chimera). PROTAC is a technique for selectively degrading a target protein by ubiquitin-proteinase system. Typically, PROTAC comprises, as heterobifunctional molecules or bifunctional compounds, an E3 ubiquitin ligase binding moiety (i.e., a ligand for E3 ubiquitin ligase) and a target protein binding moiety (i.e., a protein/polypeptide targeting ligand) such that the target protein/polypeptide is placed in proximity to ubiquitin ligase to effect degradation (and inhibition) of the protein. The difunctional compound may also contain a chemical linker that connects the two functional groups.

In certain embodiments, the PROTAC is a bifunctional compound comprising an E3 ubiquitin ligase binding moiety and an OTUB2 protein binding moiety.

In certain embodiments, the E3 ubiquitin ligase binding moiety is a small molecule. In certain embodiments, the E3 ubiquitin ligase binding moiety targets an E3 ubiquitin ligase selected from the group consisting of spell-lindera (VLM), human cerebellar protein (CLM), mouse double-micro homolog 2 (MLM), and IAP (ILM).

In certain embodiments, the OTUB2 protein binding moiety is a small molecule. In certain embodiments, the OTUB2 protein binding moiety is selected from the group of small molecule compounds described herein.

Administration of OTUB2 inhibitors

The OTUB2 inhibitors described herein may be formulated into dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration applications may include sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediamine tetraacetic acid (EDTA), buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Dosage forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil ELTM, or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption, for example, aluminum monostearate and gelatin.

The OTUB2 inhibitors described herein may be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection and injectable concentrated solutions), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. One preferred dosage form is an injection.

The OTUB2 inhibitors described herein may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. For many therapeutic uses, however, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose.

The OTUB2 inhibitors described herein may be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The subject described herein includes any individual that can develop an immune response. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

Indication of disease

The purpose of anti-tumor immunotherapy is to increase the ability of the immune system to recognize and kill tumors, and to overcome the immunosuppressive tumor microenvironment. The term "tumor" includes any form of cancer, including solid tumors or hematological tumors.

In certain embodiments, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

In certain embodiments, the tumor expresses OTUB2 or is OTUB2 positive. In this context, OTUB2 positivity can be determined at the nucleic acid level or at the protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time (real) RT-PCR. Methods for determining expression at the protein level include, but are not limited to, immunological assays.

OTUB2 small molecule inhibitors

Small molecule inhibitors specific for OTUB2 have not been developed. The invention provides a specific small molecule inhibitor targeting OTUB2 for the first time based on the OTUB2 binding pocket structure, wherein the small molecule inhibitor is selected from a compound shown in a formula (I) or pharmaceutically acceptable salt or ester or stereoisomer:

Wherein:

n is an integer selected from 1 to 12;

R ² is

Wherein m is 1, 2,3 or 4.

wherein R ¹、R^2a, m and n are as defined in formula (I).

In certain embodiments, in formula (Ia), m is 1, 2, or 3.

In certain embodiments, in formula (Ia):

In certain embodiments, the compound is selected from the group consisting of:

In one aspect, the invention provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In one aspect, the present invention provides the use of the above compound or a pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition thereof as an OTUB2 inhibitor. In certain embodiments, the OTUB2 inhibitor is used to inhibit or block the activity of an OTUB2 protein in vitro. In certain embodiments, the OTUB2 inhibitor is for non-therapeutic purposes. The invention also provides a method of inhibiting OTUB2 activity in a cell in vitro comprising contacting the cell with a compound as described above or a pharmaceutically acceptable salt or ester or stereoisomer thereof.

In one aspect, the present invention provides the use of a compound as described above or a pharmaceutically acceptable salt or ester or stereoisomer or pharmaceutical composition thereof in the manufacture of a medicament for the prevention and/or treatment of a tumor. Also provided are methods for preventing and/or treating tumors comprising administering to a subject in need thereof an effective amount of the above-described compounds or pharmaceutically acceptable salts or esters or stereoisomers or pharmaceutical compositions thereof.

In certain embodiments, the above-described compounds or pharmaceutically acceptable salts or esters or stereoisomers or pharmaceutical compositions thereof are useful in anti-tumor immunotherapy.

In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and/or reducing tumor immunosuppression.

In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell (e.g., cd8+ T cell) -mediated tumor killing in a subject. In certain embodiments, the anti-tumor immunotherapy comprises promoting immune cell function in a subject. The immune cell function is measured, for example, by one or both of (i) activation of T cells (e.g., cd8+ T cells and/or cd4+ T cells), NK cells, or any combination thereof, and (ii) proliferation of T cells (e.g., cd8+ T cells and/or cd4+ T cells), NK cells, or any combination thereof. In certain embodiments, the activation is measured as an increased level of a cytokine (e.g., IFN-gamma, IL-2, TNF-alpha, etc.). In certain embodiments, the immune cell function is measured, for example, by activation and/or proliferation of cd8+ T cells.

In certain embodiments, the tumor comprises a solid tumor or a hematological tumor.

The above-mentioned compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof may be in any form known in the medical arts, and may be in the form of, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, lyophilized powders), inhalants, sprays and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use.

The above-described compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof may be present in the pharmaceutical compositions in unit dosage form for convenient administration.

The above-described compounds, or pharmaceutically acceptable salts or esters or stereoisomers thereof, may be administered by any suitable method known in the art, including, but not limited to, oral, rectal, parenteral or topical administration.

One exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and the like. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, liquid dosage forms for oral administration can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents and the like. Solid dosage forms for oral administration include capsules, tablets, pills, troches, powders, granules and the like. In addition to the active ingredient, the solid dosage forms may contain pharmaceutically acceptable inert excipients or carriers, such as fillers (e.g., lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, crospovidone, and calcium sulfate), binders (e.g., carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g., cetyl alcohol and glyceryl monostearate), disintegrants (e.g., agar-agar, calcium carbonate, starch, alginic acid, sodium carboxymethyl cellulose, sodium carboxymethyl starch), lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof.

The above compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof may also be administered by non-oral routes.

Thus, another exemplary route of administration is parenteral, e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal and infusion. The dosage form for parenteral administration may be an injectable preparation, including injectable solutions, injectable sterile powders or injectable concentrated solutions. In addition to the active ingredient, the injectable dosage form may contain pharmaceutically acceptable carriers such as sterile water, ringer's solution and isotonic sodium chloride solution, and may also contain suitable additives such as antioxidants, buffers and bacteriostats depending on the nature of the drug.

Another exemplary route of administration is topical administration, such as transdermal administration (e.g., via a transdermal patch or iontophoresis device), intraocular administration, or intranasal or inhalation administration.

Another exemplary route of administration is rectal administration. The dosage form for rectal administration may be a suppository.

In addition, other carrier materials and modes of administration known in the pharmaceutical arts may be used. Pharmaceutical compositions comprising the above compounds, or pharmaceutically acceptable salts or esters or stereoisomers thereof, may be prepared by any well known pharmaceutical process, such as effective formulations and methods of administration. The above considerations regarding effective formulations and methods of administration are well known in the art and are described in standard textbooks.

Drug screening

In one aspect, the invention provides a method of screening for a drug for anti-tumor immunotherapy comprising the step of screening for an OTUB2 inhibitor.

In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and/or reducing tumor immunosuppression. In certain embodiments, the anti-tumor immunotherapy comprises promoting T cell mediated tumor killing in a subject.

In certain embodiments, the screening step is performed in vitro.

