WO2023213197A1 - mTOR抑制剂增强靶向蛋白降解药物功效的应用 - Google Patents

mTOR抑制剂增强靶向蛋白降解药物功效的应用 Download PDF

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WO2023213197A1
WO2023213197A1 PCT/CN2023/089697 CN2023089697W WO2023213197A1 WO 2023213197 A1 WO2023213197 A1 WO 2023213197A1 CN 2023089697 W CN2023089697 W CN 2023089697W WO 2023213197 A1 WO2023213197 A1 WO 2023213197A1
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drugs
disease
degradation
cancer
drug
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刘旸
宋天瑜
彭博
仓勇
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上海科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention belongs to the field of biomedicine and discloses the application of mTOR inhibitors to enhance the efficacy of targeted protein degradation drugs.
  • Immunomodulatory drugs mainly include the first generation of thalidomide, and thalidomide derivatives-lenalidomide and pomalidomide. And CC-122, CC-220, etc., which are under development and have not yet been clinically approved, are a kind of molecular glue drugs. In the 1990s, a series of literature studies showed that thalidomide had significant anti-myeloma efficacy, and thalidomide returned to people's attention. In order to improve the teratogenic effect of thalidomide on the fetus, lenalidomide, pomalidomide, etc. were developed based on the structure of thalidomide.
  • Thalidomide and its derivatives are collectively called immunomodulatory drugs because of their regulatory effects on the immune system.
  • Immunomodulatory drugs can induce the proliferation of T cells regulated by IL-2 (interleukin-2) by enhancing the secretion of IFN- (interferon gamma), and can also inhibit tumor growth by inhibiting TNF-, IL-1, etc.
  • IL-2 interleukin-2
  • IFN- interferon gamma
  • TNF-, IL-1 interferon gamma
  • the ubiquitination pathway involved in IMiDs drugs is an important physiological process in regulating protein post-translational modification in the body.
  • ubiquitin activating enzyme E1
  • E2 ubiquitin conjugating enzyme
  • E3ligase ubiquitin ligase
  • IMiDs are the first discovery of small molecules that can target E3 ubiquitin ligase and hijack the ubiquitin ligase to specifically degrade new substrate protein targets.
  • IMiDs recruit protein CRBN by binding to the substrate of ubiquitin ligase, forming a linker-like structure that can bind to new substrate proteins and ubiquitinate and degrade them.
  • Traditional inhibitor drugs rely on compounds that destroy the active pocket domains of enzymes, but most disease-causing proteins such as oncoproteins do not. Without a pocket structure, the revelation of the molecular mechanism of IMiDs has enabled scientists to see the potential of using them to target and degrade traditional "undruggable" proteins.
  • PROTAC drugs By connecting the target protein at one end and the ubiquitination degradation system at the other end, the target protein can be continuously and thoroughly degraded, which greatly reduces the required drug concentration and has high selectivity and speed. As a result, a series of PROTAC drugs have been developed, and a considerable number of them have entered clinical trials. Some once complex or difficult-to-drug target proteins, such as KRAS, also have targeted PROTAC drugs.
  • the mTOR signaling pathway is related to the material exchange process between cells and the outside world, and is an important signaling pathway that cells rely on for normal survival and metabolism.
  • the mTOR signaling pathway is involved in many basic life processes, from protein synthesis to autophagy, and is involved in the regulation.
  • Two complexes, mTORC1 and mTORC2 play key roles in the mTOR signaling pathway.
  • mTORC1 can phosphorylate the downstream substrate S6K1, thereby further phosphorylating CAD (carbamoyl phosphatase) and promoting the synthesis of pyrimidines.
  • mTORC1 can also phosphorylate the substrate 4EBP to unbind it from eIF4E, thereby promoting the translation of mRNA.