In certain embodiments, the screening step comprises:

(1) Determining the expression level or biological activity of OTUB2 in the presence of a test agent;

(2) Comparing the assay result of step (1) with the expression level or biological activity of OTUB2 measured in the absence of the test agent;

Wherein if the assay result of step (1) is reduced compared to the assay result in the absence of the test agent, it indicates that the test agent is an inhibitor of OTUB2 and is a candidate for anti-tumor immunotherapy.

In certain embodiments, the biological activity of OTUB2 comprises deubiquitinase activity. In certain embodiments, the biological activity of the OTUB2 comprises binding to a substrate protein and removing ubiquitination thereof (i.e., interacting with the substrate protein to stabilize the substrate protein from ubiquitination). In certain embodiments, the substrate protein is PD-L1. Those skilled in the art understand that OTUB2 exists in a variety of substrate proteins, and PD-L1 is only one exemplary substrate protein for use in detecting the ability of an OTUB2 inhibitor to inhibit OTUB2 biological activity. However, the growth of the knocked-out OTUB2 tumor cells was inhibited regardless of whether PD-L1 expression was high or low, indicating that the modulation of tumor immunity by OTUB2 inhibitors is not limited by the PD-L1 pathway, i.e., the use of OTUB2 inhibitors is not limited by the expression of PD-L1.

In certain embodiments, the screening step comprises:

(i) Reagents that interact with the OTUB2 protein are screened as test reagents, for example by BLI techniques (e.g. Octet apparatus) or SPR techniques (e.g. Biacore apparatus);

(ii) Detecting the deubiquitinase activity of an OTUB2 protein on a substrate protein in the presence of the test agent obtained in step (i);

(iii) Comparing the assay result of step (ii) with the activity determined in the absence of the test agent;

wherein if the assay result of step (ii) is reduced compared to the assay result in the absence of the test agent, it indicates that the test agent is an inhibitor of OTUB2 and is a candidate for anti-tumor immunotherapy.

In certain embodiments, the detecting the deubiquitinase activity of an OTUB2 protein on a substrate protein comprises:

(1) Contacting a test agent with an OTUB2 protein and a ubiquitinated substrate protein;

(2) Determining the level of ubiquitinated substrate protein;

(3) Comparing the above assay result with the level measured in the absence of the test agent;

(4) Selecting a test agent capable of increasing the level of the ubiquitinated substrate protein.

(1) Contacting the test agent with a cell (e.g., a mammalian cell, such as LL/2 or B16-F10) capable of expressing the substrate protein;

(2) Determining the protein level of the substrate protein;

(3) Comparing the assay result of step (2) with the level determined in the absence of the test agent;

(4) Selecting a test agent capable of inhibiting the level of a substrate protein.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. For a better understanding of the present invention, definitions and explanations of the relevant terms are provided below.

As used herein, the term "Otubin-2 (OTUB 2)" may be of human origin or may be a homologous gene from other species (e.g., non-human mammals, fish, reptiles, or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, zebras, etc.). An exemplary amino acid sequence of human OTUB2 can be found in SEQ ID NO. 7.

As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated). For "anti-tumor effect" includes, but is not limited to, for example, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, a decrease in cancer cell survival, or an improvement in a variety of physiological symptoms associated with a cancer condition.

As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount of any toxic or adverse effect of the treatment that is beyond the therapeutic benefit. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of a disease, including biochemical, histological and/or behavioral symptoms of a disease, complications thereof, and intermediate pathological phenotypes exhibited during disease formation. For therapeutic use, beneficial or desired results include clinical results, such as reducing one or more symptoms derived from a disease, improving the quality of life of those subjects suffering from a disease, reducing the dosage of other drugs needed to treat a disease, enhancing the effect of another drug (such as via targeting), delaying the progression of a disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have an effect in reducing the number of cancer cells, reducing the size of the tumor, inhibiting (i.e., slowing or desirably stopping to some extent) infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing and desirably stopping to some extent) tumor metastasis, inhibiting to some extent tumor growth, and/or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment.

As used herein, the terms "cancer," "tumor," and "tumor" are used interchangeably to refer to a broad class of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant sites of the body through the lymphatic system or blood flow. Cancers include benign and malignant cancers, dormant tumors or micrometastases. Cancers also include hematological malignancies, such as lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors.

As used herein, "alkyl" refers to a straight (i.e., unbranched) or branched, substituted or unsubstituted, fully saturated hydrocarbon chain. As used herein, an alkyl group has 1 to 20 carbon atoms (i.e., C1-20 alkyl), 1 to 12 carbon atoms (i.e., C1-12 alkyl), 1 to 8 carbon atoms (i.e., C1-8 alkyl), 1 to 6 carbon atoms (i.e., C1-6 alkyl), 1 to 4 carbon atoms (i.e., C1-4 alkyl), 1 to 3 carbon atoms (i.e., C1-3 alkyl), or 1 to 2 carbon atoms (i.e., C1-2 alkyl). Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. When an alkyl residue having a particular carbon number is named by chemical name or identified by molecular formula, all positional isomers having that carbon number are contemplated, thus, for example, "butyl" includes n-butyl (i.e., - (CH 2) 3CH 3), sec-butyl (i.e., -CH (CH 3) CH2CH 3), isobutyl (i.e., -CH2CH (CH 3) 2) and tert-butyl (i.e., -C (CH 3) 3), and "propyl" includes n-propyl (i.e., (CH 2) 2CH 3) and isopropyl (i.e., -CH (CH 3) 2). The term "C1-C4 alkyl" refers to a straight or branched alkane containing 1 to 4 carbon atoms from which one hydrogen atom has been removed and includes, for example, "C _1-C₂ alkyl", "C _1-C₃ alkyl", "C _2-C₃ alkyl", "C _2-C₄ alkyl", "C _3-C₄ alkyl", and the like, with specific examples including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

As used herein, "alkenyl" refers to a monovalent straight or branched hydrocarbon radical containing one or more carbon-carbon double bonds, such as an aliphatic radical containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms (i.e., C2-20 alkenyl), 2 to 8 carbon atoms (i.e., C2-8 alkenyl), 2 to 6 carbon atoms (i.e., C2-6 alkenyl), or 2 to 4 carbon atoms (i.e., C2-4 alkenyl). Examples of alkenyl groups include ethenyl, propenyl, butadienyl (including 1, 2-butadienyl and 1, 3-butadienyl).

As used herein, "alkoxy" refers to the group "alkyl-O-". Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1, 2-dimethylbutoxy.

As used herein, "aryl" refers to an aromatic carbocyclic group having a single ring (e.g., a single ring) or multiple rings (e.g., bicyclic or tricyclic) comprising a fused system. As used herein, aryl has 6 to 20 ring carbon atoms (i.e., C6-20 aryl), 6 to 12 carbon ring atoms (i.e., C6-12 aryl), or 6 to 10 carbon ring atoms (i.e., C6-10 aryl). Examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracyl. However, aryl does not encompass or overlap in any way with heteroaryl as defined below. If one or more aryl groups are fused to a heteroaryl ring, the resulting ring system is heteroaryl. As used herein, the term "C ₅-C₆ aryl" refers to an aromatic group containing 5 to 6 ring members, specific examples include, but are not limited to, phenyl and the like.