  • mTOR signaling pathway is likely to be involved in the regulation of resistance to many tumor drugs. Rapamycin and its derivatives such as everolimus can inhibit the mTOR signaling pathway. Everolimus has better water solubility than rapamycin and has the effect of inhibiting the proliferation of vascular endothelial cells. It has been approved for the treatment of melanoma, rectal cancer, etc. There have been no reports of mTOR inhibitors enhancing the efficacy of targeted protein degradation drugs.
  • the purpose of the present invention is to provide an application of an mTOR inhibitor to enhance the efficacy of targeted protein degradation drugs to solve the problems of disease resistance or recurrence in the prior art.
  • the purpose of the present invention is to The technical problem to be solved is how to further improve the ability of clinical first-line targeted protein degradation drugs such as immunomodulatory drugs IMiDs to degrade substrate proteins. power, thereby improving its efficacy.
  • mTOR signaling pathway inhibitors such as rapamycin and everolimus can enhance the degradation of substrate proteins by molecular glue drugs and PROTAC drugs. Combining mTOR signaling pathway inhibitors is promising. It can improve the therapeutic effect of protein degradation agents such as molecular glue drugs represented by IMiDs and PROTAC drugs in clinical treatment.
  • One of the purposes of the present invention is to provide an application of an mTOR inhibitor in the preparation of products that enhance the efficacy of targeted protein degradation drugs.
  • Another object of the present invention is to provide a pharmaceutical composition, which contains molecular glue, a PROTAC protein degradation agent and an mTOR inhibitor, as well as pharmaceutically acceptable excipients.
  • the mTOR inhibitor proposed by the present invention can significantly enhance the degradation of substrate proteins by small molecule drugs targeting protein degradation such as molecular glues and PROTAC, thereby achieving the purpose of treating diseases.
  • small molecule drugs targeting protein degradation such as molecular glues and PROTAC
  • the combination of mTOR inhibitors can help treat such drug-resistant or relapsed patients and improve current outcomes. It has the therapeutic effect of clinical drugs.
  • a variety of next-generation molecular glue and PROTAC protein-degrading drugs targeting IKZF are under development and even in clinical trials. They are hailed as a new generation of drugs that break the traditional small molecule inhibitors.
  • the combination of mTOR inhibitors will have enhanced its therapeutic potential.
  • FIG. 1 Activation of the mTOR pathway can make myeloma cells OPM2 acquire resistance to immunomodulatory drugs.
  • A is the DEPDC5 knockout cell line constructed in the myeloma cell line OPM2;
  • B-E are the immunomodulatory drugs pomalidomide (B), lenalidomide (C), CC-220 (D), and CC-122 respectively.
  • E Killing curves of DEPDC5 knockout OPM2 respectively.
  • Figure 2 The combination of pomalidomide and the mTOR signaling pathway inhibitor rapamycin has a stronger killing effect on myeloma cells.
  • A-E are the effects of pomalidomide (Poma), rapamycin (Rapa) and their combination on myeloma cell lines MM1S (A), H929 (B), OPM2 (C), U266 (D) and RPMI- Kill curve in 8226(E).
  • FIG. 3 Combined use of rapamycin can enhance the degradation of IKZF3 and IKZF1 by pomalidomide.
  • A-C are Western blotting methods. In myeloma cell lines OPM2 (A), RPMI-8226 (B) and U266 (C), pomalidomide (Pomalidomide), rapamycin (Rapamycin) and the two-drug combination The protein level expression of substrate protein IKZF3 after use.
  • FIG. 4 shows the protein level expression of CC90009, rapamycin and the substrate protein GSPT1 after the combination of the two drugs in HL60 (A) and MOLT-4 (B).
  • AC is the sulfonamide drug Indisulam (A) and PROTAC drugs ZNL-02-096 (B) and ARV-771 (C) in OPM2 in single and combined use. Changes in protein levels of its substrate proteins after administration of paromycin.
  • FIG. 6 Combined use of the mTOR signaling pathway inhibitor everolimus can enhance the degradation of IKZF3 and IKZF1 by pomalidomide.