As used herein, "cycloalkyl" refers to a saturated or partially saturated cyclic alkyl group having a single ring or multiple rings including fused, bridged, and spiro ring systems. The term "cycloalkyl" includes cycloalkenyl groups (i.e., cyclic groups having at least one double bond). As used herein, cycloalkyl has 3 to 20 ring carbon atoms (i.e., C3-20 cycloalkyl), 3 to 12 ring carbon atoms (i.e., C3-12 cycloalkyl), 3 to 10 ring carbon atoms (i.e., C3-10 cycloalkyl), 3 to 8 ring carbon atoms (i.e., C3-8 cycloalkyl), or 3 to 6 ring carbon atoms (i.e., C3-6 cycloalkyl). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, "bridging" refers to ring fusion in which non-adjacent atoms on the ring are connected by a divalent substituent (such as an alkylene group, an alkylene group containing one or two heteroatoms) or a single heteroatom. Quinuclidinyl and adamantyl are examples of bridging ring systems.

As used herein, "fused" refers to a ring that binds to an adjacent ring.

As used herein, "spiro" refers to a ring substituent attached at the same carbon atom through two bonds. Examples of spiro groups include 1, 1-diethylcyclopentane, dimethyl-dioxolane, and 4-benzyl-4-methylpiperidine, wherein cyclopentane and piperidine are each spiro substituents.

As used herein, "halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

As used herein, "heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl includes 1 to 20 carbon ring atoms (i.e., C1-20 heteroaryl), 3 to 12 carbon ring atoms (i.e., C3-12 heteroaryl), or 3 to 8 carbon ring atoms (i.e., C3-8 heteroaryl), and 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include pyrimidinyl, purinyl, pyridinyl, pyridazinyl, benzothiazolyl, and pyrazolyl. Heteroaryl does not encompass or overlap with aryl as defined above. As used herein, the term "C ₅-C₆ heteroaryl" refers to an aryl group containing 5 to 6 ring members and containing heteroatoms selected from N, O, S and P in its ring structure, specific examples include, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, and the like.

As used herein, "heterocyclyl" or "heterocycle" refers to a non-aromatic cyclic alkyl group in which one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. As used herein, unless otherwise indicated, "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated ring, e.g., in some embodiments, "heterocyclyl" or "heterocycle" refers to a ring that is partially saturated under the specified circumstances. The term "heterocyclyl" or "heterocycle" includes heterocyclyl groups (i.e., heterocyclyl groups having at least one double bond). The heterocyclyl may be a single ring or multiple rings, wherein the multiple rings may be fused, bridged or spiro. As used herein, heterocyclyl has 2 to 20 carbon ring atoms (i.e., C2-20 heterocyclyl), 2 to 12 carbon ring atoms (i.e., C2-12 heterocyclyl), 2 to 10 carbon ring atoms (i.e., C2-10 heterocyclyl), 2 to 8 carbon ring atoms (i.e., C2-8 heterocyclyl), 3 to 12 carbon ring atoms (i.e., C3-12 heterocyclyl), 3 to 8 carbon ring atoms (i.e., C3-8 heterocyclyl), or 3 to 6 carbon ring atoms (i.e., C3-6 heterocyclyl), 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms, or 1 ring heteroatom independently selected from nitrogen, sulfur, or oxygen. Examples of heterocycles include, but are not limited to, a single ring of 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothienyl, 3-tetrahydrothienyl, 2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropyrazinyl, 2-tetrahydropyrazinyl, 3-tetrahydropyrazinyl, 1-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, oxetanyl, dioxolanyl, azetidinyl.

As used herein, "oxo" refers to a group (=o) or (O).

As used herein, the term "pharmaceutically acceptable salt" refers to salts of (i) acidic functional groups (e.g., -COOH) present in the compounds provided herein with suitable inorganic or organic cations (bases) and includes, but is not limited to, alkali metal salts such as sodium, potassium, lithium, etc., alkaline earth metal salts such as calcium, magnesium, etc., other metal salts such as aluminum, iron, zinc, copper, nickel, cobalt, etc., inorganic base salts such as ammonium salts, organic base salts such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N' -dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris (hydroxymethyl) aminomethane salts. And (ii) salts of basic functional groups (e.g., -NH ₂) present in the compounds provided herein with suitable inorganic or organic anions (acids) and include, but are not limited to, hydrohalates such as hydrofluoric acid salts, hydrochloric acid salts, hydrobromides, hydroiodides, and the like, inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate, and the like, lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, and the like, aryl sulfonates such as benzenesulfonate, p-benzenesulfonate, and the like, organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, and the like, amino acid salts such as glycinate, trimethylglycinate, arginine, ornithine, glutamate, aspartate, and the like.

As used herein, the term "pharmaceutically acceptable ester" refers to an ester of-COOH present in a compound provided herein with a suitable alcohol, or an ester of-OH present in a compound provided herein with a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethylsuccinate, hard fatty acid ester, or palmitate. The esters can undergo hydrolysis in the presence of an acid or base to form the corresponding acid or alcohol.

As used herein, the term "stereoisomers" includes conformational isomers and configurational isomers, wherein the configurational isomers include predominantly cis-trans isomers and optical isomers. The compounds described herein may exist in stereoisomeric forms and thus encompass all possible stereoisomeric forms, as well as any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer, or a mixture thereof.

As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: disintegrants, binders, surfactants, glidants, lubricants, pH adjusters, ionic strength enhancers, agents to maintain osmotic pressure, agents to delay absorption, diluents, antioxidants, colorants, flavoring agents, preservatives, taste masking agents, and the like, non-limiting examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate; non-limiting examples of diluents include lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate, non-limiting examples of surfactants include sodium lauryl sulfate and polysorbate 80, non-limiting examples of glidants include silicon dioxide and talc, non-limiting examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, magnesium oxide, magnesium and magnesium, magnesium and magnesium, sodium stearyl fumarate and mixtures of magnesium stearate with sodium lauryl sulfate. Non-limiting examples of pH adjusters include, but are not phosphate buffers. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.

Advantageous effects

The invention provides the tumor immunotherapy targeting OTUB2 for the first time, and the tumor microenvironment is improved and the anti-tumor immunity is improved by inhibiting the OTUB2, so that the tumor treatment effect is exerted. Furthermore, based on the OTUB2 binding pocket structure, the present invention provides for the first time specific small molecule inhibitors targeting OTUB2, which can inhibit tumor growth by enhancing the anti-tumor immune response and reducing immunosuppression.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 effect of knockdown, knockdown or over-expression of OTUB2 on proliferation of tumor cells in vitro and in vivo. Wherein:

FIG. 1A shows in vitro growth proliferation curves of MC38 cells knocked down with OTUB2 and control MC38 cells;

FIG. 1B shows tumor growth curves of the knockdown OTUB2 MC38 cells and control MC38 cells in C57BL/6J immunized healthy mice;

FIG. 1C shows the survival curves of mice transplanted with MC38 tumors knocked down with OTUB2 and control MC38 tumors;

FIG. 1D shows that blocking CD8+ T cells in vivo reverses growth inhibition of MC38 tumor knockdown OTUB2 in vivo;

FIG. 1E shows tumor growth curves of MC38 cells knocked out of OTUB2 and control MC38 cells in C57BL/6J immunized healthy mice;

FIG. 1F shows tumor growth curves of the knocked out OTUB2 MC38 cells and control MC38 cells in NOD-SCID immunodeficient mice;

FIG. 1G shows tumor growth curves of OTUB2 knocked-out LL/2 cells and control LL/2 cells in C57BL/6J immunosound mice;

FIG. 1H shows tumor growth curves of B16-F10 cells with knocked out OTUB2 and control B16-F10 cells in C57BL/6J immunosound mice;

FIG. 1I shows tumor growth curves of B16-F10 cells over-expressing OTUB2 and control B16-F10 cells in C57BL/6J immunosound mice.

FIG. 2 effect of targeted OTUB2 on tumor-specific T cell killing on tumor cells. Wherein:

FIG. 2A shows that OVA-specific murine CD8+ T cells have significantly improved killing activity against OTUB2 knockdown LL/2 tumor cells compared to control LL/2 cells;

FIG. 2B shows that OVA-specific murine CD8+ T cells have significantly improved killing activity against B16-F10 tumor cells that were otUB2 knocked out as compared to control B16-F10 cells;

FIG. 2C shows that human PBMCs have significantly improved killing activity against the NCI-H358 tumor cells of the OTUB2 knockout compared to control NCI-H358 cells.

FIG. 3 identification of OTUB2 substrate protein. Wherein:

FIG. 3A shows the interaction between intracellular OTUB2 and a substrate protein;

FIG. 3B shows that knockout of B16-F10 cells from OTUB2 reduces the expression level of the substrate protein;

FIG. 3C shows that knock-down of NCI-H358 cells from OTUB2 reduces the expression level of the substrate protein;

Fig. 3D shows that knocking down the LoVo cells of OTUB2 reduces the expression level of the substrate protein.

FIG. 4 anti-tumor immunity of novel inhibitors OTUB2-IN-1 targeting OTUB 2. Wherein:

FIG. 4A schematically shows the chemical name and chemical formula of OTUB 2-IN-1;

FIG. 4B schematically shows a graph of modes of action of OTUB2 with OTUB 2-IN-1;

FIG. 4C shows the affinity between OTUB2-IN-1 and recombinant OTUB2 protein;

FIG. 4D shows Western Blot identification of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 4E shows a response curve of OTUB2-IN-1 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 4F shows a decrease IN the level of OTUB2-IN-1 on substrate protein IN LL/2 cells;

FIG. 4G shows a decrease IN the level of OTUB2-IN-1 on substrate protein IN B16F10 cells;

FIG. 4H shows the therapeutic effect of OTUB2-IN-1 on a murine lung carcinoma (LL/2) subcutaneous tumor mouse model;

FIG. 4I shows the safety assessment of OTUB2-IN-1 on a murine lung carcinoma (LL/2) subcutaneous tumor mouse model.

FIG. 5 anti-tumor immunity of novel inhibitors OTUB2-IN-2 targeting OTUB 2. Wherein:

FIG. 5A schematically shows the chemical name and chemical formula of OTUB 2-IN-2;

FIG. 5B shows the affinity between OTUB2-IN-2 and recombinant OTUB2 proteins;

FIG. 5C shows Western Blot identification of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 5D shows a response curve of OTUB2-IN-2 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 5E shows the reduction of substrate protein levels by OTUB2-IN-2 IN B16F10 cells.

FIG. 6 anti-tumor immunity of novel inhibitors OTUB2-IN-3 targeting OTUB 2. Wherein:

FIG. 6A schematically shows the chemical name and chemical formula of OTUB 2-IN-3;

FIG. 6B shows the affinity between OTUB2-IN-3 and recombinant OTUB2 protein;

FIG. 6C shows Western Blot identification of OTUB2-IN-3 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 6D shows a response curve of OTUB2-IN-3 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein.

FIG. 7 anti-tumor immunity of novel inhibitors OTUB2-IN-4 targeting OTUB 2. Wherein:

FIG. 7A schematically shows the chemical name and chemical formula of OTUB 2-IN-4;

FIG. 7B shows the affinity between OTUB2-IN-4 and recombinant OTUB2 protein;

FIG. 7C shows Western Blot identification of OTUB2-IN-4 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 7D shows a response curve of OTUB2-IN-4 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein.

FIG. 8 anti-tumor immunity of novel inhibitors OTUB2-IN-5 targeting OTUB 2. Wherein:

FIG. 8A schematically shows the chemical name and chemical formula of OTUB 2-IN-5;

FIG. 8B shows the affinity between OTUB2-IN-5 and recombinant OTUB2 protein;

FIG. 8C shows Western Blot identification of OTUB2-IN-5 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein;

FIG. 8D shows a response curve of OTUB2-IN-5 inhibiting the deubiquitination activity of OTUB2 on its ubiquitinated substrate protein.

Description about sequence information

Information on the sequences to which the present invention relates is provided in table 1.

TABLE 1 sequence information

Examples

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Those skilled in the art will appreciate that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are all conventional methods unless otherwise specified. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 knockdown or knockout of OTUB2 inhibits tumor cell growth

This example prepares tumor cell lines knocked down, knocked out or overexpressed OTUB2 by lentiviral techniques and evaluates the growth proliferation capacity of these cell lines in vitro and in vivo.

1.1 Construction of plasmids and cell lines

Knock-down shRNA nucleotide fragment of target mouse OTUB2 (NCBI reference sequence number: NM_001177841.2,Gene ID:68149) and shRNA nucleotide fragment of target irrelevant sequence (SEQ ID NO:1 and SEQ ID NO:2; synthesized by Production company) are respectively connected into pLKO.1 vector (Addgene company, product number 10878) to construct shRNA plasmid capable of knocking down mouse OTUB2, named pLKO.1-sh-OTUB2 and pLKO.1-sh-Scramble. The plko.1-sh-OTUB2 or plko.1-sh-Scramble plasmid and packaging plasmid psPAX (adedge corporation, cat# 12260) and pmd2.g (adedge corporation, cat# 12259) were co-transfected into 293T cells (purchased from ATCC corporation, usa, cat# CRL-3216) using lipofectamine 2000 transfection reagent (Invitrogen corporation, cat# 11668019). After 72 hours, MC38 cells were infected with the recombinant virus to express the corresponding shRNA, and the MC38 cell line (sh-OTUB 2) with stable mouse OTUB2 knocked down and the control cell line (Scramble) were obtained by pressurizing with a medium containing 1. Mu.g/mL puromycin (InvivoGen, cat# ant-pr-5).

The shRNA nucleotide fragments (SEQ ID NO:3 and SEQ ID NO:4; synthesized by Productivity Co.) of targeted human OTUB2 (NCBI reference sequence No. NM-023112.4,Gene ID:78990) were ligated into pLKO.1 vector to construct shRNA plasmids capable of knocking down mouse OTUB2, designated pLKO.1-shOTUB2#1 and pLKO.1-shOTUB2#2, respectively. The plko.1-shOTUB2#1 and plko.1-shOTUB2#2 plasmids and the packaging plasmids psPAX2 and pmd2.G were co-transfected into 293T cells using lipofectamine 2000 transfection reagent. After 72 hours, NCI-H358 cells were infected with recombinant virus to express the corresponding shRNA, and the NCI-H358 cell line (shOTUB 2#1 and shOTUB 2#2) with stable knockdown of human OTUB2 was obtained by pressurizing with medium containing 1. Mu.g/mL puromycin.