  • A is the protein level expression of Pomalidomide, Everolimus and their substrate protein IKZF3 in OPM2;
  • B is the combination of CC90009 and CC885 in KP4 with rapamycin ( The protein level expression of substrate protein GSPT1 after combined use of Rapamycine and Everolimus.
  • Figure 7 Combined use of rapamycin can enhance the degradation of IKZF3 by CC-92480 and improve the killing of myeloma cells by CC-92480.
  • A is the protein expression level of CC-92480, rapamycin and the substrate protein IKZF3 after the combination of the two drugs in the pomalidomide-resistant myeloma cell line OPM2-P5000;
  • B is the protein expression level of the substrate protein IKZF3 in the pomalidomide-resistant myeloma cell line OPM2-P5000.
  • C is pomalidomide, CC-92480 , rapamycin, and the killing curves of the above two drugs combined with rapamycin in the myeloma cell line OPM2-P5000.
  • the present invention is tested in various human myeloma cell models. Through genome-wide CRISPR in vitro library screening, the present invention found that the activity of the mTOR pathway plays a crucial role in the resistance of myeloma cells to the immunomodulatory drugs IMiDs.
  • mTOR pathway inhibitors can significantly enhance immunomodulatory drugs and other molecular glue drugs (pomalidomide, CC-90009, CC-885, CC -92480 (Mezigdomide) and sulfonamide drugs, etc.) and PROTAC drugs (ZNL-02-096, ARV-771, etc.) and other corresponding protein degradation agents have a substrate protein degradation effect to improve their efficacy, prompt Clinically, the combined use of mTOR inhibitors (rapamycin, everolimus, etc.) can effectively increase the therapeutic effect of protein-degrading drugs.
  • molecular glue drugs pomalidomide, CC-90009, CC-885, CC -92480 (Mezigdomide) and sulfonamide drugs, etc.
  • PROTAC drugs ZNL-02-096, ARV-771, etc.
  • the present invention provides the use of mTOR inhibitors in the preparation of products that enhance the efficacy of targeted protein degradation drugs.
  • the mTOR inhibitor refers to a protein allosteric inhibitor or catalytic inhibitor that can inhibit the mTOR signaling pathway; preferably, the mTOR inhibitor includes rapamycin (Sirolimus), everolimus, One of Temsirolimus, Ridaforolimus, deforolimus, MK-8669, Sapanisertib, MLN0128, Vistusertib, AZD2014, CC-115, or Several kinds.
  • rapamycin Sirolimus
  • everolimus One of Temsirolimus, Ridaforolimus, deforolimus, MK-8669, Sapanisertib, MLN0128, Vistusertib, AZD2014, CC-115, or Several kinds.
  • the catalytic inhibitor is a kinase inhibitor, such as an AKT inhibitor.
  • the mTOR inhibitor inhibits activation of mTORCl and mTORC2.
  • the mTOR inhibitor inhibits the activation of one or more of the PI3K protein, AKT protein, mTOR protein, S6K1 protein, and 4EBP1 protein in the PI3K/Akt/mTOR signaling pathway.
  • the mTOR inhibitor is an anti-tumor drug that acts through the mTOR signaling pathway, and/or a drug that treats diabetes, and/or a drug that treats Alzheimer, and/or a drug that delays aging. .
  • the mTOR inhibitor is an anti-tumor drug that acts through the mTOR signaling pathway, and inhibits at least PI3K protein, AKT protein, mTOR protein, S6K1 protein, and 4EBP1 in the PI3K/Akt/mTOR signaling pathway. Activation of one or more proteins.
  • the targeted protein degradation drug is selected from at least one of molecular glue drugs and PROTAC drugs; preferably, the targeted protein degradation drug is a molecular glue drug; more preferably, the targeted protein degradation drug is a molecular glue drug;
  • the targeted protein degradation drugs are immunomodulatory drugs.