Knockout-the sgRNA nucleotide fragment of the targeted mouse OTUB2 and the sgRNA nucleotide fragment of the targeted unrelated sequence (SEQ ID NO:5 and SEQ ID NO:6; synthesized by the Producter company) are respectively connected into LENTICRISPR V vectors (Addgene company, cat. No. 52961) to construct CRISPR plasmids which can knock out the mouse OTUB2 and are named LENTICRISPR-sg-mOTUB2 and LENTICRISPR-sg-Scramble. LENTICRISPR-sg-mOTUB2 or LENTICRISPR-sg-Scramble plasmids and packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells using lipofectamine 2000 transfection reagents. After 72 hours, MC38, LL/2 and B16-F10 cells were infected with recombinant virus, respectively, to express the corresponding sgRNA, and the stable integrated tumor cell line was obtained by pressurizing with medium containing 1. Mu.g/mL puromycin. Individual clones were obtained by limiting dilution. Finally, anti-OTUB 2 antibody (division No. D199590) was used to screen the monoclonal tumor cell line with the OTUB2 completely knocked out for subsequent experiments. The MC38, LL/2 and B16-F10 cell lines (OTUB 2 KO) and Control cell lines (Control) were finally obtained from the complete knockout mouse OTUB 2.

Overexpression, namely, gene fragments (SEQ ID NO:7 and SEQ ID NO:8; synthesized by the manufacturing company) expressing OTUB2 and PD-L1 are respectively connected into LENTICRISPR V vectors (Addgene company, product number 52961) to construct plasmids capable of stably expressing corresponding genes, and the plasmids are named pLenti-OTUB2-Flag (with Flag tag) and pLenti-PD-L1. pLenti-OTUB2 or pLenti was co-transfected into 293T cells with packaging plasmid psPAX and pMD2.G, respectively, using lipofectamine 2000 transfection reagent. After 72 hours, B16-F10 cells were infected with the recombinant virus to express the corresponding genes, and B16-F10 cell lines (OTUB 2 OE) stably expressing OTUB2 and Control cell lines (Control) were obtained by pressurizing with a medium containing 1. Mu.g/mL puromycin.

Over-expressing chicken Ovalbumin (OVA) on cells of Control, LL/2 and B16-F10 OTUB2 KO, respectively, by ligating a gene fragment (SEQ ID NO:9; synthesized by Bio-Rad corporation) expressing OVA into lentiCas-Blast vector (Addgene corporation, cat. No. 52962) to construct a plasmid capable of stably expressing the corresponding gene, designated pLenti-OVA-Blast. pLenti-OVA-Blast or pLenti-Blast was co-transfected with packaging plasmid into 293T cells using lipofectamine 2000 transfection reagents, respectively. After 72 hours, B16-F10, LL/2 cells were again infected with the recombinant virus to express the corresponding gene, and the OTUB2 knockout tumor cell line (OTUB 2 KO-OVA) and the Control cell line (OTUB 2 KO-Control) stably expressing OVA were obtained by pressurizing with a medium containing 6. Mu.g/mL (B16-F10) or 2. Mu.g/mL (LL/2) blasticidin (InvivoGen, cat# ant-pr-5).

1.2CCK-8 method for detecting growth and proliferation ability of OTUB2 knockout tumor cells

MC38-Scramble and MC38-sh-OTUB2 cells were plated in 96-well plates at a number of 2X 10 ³ cells/well, respectively. 10 μl of CCK-8 cell proliferation assay reagent (MCE company, cat. No. HY-K-03001) was added to each well on days 0,1,2,3 after cells were completely adherent. After incubation at 37 ℃ for 4 hours, the 96-well plate was placed in a microplate reader (Thermo FISHER SCIENTIFIC, model Multiskan Go 1510) for data reading at 450nm wavelength and cell proliferation curves were plotted.

The results in fig. 1A show that MC38 knockdown OTUB2 had no effect on cell proliferation compared to the control group.

1.3 Evaluation of tumor growth of MC38 cells knocked down OTUB2 and control MC38 cells on C57BL/6J immunocompetent mice

C57BL/6J mice (Jiangsu Ji Kangsu Biotech Co., ltd.) of 6 weeks old were selected as experimental animals, and 100. Mu.L of 5X 10 ⁵ MC38 cell suspension (sh-OTUB 2 and Scramble) was inoculated subcutaneously, and tumor size was monitored 8 days after tumor inoculation. The length (L) and width (W) of the tumor were measured with a vernier caliper, and the Volume of the tumor was calculated with the formula volume= (l× (W) ²)/2. Tumor volume was measured continuously once every two days, tumor volume 1000mm ³ was used as ethical endpoint, and survival time of mice was recorded.

The experimental results of fig. 1B and 1C show that, compared to the control group, knocking down OTUB2 significantly inhibited MC38 tumor growth (P < 0.05) and significantly prolonged the survival of tumor-bearing mice (p=0.0034) in immune sound mouse models.

1.4 Reduction of MC38 from OTUB2 tumor growth inhibition in C57BL/6J immunocompetent mice was dependent on CD8 ⁺ T cells

C57BL/6J mice of 6 weeks of age were selected as experimental animals, and treated with anti-CD8 antibody (BioXcell Corp., cat# BE 0061) and control antibody, respectively, 2 days before tumor inoculation, once every 3 days until the end of the experiment. Mice were then subcutaneously inoculated with 100 μl of 5×10 ⁵ MC38 cell suspension (sh-OTUB 2 and Scramble), tumor size was monitored beginning 7 days after tumor inoculation, tumor was measured every two days, and tumor volume was measured continuously until day 22.

The experimental results of fig. 1D show that knocking down OTUB2 significantly inhibited MC38 tumor growth (p=0.0059) in an immune-robust mouse model compared to the control group. Whereas when the function of CD8 cells was blocked, the effect of knockdown OTUB2 on inhibition of MC38 tumor growth was lost, suggesting that tumor inhibition by targeted OTUB2 was immune system dependent.

1.5 Evaluation of tumor growth of MC38 cells from which OTUB2 was knocked out and control MC38 cells on C57BL/6J immunosound mice and NOD-SCID immunodeficient mice

C57BL/6J mice and NOD-SCID mice (Jiangsu Ji Kangyaokang Biotechnology Co., ltd.) of 6 weeks old were selected as experimental animals, and 100. Mu.L of 5X 10 ⁵ MC38 cell suspensions (OTUB 2 KO and Control) were inoculated subcutaneously, respectively, and tumor size was monitored after 8 days of tumor inoculation. Tumors were measured every two days, and tumor volumes were measured continuously until day 20 or day 28.

The experimental results of fig. 1E and 1F show that knockout of OTUB2 significantly inhibited the growth of MC38 tumors in immunocompetent mice (P < 0.0001), but not in immunodeficient mice, compared to the control. It is shown that the anti-tumor immunomodulating effect exerted by OTUB2 is dependent on the immune system.

1.6 Evaluation of tumor growth of OTUB2 on LL/2 cells and control LL/2 cells on C57BL/6J immunized healthy mice

C57BL/6J mice at 6 weeks of age were selected as experimental animals, inoculated subcutaneously with 100. Mu.L of 5X 10 ⁵ LL/2 cell suspension (OTUB 2 KO and Control), and tumor size was monitored 8 days after tumor inoculation. Tumors were measured every two days, and tumor volumes were measured continuously until day 20.

The experimental results of fig. 1G show that knockout of OTUB2 significantly inhibited the growth of LL/2 tumors in immunocompetent mice (P < 0.0001) compared to controls.

1.7 Evaluation of tumor growth on C57BL/6J immunocompetent mice by knocking out B16-F10 cells of OTUB2 and control B16-F10 cells

C57BL/6J mice at 6 weeks of age were selected as experimental animals, inoculated subcutaneously with 100. Mu.L of 3X 10 ⁵ B16-F10 cell suspension (OTUB 2 KO and Control), and tumor size was monitored 7 days after tumor inoculation. Tumor volume was measured once every other day, and tumor volume was measured continuously, from day 17.

The experimental results of fig. 1H show that knockout of OTUB2 significantly inhibited the growth of B16-F10 tumors in immunocompetent mice (p=0.0036) compared to the control.

1.8 Evaluation of tumor growth of B16-F10 cells overexpressing OTUB2 and control B16-F10 cells on C57BL/6J immunocompetent mice

C57BL/6J mice at 6 weeks of age were selected as experimental animals, inoculated subcutaneously with 100. Mu.L of 3X 10 ⁵ B16-F10 cell suspension (OTUB 2 OE and Control) and tumor size was monitored 7 days after tumor inoculation. Tumors were measured every two days, and tumor volumes were measured continuously, from tumor volume to day 16.

The experimental results of fig. 1I show that over-expression of OTUB2 significantly promotes the growth of B16-F10 tumors in immunocompetent mice (p=0.0097) compared to the control.

The result shows that OTUB2 is used as a potential tumor immunity negative regulation target to promote tumor generation immune escape.

Example 2 tumor-targeting OTUB2 promotes killing of tumor cells by cd8+ T cells

In this example, it was verified by T cell in vitro killing experiments that OTUB2 in targeted tumor cells can increase the response of CD8 ⁺ T cells to tumor cells.

2.1 Tumor-specific T cell specific killing experiments on tumor cells

OT-1T cells isolated from the spleen of an OT-1 transgenic mouse (Jackson Laboratory, cat. No. 003831) were cultured at a density of 2X 10 ⁶ cells/ml in RPMI 1640 medium containing 1. Mu.g/ML SIINFEKL polypeptide (Bio Inc.) and 50U/ml mouse recombinant IL-2 protein (Sino Biological, 51061-MNAE-20). After 7 days of OT-1T cell activation, it was added to tumor cells (OTUB 2 KO and Control cells) pretreated with 1ng/ml IFN-. Gamma.in a ratio of 1:1 for co-cultivation for 24 hours. And finally, detecting the survival rate of the tumor cells by using flow cytometry.

The experimental results of FIGS. 2A-2B show that OTUB2 knockdown results in enhanced killing of B16-F10 and LL/2 tumor cells by tumor-specific OT-1T cells, as compared to control cells.

2.2 Experiments of specific killing of tumor cells by tumor-specific T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy Human whole blood by Ficoll-Paque PLUS (GE HEALTHCARE, cat# 17-1440-02) density gradient centrifugation, 10 ⁶ cells were added to microwells, 0.1 μg/well Dynabeads ^TM Human T-Activator CD3/CD28 (Thermo FISHER SCIENTIFIC, cat# 11131D) and 50U/ml Human recombinant IL-2 protein (STEMCELL, cat# 78036) were added. After 3 days of PBMCs stimulation, they were co-cultured with tumor cells (shOTUB and control Scramble cells) pre-treated with 1ng/ml IFN-. Gamma.in a 5:1 ratio of activated cells (effector cells) to tumor cells (target cells) for 120 hours. And finally, detecting the survival rate of the tumor cells by using flow cytometry.

The results of the experiment in FIG. 2C show that the killing capacity of PBMCs against tumor cells is increased after the OTUB2 knockdown of tumor cells compared to control cells.

In conclusion, targeting OTUB2 enhances the killing capacity of tumor-specific T cells against tumor cells, thereby exerting an effect of inhibiting tumor growth.

Example 3 novel inhibitors of OTUB2-IN-1 targeting OTUB2 are capable of promoting anti-tumor immunity

This example was prepared by developing novel, specific inhibitors against OTUB2 and evaluating the inhibition of tumor growth by the inhibitors in vivo.

3.1 Identification of substrate proteins

OTUB2 is a deubiquitinase that binds to a substrate protein and removes its ubiquitination, i.e., interacts with the substrate protein to stabilize the substrate protein from ubiquitination degradation. The enzymatic activity of OTUB2 can thus be determined by verifying the ubiquitination and degradation of the OTUB2 substrate protein. In this experiment, a substrate protein of OTUB2 was identified, and the effect of an OTUB2 inhibitor on OTUB2 enzyme activity was verified by taking this substrate protein as an example.

3.1.1 Co-immunoprecipitation to detect interactions between OTUB2 and substrate protein

The pLenti-OTUB2-Flag or pLenti and pLenti-PD-L1 plasmids were co-transfected into 293T cells using lipofectamine 2000 transfection reagent. Collecting total protein sample after 72 hr, and usingM2AFFINITY GEL (Sigma-Aldrich Co., cat. No. A2220) was subjected to an immunoprecipitation test, and the coprecipitated protein and total protein samples were subjected to Western immunoblotting, and PD-L1 and OTUB2 were detected with the corresponding antibodies, the PD-L1 antibody was Abcam, ab213480, and OTUB2 was detected with the tag antibody anti-Flag (Sigma-Aldrich, F1804). The experimental results of FIG. 3A show that capture of OTUB2 protein enables detection of PD-L1 protein compared to control, indicating the presence of direct interaction between OTUB2 and PD-L1.

3.1.2 Knockout of OTUB2 in tumor cells can reduce substrate protein levels

Tumor cells from OTUB2 knockdown and control in the logarithmic growth phase were inoculated into 6-well plates at a density of 5×10 ⁵/well, treated with fresh medium or with 0.5ng/ml IFN- γ (R & D system company, cat# 285-IF) medium, respectively, and 24 hours later stained with APC-labeled anti-mouse PD-L1 antibody (BioLegend company, cat# 124312) and APC-labeled anti-human PD-L1 antibody (BioLegend company, cat# 329708), and finally the expression level of PD-L1 protein was detected by flow cytometry. The experimental results in FIGS. 3B-3D show that knocking out OTUB2 in B16-F10 cells or knocking out OTUB2 in NCI-H358 and LoVo cells reduces PD-L1 expression levels in tumor cells as compared to controls.

The above results indicate that a substrate protein in which PD-L1 is OTUB2 can be used for the detection of the enzymatic activity of OTUB 2.

3.2OTUB2-IN-1 inhibitors are capable of targeting the catalytic center of OTUB2

Recombinant OTUB2 protein was coupled to CM5 chip (GE HEALTHCARE, cat# BR 100530) and tested and analyzed with Biacore 8K as mobile phase with 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M OTUB2-IN-1 inhibitor (LIFECHEMICAL, cat# F0444-0064), respectively, and experimental data were processed with Biacore Insight Evaluation Software.

FIG. 4A shows that by virtual screening we identified that an OTUB2-IN-1 inhibitor is capable of binding specifically to an OTUB2 protein. The OTUB2-IN-1 inhibitor is CAS No.:300558-22-9, and has the chemical formula 6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}hexanoic acid.

FIG. 4B shows a schematic representation of the docking interactions between the OTUB2-IN-1 inhibitor and the amino acid residues IN the catalytic pocket of OTUB 2.

FIG. 4C shows that the interaction affinity between the OTUB2-IN-1 inhibitor and OTUB2 as determined by SPR method is 12. Mu.M.