  • the molecular glue drugs include one or more of thalidomide, thalidomide derivatives, CC-90009, CC-885, indisulfonamide or CC-92480; preferably, the thalidomide
  • the ledomide derivative is selected from at least one of lenalidomide, pomalidomide, CC-122 or CC-220.
  • the PROTAC drugs include one or more of ZNL-02-096, ARV-771, ARV-110, ARV-471, KT-474, and NX-2127.
  • the mTOR inhibitor is used to enhance the ability of targeted protein degradation drugs to degrade substrates.
  • the mTOR inhibitor is used to enhance the sensitivity of substrate proteins to the targeted protein degradation drug.
  • the mTOR inhibitor is used to reduce the resistance of the targeted protein degradation drug.
  • the mTOR inhibitor is used to reduce the viability of tumor cells.
  • the mTOR inhibitor is used to treat diseases that are symptomatic with targeted protein degradation drugs.
  • the disease is selected from one or more of tumors, neurological diseases, autoimmune diseases, infectious diseases or inflammatory diseases.
  • the tumor is selected from lymphoma, hematoma or solid tumor; preferably, it is selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, Lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute Myeloid leukemia, cerebral low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pan
  • the neurological disease is selected from Parkinson's disease, Huntington's disease, multiple system atrophy, motor neuron disease, Alzheimer's disease, brain trauma, ischemic stroke, spinal cord disease, amyotrophic lateral sclerosis, multiple One or more of sclerosis or epileptic seizures.
  • the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, and polymyositis.
  • the tumor is myeloma; preferably, the myeloma cells include MM1S, NCI-H929, OPM2, U266 and RPMI-8226.
  • the rapamycin can increase the killing effect of pomalidomide on myeloma cells; more preferably, the myeloma cells are selected from myeloma cells that are sensitive to the immunomodulatory drug pomalidomide. At least one of the cell lines MM1S, H929, OPM2 and the pomalidomide-resistant myeloma cell lines U266 and RPMI-8226.
  • the rapamycin can improve the degradation effect of pomalidomide on the substrate proteins IKZF3 and IKZF1; more preferably, the rapamycin can enhance the degradation of the myeloma cell lines OPM2 and RPMI- 8226 or U266 improves the degradation effect of pomalidomide on the substrate proteins IKZF3 and IKZF1.
  • the rapamycin can improve the degradation of the substrate protein GSPT1 by CC-90009 and CC-885; more preferably, the rapamycin can enhance the degradation of the substrate protein GSPT1 in leukemia cell lines HL60 and MOLT-4. Moderately improve the degradation of substrate protein GSPT1 by CC-90009 and CC-885.
  • the rapamycin can improve the degradation of substrate protein by the sulfonamide drug indisulfonamide; more preferably, the rapamycin can improve the degradation of the sulfonamide drug in the myeloma cell line OPM2. Degradation of substrate protein RBM39 by indisulfonamide.
  • the rapamycin can improve the degradation of substrate proteins by PROTAC drugs ZNL-02-096 and ARV-771; more preferably, the rapamycin can improve the degradation of PROTAC drugs ZNL -02-096 degrades the substrate protein Wee1 and improves the degradation of the substrate BRD4 by the PROTAC drug ARV-771.
  • the everolimus can improve the degradation of substrate proteins by the protein degrading agent pomalidomide; more preferably, the everolimus can improve protein degradation in the myeloma cell line OPM2
  • the agent pomalidomide degrades the substrate proteins IKZF3 and IKZF1.
  • the rapamycin can increase the effect of the protein degrading agent CC-90009 or CC-885 on the substrate. Degradation of proteins; more preferably, the rapamycin can improve the degradation of the substrate protein GSPT1 by the protein degradation agent CC-90009 or CC-885 in the solid tumor cell line human pancreatic cancer cell line KP4.
  • the everolimus can improve the degradation of substrate proteins by the protein degrading agent CC-90009 or CC-885; preferably, the everolimus can increase the activity of human pancreatic cancer in a solid tumor cell line.
  • the degradation of the substrate protein GSPT1 by the protein degradation agent CC-90009 or CC-885 was improved.
  • the rapamycin can enhance the degradation of substrate proteins by CC-92480. In some preferred embodiments, the rapamycin can enhance the degradation of the substrate protein IKZF3 by CC-92480. In some embodiments, the rapamycin is capable of increasing degradation of the substrate protein IKZF3 by CC-92480 in a myeloma cell line. In some preferred embodiments, the rapamycin is capable of increasing the degradation of the substrate protein IKZF3 by CC-92480 in pomalidomide-sensitive or pomalidomide-resistant myeloma cell lines.
  • the rapamycin is capable of increasing the response of CC-92480 to the substrate protein IKZF3 in the pomalidomide-sensitive myeloma cell line OPM2 and the pomalidomide-resistant OPM2-P5000. degradation.
  • the rapamycin can enhance the killing effect of CC-92480 on myeloma cells. In some preferred embodiments, the rapamycin can enhance the killing effect of CC-92480 on pomalidomide-sensitive or pomalidomide-resistant myeloma cells. In some more preferred embodiments, the rapamycin can enhance the killing effect of CC-92480 on the pomalidomide-sensitive myeloma cell line OPM2 or the pomalidomide-resistant myeloma cell line OPM2-P5000. .
  • the present invention also provides a pharmaceutical composition, which contains a targeted protein degradation drug and an mTOR inhibitor, as well as pharmaceutically acceptable excipients.
  • the diseases that the pharmaceutical composition of the present invention can be used to treat are selected from one or more of tumors, neurological diseases, autoimmune diseases, infectious diseases or inflammatory diseases.
  • the content of the active components targeted protein degradation drugs and mTOR inhibitors is usually a safe and effective amount.
  • the safe and effective amount should be adjustable to those skilled in the art.
  • the activity The dosage of the components usually depends on the patient's weight, the type of application, the condition and severity of the disease.
  • the dosage of the active ingredient can usually be 1 to 1000 mg/kg/day, 20 to 200 mg/kg/day, 1 ⁇ 3mg/kg/day, 3 ⁇ 5mg/kg/day, 5 ⁇ 10mg/kg/day, 10 ⁇ 25mg/kg/day, 25 ⁇ 30mg/kg/day, 30 ⁇ 40mg/kg/day, 40 ⁇ 60mg/kg/day, 60 ⁇ 80mg/kg/day, 80 ⁇ 100mg/kg/day, 100 ⁇ 150mg/kg/day, 150 ⁇ 200mg/kg/day, 200 ⁇ 300mg/kg/day, 300 ⁇ 500mg/ kg/day, or 500 ⁇ 1000mg/kg/day.
  • the effective amount to be administered based on the severity of the condition and the health and age of the subject.
  • the effective amount usually ranges from 0.05 ng/kg body weight to about 100 mg/kg body weight.
  • the pharmaceutical composition can be adapted to any form of administration, which can be oral, nasal, rectal, Intravenous, parenteral administration, etc.
  • the pharmaceutical composition can be made into injections, sterile powder for injection, tablets, pills, capsules, lozenges, liquors, powders, granules, syrups, solutions, tinctures, aerosols, powder sprays, or suppositories. .
  • the targeted protein degradation drug and the mTOR inhibitor are administered together.
  • “Co-administered” means simultaneous administration by the same or different routes in the same formulation or in two different formulations, or sequential administration by the same or different routes.
  • “Sequential” administration means that there is a time difference in seconds, minutes, hours, or days between the targeted protein degradation drug and the mTOR inhibitor.
  • the targeted protein degradation drugs, mTOR inhibitors, and pharmaceutical compositions of the present invention can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.
  • the targeted protein degradation drugs, mTOR inhibitors and pharmaceutical compositions of the present invention are mainly targeted at mammals or their isolated cancer cells.
  • the mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc.