Effect of 3.3OTUB2-IN-1 inhibitors on ubiquitination of substrate proteins

10Ng of recombinant OTUB2 protein was incubated with different gradients of OTUB2-IN-1 inhibitor (0. Mu.M, 0.39. Mu.M, 1.56. Mu.M, 6.25. Mu.M, 25. Mu.M, 100. Mu.M) followed by addition of 100ng of ubiquitinated PD-L1 protein, and after incubation for 4 hours, the ubiquitinated PD-L1 protein was detected by Western blotting and experimental data were treated with Image J software (NIH).

Figures 4D and 4E show that with increasing concentrations of OTUB2-IN-1 inhibitor added, the level of ubiquitinated substrate protein increases significantly, indicating that the activity of OTUB2 enzyme is significantly inhibited.

Effect of 3.4OTUB2-IN-1 inhibitors on degradation of substrate proteins IN cells

LL/2 and B16-F10 cells in log phase were selected and seeded at a density of 5X 10 ⁵ cells/well in 6-well plates. After the cell density reached 70%, the OTUB2-IN-1 inhibitor (0,0.15,0.6,2.5,10,40 μm) was diluted IN a gradient with fresh medium and the cells were subjected to drug treatment. After 24 hours of drug treatment, the corresponding samples were collected for western blotting. The expression of the substrate proteins PD-L1 and OTUB2 was detected with specific antibodies.

FIGS. 4F and 4G show that the amount of substrate protein decreased IN a gradient with increasing concentration of OTUB2-IN-1 inhibitor added, without significant change IN the amount of OTUB2, IN either LL/2 cells or B16-F10 cells. It is shown that the OTUB2-IN-1 inhibitor inhibits the function of OTUB2 by binding to OTUB2 without altering the stability of OTUB2, thereby exerting a degradation effect on the substrate protein.

Anti-tumor effects of 3.5OTUB2-IN-1 inhibitors IN tumor-bearing mice

C57BL/6J mice of 6 weeks of age were selected as experimental animals, and 100. Mu.L of a 5X 10 ⁵ LL/2 cell suspension was inoculated subcutaneously IN the mice, and when the tumor had grown to 50mm ³, the mice were treated with an OTUB2-IN-1 inhibitor and a control drug, once daily for a total of 6 times until the end of the experiment. Tumor size was monitored after treatment, tumor volume was measured every two days, tumor volume was measured continuously, and 1000mm ³ tumor volume was used as the ethical endpoint.

Figures 4H and 4I show that OTUB2-IN-1 inhibitors significantly inhibited tumor growth (P < 0.001) compared to the control group, without significant effect on mouse body weight, indicating that OTUB2-IN-1 has better anti-tumor effect IN vivo and shows a certain safety.

Example 4 other novel inhibitors targeting OTUB2 are capable of inhibiting the activity of OTUB2

This example demonstrates the inhibition of OTUB2 enzyme activity by developing a novel, specific series of inhibitors against OTUB2 and evaluating the inhibition in vitro. The experimental procedure was the same as in example 3.

4.1OTUB2-IN-2 is effective IN inhibiting OTUB2 activity

FIG. 5A shows that by virtual screening we identified that the inhibitor OTUB2-IN-2 is capable of binding specifically to the OTUB2 protein. The chemical formula ：2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3-dihydro-1H-indol-1-yl}acetic acid( is obtained from OTAVAchemicals company under the trade designation 1663785).

FIG. 5B shows that the interaction affinity between the OTUB2-IN-2 inhibitor and OTUB2 as determined by SPR method is 41. Mu.M.

Figures 5C and 5D show that with increasing concentrations of OTUB2-IN-2 inhibitor added, the level of ubiquitinated substrate protein increases significantly, indicating that the activity of OTUB2 enzyme is significantly inhibited.

FIG. 5E shows that IN B16-F10 cells, the substrate protein content was graded down with increasing concentration of OTUB2-IN-2 inhibitor added, while the OTUB2 content was not significantly changed. It is shown that the OTUB2-IN-2 inhibitor inhibits the function of OTUB2 by binding to OTUB2 without altering the stability of OTUB2, thereby exerting a degradation effect on the substrate protein.

4.2OTUB2-IN-3 is effective IN inhibiting OTUB2 activity

FIG. 6A shows that by virtual screening we identified that the inhibitor OTUB2-IN-3 was able to bind specifically to the OTUB2 protein. The OTUB2-IN-3 inhibitor is CAS No.:313646-95-6, having the formula ：2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid(, available from LIFECHEMICALS under the trade designation F3224-0128.

FIG. 6B shows that the interaction affinity between the OTUB2-IN-3 inhibitor and OTUB2 as determined by SPR method is 32.6. Mu.M.

Figures 6C and 6D show that with increasing concentrations of OTUB2-IN-3 inhibitor added, the level of ubiquitinated substrate protein increases significantly, indicating that the activity of OTUB2 enzyme is significantly inhibited.

4.3OTUB2-IN-4 inhibitor can effectively inhibit activity of OTUB2

FIG. 7A shows that by virtual screening we identified that the inhibitor OTUB2-IN-4 was able to bind specifically to the OTUB2 protein. The OTUB2-IN-4 inhibitor is CAS No.:374543-72-3, having the formula ：11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid(, available from OTAVAchemicals under the trade designation 4349982.

FIG. 7B shows that the interaction affinity between the OTUB2-IN-4 inhibitor and OTUB2 as determined by SPR method is 25.9. Mu.M.

Figures 7C and 7D show that with increasing concentrations of OTUB2-IN-4 inhibitor added, the level of ubiquitinated substrate protein increases significantly, indicating that the activity of OTUB2 enzyme is significantly inhibited.

4.4OTUB2-IN-5 inhibitor can effectively inhibit activity of OTUB2

FIG. 8A shows that by virtual screening we identified that the inhibitor OTUB2-IN-5 was able to bind specifically to the OTUB2 protein. The chemical formula ：11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene-1,3-thiazolidin-3-yl}undecanoic acid( is obtained from OTAVAchemicals company under the trade designation 4349908).

FIG. 8B shows that the interaction affinity between the OTUB2-IN-5 inhibitor and OTUB2 as determined by SPR method is 3.91. Mu.M.

Figures 8C and 8D show that with increasing concentrations of OTUB2-IN-4 inhibitor added, the level of ubiquitinated substrate protein increases significantly, indicating that the activity of OTUB2 enzyme is significantly inhibited.

The results of FIGS. 5-8 above demonstrate that these small molecule compounds are capable of acting as OTUB inhibitors to modulate tumor immunity.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. Use of OTUB2 inhibitors in the preparation of drugs for anti-tumor immunotherapy.

2. The use according to claim 1, wherein the drug is used to enhance anti-tumor immune response and/or reduce tumor immunosuppression in a subject;

Preferably, the medicament is used to promote T cell-mediated tumor killing in a subject;

Preferably, the subject suffers from a tumor.

3. The use according to claim 1 or 2, wherein the inhibitor can inhibit or down-regulate the expression of the OTUB2 gene, inhibit or block the activity of the OTUB2 protein, or degrade the OTUB2 protein.

4. The use of claim 3, wherein the inhibitor is selected from an antibody, an RNA interfering agent (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA) or microRNA (miRNA)) or an antisense oligonucleotide, a gene editing system (e.g., a CRISPR-Cas system), a small molecule compound, or a PROTAC (protein degradation targeting chimera).

5. The use according to claim 3, wherein the inhibitor is an RNA interfering agent, for example, small interfering RNA (siRNA), small hairpin RNA (shRNA) or micro RNA (miRNA)) or an antisense oligonucleotide;

Preferably, the inhibitor is shRNA.