  • the primate is preferably a monkey, ape or human.
  • the thalidomide derivatives of the present invention include pharmaceutically acceptable salts, esters, isomers, prodrugs, polymorphs or solvates of thalidomide.
  • the pharmaceutically acceptable salts and esters include the thalidomide, salts or esters formed with the following acids: hydrochloric acid, hydrobromic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, rich Maleic acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, sulfuric acid, citric acid, tartaric acid, benzenesulfonic acid, or isethionic acid.
  • prodrug means that when taken with an appropriate method, the “prodrug” undergoes metabolism or chemical reactions in the human body and is converted into the active drug, or its pharmaceutically acceptable salts, esters, isomers, etc. Conforms, prodrugs, polymorphs or solvates.
  • the present invention also provides a method for treating diseases, which involves administering a therapeutically effective amount of the targeted protein degradation drug and mTOR inhibitor, or the above pharmaceutical composition to an individual or subject in need.
  • the disease is selected from one or more of tumors, neurological diseases, autoimmune diseases, infectious diseases or inflammatory diseases.
  • treatment refers to the administration of one or more targeted protein degradation drugs, mTOR, as described herein, to a subject, such as a mammal, such as a human, suffering from the disease, or having symptoms of the disease.
  • mTOR targeted protein degradation drugs
  • Inhibitors or the above pharmaceutical compositions are used to cure, alleviate, reduce or affect the disease or the symptoms of the disease.
  • the disease is a tumor or cancer as defined below.
  • the tumor may be selected from lymphoma, hematological tumor, or solid tumor; preferably, the tumor is selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical carcinoma, Adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid neoplasm, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal hyaline renal cell carcinoma, renal papillary cell carcinoma, Acute myeloid leukemia, low-grade cerebral glioma, hepatocellular carcinoma, lung adenocarcinoma,
  • the term "neurological disease” refers to any disorder of the brain, spinal cord, or other nervous system that produces clearly identifiable structural damage.
  • the neurological disease may be selected from Parkinson's disease, Huntington's disease, multiple system atrophy, motor neuron disease, Alzheimer's disease, brain trauma, ischemic stroke, spinal cord disease, myocardial infarction, One or more of atrophic lateral sclerosis, multiple sclerosis, or epileptic seizures.
  • the autoimmune disease may be selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, and polymyositis.
  • the mTOR inhibitor refers to a protein allosteric inhibitor or a catalytic inhibitor capable of inhibiting the mTOR signaling pathway; preferably, the mTOR inhibitor includes rapamycin (Sirolimus), Iveline Everolimus, Temsirolimus, Ridaforolimus, deforolimus, MK-8669, Sapanisertib, MLN0128, Vistusertib, AZD2014, CC- One or more of 115.
  • rapamycin Sirolimus
  • Iveline Everolimus Temsirolimus
  • Ridaforolimus Ridaforolimus
  • deforolimus MK-8669
  • Sapanisertib MLN0128
  • Vistusertib AZD2014
  • CC- One or more of 115 CC- One or more of 115.
  • the targeted protein degradation drug is selected from at least one of molecular glue drugs and PROTAC drugs; preferably, the targeted protein degradation drug is a molecular glue drug; more preferably, the targeted protein degradation drug is a molecular glue drug; Targeted protein degradation drugs are immunomodulatory drugs.
  • the molecular glue drug may include one or more of thalidomide, thalidomide derivatives, CC-90009, CC-885, indisulfonamide or CC-92480; preferably Preferably, the thalidomide derivative is selected from at least one of lenalidomide, pomalidomide, CC-220 or CC-122.
  • the PROTAC drug may include one or more of ZNL-02-096, ARV-771, ARV-110, ARV-471, KT-474, and NX-2127.
  • a “therapeutically effective amount” generally refers to an amount that, after an appropriate administration period, is capable of achieving the effect of treating the diseases listed above.
  • therapeutic should be understood in its broadest sense.