6. The use according to claim 3, wherein the inhibitor is selected from small molecule compounds.

7. The use according to claim 6, wherein the small molecule compound is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt or ester or stereoisomer thereof:

in:

^R1 is -C(O)X, X is hydroxy or _C1 - _C4 alkoxy; or is C3-7 monocyclic cycloalkyl, 4-6-membered monocyclic heterocyclyl or 5-6-membered monocyclic heteroaryl, wherein the 4-6-membered monocyclic heterocyclyl and the 5-6-membered monocyclic heteroaryl each have 1 to 3 ring heteroatoms independently selected from N, O and S;

n is an integer selected from 1 to 12;

^R2 is

Wherein, R ^2a is selected from (C1-C4) alkyl, (C2-C4) alkenyl, or -C(O)Y, and Y is hydroxy or (C1-C4) alkoxy;

wherein R ^2b , R ^2c , and R ^2d are each independently selected from halogen (e.g., -F, -Cl, -Br, or -I), nitro, amino, hydroxy, thiol, (C1-C4) alkyl, or (C1-C4) alkoxy; preferably, R ^2b , R ^2c , and R ^2d are different from each other;

Here, m is 1, 2, 3 or 4.

8. The use according to claim 7, wherein R ¹ is -C(O)X, X is hydroxyl or (C1-C4) alkoxy, and n is an integer selected from 1 to 12;

Preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

9. The use according to claim 8, wherein R ¹ is -C(O)X, and X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy;

Preferably, X is hydroxy, methoxy or ethoxy.

Preferably, X is hydroxy.

10. The use according to claim 7, wherein R ¹ is a C3-7 monocyclic cycloalkyl group, a 4- to 6-membered monocyclic heterocyclic group, or a 5- to 6-membered monocyclic heteroaryl group, and n is 1, 2, 3 or 4;

Preferably, n is 1 or 2; preferably, n is 1.

11. The use according to claim 10, wherein R ¹ is a C3-7 monocyclic cycloalkyl, a 4- to 6-membered monocyclic heterocyclic group, or a 5- to 6-membered monocyclic heteroaryl, wherein the 4- to 6-membered monocyclic heterocyclic group and the 5- to 6-membered monocyclic heteroaryl each have 1 ring heteroatom selected from N, O and S;

Preferably, R ¹ is a C3-7 monocyclic cycloalkyl group or a 4 to 6-membered monocyclic heterocyclic group;

Preferably, R ¹ is a C3-6 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclyl group (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl);

Preferably, R ¹ is tetrahydrofuranyl or pyrrolidinyl;

Preferably, R ¹ is tetrahydrofuran.

12. The use according to any one of claims 7 to 11, wherein R ² is wherein R ^2a is selected from methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, or -C(O)Y, and Y is hydroxy, methoxy, ethoxy, n-propoxy, or isopropoxy;

Preferably, R ^2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, where Y is hydroxy, methoxy or ethoxy;

Preferably, R ^2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

13. The use according to any one of claims 7 to 11, wherein R ² is wherein R ^2b , R ^2c , and R ^2d are each independently selected from nitro, hydroxy, (C1-C4) alkyl, or (C1-C4) alkoxy;

Preferably, R ^2b , R ^2c , and R ^2d are each independently selected from nitro, hydroxy, methyl, ethyl, n-propyl, isopropyl, methoxy, and ethoxy;

Preferably, R ^2b , R ^2c , and R ^2d are each independently selected from nitro, hydroxy, methyl, ethyl, methoxy, and ethoxy;

Preferably, R ^2b , R ^2c , and R ^2d are different from each other;

Preferably, R ^2b , R ^2c , and R ^2d are nitro, hydroxy, and methoxy, respectively.

14. The use according to any one of claims 7 to 13, wherein the compound has a structure represented by formula (Ia):

wherein R ¹ , R ^2a , m and n are as defined in formula (I).

15. The use according to claim 14, wherein R ¹ has a feature selected from the following:

(1) R ¹ is -C(O)X, X is hydroxy, methoxy, ethoxy, n-propoxy or isopropoxy (preferably, X is hydroxy, methoxy or ethoxy); preferably, n is an integer selected from 1 to 11, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or

(2) R ¹ is a C 3-7 monocyclic cycloalkyl group (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) or a 4- to 6-membered monocyclic heterocyclic group having one ring heteroatom selected from N, O and S (e.g., oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl or piperidinyl); preferably, R ¹ is tetrahydrofuranyl or pyrrolidinyl; preferably, n is 1 or 2, for example, 1.

16. The use according to claim 14 or 15, wherein R ^2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)Y, and Y is hydroxy, methoxy or ethoxy;

Preferably, R ^2a is selected from methyl, ethyl, vinyl, propenyl, or -C(O)OH.

17. The use according to any one of claims 7 to 16, wherein the compound is selected from the following compounds or pharmaceutically acceptable salts or esters or stereoisomers thereof:

6-{5-[(3Z)-1-(carboxymethyl)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3 -yl}hexanoic acid;

2-{2-oxo-3-[(5Z)-4-oxo-3-[(oxolan-2-yl)methyl]-2-sulfanylidene-1,3-thiazolidin-5-ylidene]-2,3- dihydro-1H-indol-1-yl}acetic acid;

2-[(5Z)-5-[(4-hydroxy-3-methoxy-5-nitrophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid;

11-{5-[(3Z)-1-butyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl }undecanoic acid;

11-{4-oxo-5-[(3Z)-2-oxo-1-(prop-2-en-1-yl)-2,3-dihydro-1H-indol-3-ylidene]-2-sulfanylidene -1,3-thiazolidin-3-yl}unddecanoic acid.

18. The use according to any one of claims 1 to 17, wherein the tumor comprises a solid tumor or a blood tumor;

Preferably, the tumor is selected from lung cancer, cervical cancer, breast cancer, head and neck cancer, liver cancer, bladder cancer, esophageal cancer, pancreatic cancer, kidney cancer, melanoma, ovarian cancer, gastric cancer, colorectal cancer.

19. Use of a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof as an OTUB2 inhibitor, wherein the compound is defined in any one of claims 7 to 17;

Preferably, the OTUB2 inhibitor is used to inhibit or block the activity of OTUB2 protein in vitro;

Preferably, the OTUB2 inhibitor is used for non-therapeutic purposes.

20. Use of a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof in the preparation of a medicament for preventing and/or treating tumors, wherein the compound is defined in any one of claims 7 to 17;

Preferably, the drug is used for anti-tumor immunotherapy;

Preferably, the medicament is used to enhance anti-tumor immune response and/or reduce tumor immunosuppression in a subject;

Preferably, the tumor comprises a solid tumor or a blood tumor;

21. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt or ester or stereoisomer thereof, and a pharmaceutically acceptable carrier and/or excipient; wherein the compound is as defined in any one of claims 7 to 17.

22. A method for screening drugs for anti-tumor immunotherapy, comprising the step of screening OTUB2 inhibitors.

23. The method of claim 22, wherein the method comprises:

(i) screening agents that interact with the OTUB2 protein as test agents, for example, by measuring using BLI or SPR technology;

(ii) detecting the deubiquitinase activity of the OTUB2 protein on the substrate protein in the presence of the test agent obtained in step (i);

(iii) comparing the result of step (ii) with the activity measured in the absence of the test agent.