  • the term “therapeutic” does not necessarily imply that the mammal is treated until full recovery is achieved. Therefore, treatment involves alleviating the symptoms of a specific condition. Treatment can also reduce the severity of existing conditions or the frequency of flare-ups.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology.
  • the present invention activates the mTOR pathway by genetically knocking out the DEPDC5 gene (marked by the phosphorylation of downstream S6K1), which can make myeloma cells OPM2 acquire resistance to molecular glue protein degradation agents such as immunomodulatory drugs (see Figure 1 ).
  • the present invention further found that mTOR inhibitors represented by the clinical drug rapamycin can significantly improve a variety of myeloma cells with different sensitivities to IMiDs (MM1S, NCI-H929, OPM2, U266 and RPMI-8226) (see Figure 1).
  • the present invention conducts research on the molecular mechanism by which mTOR inhibitors increase the sensitivity of myeloma to immunomodulatory drugs IMiDs.
  • the present invention designs experiments to explore whether mTOR inhibitors can improve the degradation of substrate proteins by immunomodulatory drugs IMiDs.
  • Experimental results show that the present invention can effectively improve the degradation efficiency of the substrate protein IKZF of the immunomodulatory drug IMiDs when used in combination with rapamycin in myeloma cells such as OPM2, RPMI-8226 and U266. This protein is important for the survival of myeloma cells. is crucial (see Figure 3).
  • the present invention expanded the range of combined targeted protein degradation drugs and the types of diseases they are suitable for, and explored whether mTOR inhibitors can widely improve PROTAC and molecular glue-targeted proteins developed from IMiDs drugs in a variety of diseases. Degradation of drug efficacy.
  • the present invention combines rapamycin with molecular glue drugs CC-90009 and CC-885 in human leukemia granulocyte cell line HL-60, human acute lymphoblastic leukemia cell MOLT-4 and human pancreatic cancer cell line KP4. It was found that rapamycin still has the effect of assisting this type of targeted protein degradation drugs to degrade more of the substrate protein GSPT1 (see Figure 4).
  • the present invention tested the combined effect of other molecular glue drugs indisulfonamide and PROTAC drugs ZNL-02-096 (CRBN-based) and ARV771 (VHL-based) with rapamycin respectively, and observed that the substrate protein The degradation is enhanced (see Figure 5).
  • the present invention also detects the combined effect of the new generation mTOR inhibitor Everolimus on the immunomodulatory drugs IMiDs and molecular glue drugs.
  • the results show that the combination of the new generation mTOR inhibitor everolimus can also enhance the degradation of proteins critical for cell survival such as IKZF and GSPT1 (see Figure 6).
  • the present invention tested the drug combination effect of rapamycin and the new generation IKZF3 degradation agent CC-92480. It was found that rapamycin can enhance the degradation of substrate proteins by CC-92480 and enhance the killing effect of CC-92480 on myeloma cells (see Figure 7).
  • the above sgRNA was synthesized, diluted 100 times after annealing, and connected to the lentiCRISPR-V2 plasmid. After transformation and single clone selection to verify that the connection was successful and correct, plasmid extraction was performed. Viruses packaged in 15cm dish. Mix the successfully extracted plasmid (15ug) with the lentiviral packaging vector pVSV-G (7.5ug) and psPAX2 (11.25ug), and add it to the cell transfection solution EZ Trans (Liji Biotechnology) and plasmid mixture at a ratio of 3:1. Incubate for a total of 20 minutes at the appropriate ratio.
  • Seed OPM2 cells in a 12-well plate keep the cell density at 2M/well, add 300-400 ⁇ l of concentrated virus solution dropwise, and add polybrene with a final concentration of 10 ⁇ g/ ⁇ l. After balancing, centrifuge at 36°C, 800g, 120min. After centrifugation, transfer all the liquid in the 12-well plate to T 12.5 . Change the fluid the next day. On the third day, Puromycin was added to test the success of transduction. Compared with the control group, almost all the cells in the control group were killed by Puromycin due to lack of resistance in about three days, while the viable cells in the virus experimental group were the cells that were successfully transduced.
  • Figure 1A shows the detection of DEPDC5 gene knockout in the myeloma cell line OPM2 by Western blotting.
  • the protein level of DEPDC5 was Partially decreased, and the expression level of mTOR downstream pathway p-S6K1 increased, suggesting the activation of the mTOR pathway in the constructed DEPDC5 cell line, which also means that the knockout was successful.
  • the CellTiter-Lumi luminescence method cell viability detection kit (Beyotime) is used, and the chemiluminescence intensity reflects the cell viability with the help of the fluorescein luminescence reaction catalyzed by ATP in the cell culture medium.
  • Activation of the mTOR pathway can make myeloma cells OPM2 acquire resistance to immunomodulatory drugs.
  • DEPDC5 is part of the GATOR1 complex, and the GATOR-RagGTPase pathway inhibits mTORC1 activation.
  • the results in Figure 1B to E of this example show that after DEPDC5 knockout, the mTOR pathway can be activated.
  • the immunomodulatory drugs lenalidomide (Pom), pomalidomide (Len), and the new generation molecular glue drugs CC-122 and CC-220 respectively. You can see Compared with the control group, DEPDC5 knockout myeloma cells were resistant to molecular glue drugs such as immunomodulatory drugs.
  • Example 2 The killing effect of immunomodulatory drug pomalidomide in combination with rapamycin on myeloma cells
  • the mTOR signaling pathway inhibitor Ray was set up in the myeloma cell lines MM1S, H929, and OPM2 that are sensitive to the immunomodulatory drug pomalidomide and the myeloma cell lines U266 and RPMI-8226 that are resistant to pomalidomide.
  • cell viability detection was performed on days 0, 2, 4 and 5 respectively (the detection method is the same as in Example 1).
  • Example 3 Combined use of rapamycin to enhance the degradation effect of pomalidomide on IKZF3 and IKZF1
  • pomalidomide 25 nM pomalidomide and different concentration gradients of rapamycin were added to the myeloma cell lines OPM2, RPMI-8226 and U266 respectively, and the samples were collected after co-treatment for 24 hours. After the cell samples were lysed by RIPA, the obtained protein samples were Protein levels were detected using Western blotting.
  • Example 4 Combined use of rapamycin can enhance the degradation of CC90009 and CC-885 to its substrate GSPT1
  • Example 5 Combined use of rapamycin can enhance the degradation of sulfa drugs and PROTAC drugs to their substrates
  • Indisulam (Indisulam) at different concentrations (0, 0.1, 1 ⁇ M) and rapamycin (Rapamycin) at a fixed concentration of 20 nM were added to the myeloma cell line OPM2. After 24 hours, the protein level was measured using the same method as in Example 3. The results of the test are shown in Figure 5a. At a working concentration of 1 ⁇ M indisulfonamide, the combination of rapamycin has a stronger effect on degrading the indisulfonamide substrate RBM39 (Vinculin is the internal reference).
  • Indisulfonamide used in this example is a type of sulfonamide drug and a molecular glue drug, which can hold dcaf15 hostage and cause it to degrade RBM39.
  • ZNL-02-096 is a type of PROTAC drug.
  • the substrate protein is Wee1 (synthesized at Dana-Farber Cancer Institute in 2020.
  • the structure is known.
  • the reference is as follows: Li et al (2020) Development and characterization of a Wee1 kinase degrader.Cell Chem.Biol.27 57 PMID:31735695.ARV-771 is One of the PROTAC drugs, which can hijack VHL and cause it to degrade BET proteins, such as BRD4, etc.
  • the structure is known, and the reference is as follows Raina et al (2016) PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc Natl Acad SciU SA.1137124PMID:27274052
  • Example 6 Combined use of mTOR signaling pathway inhibitor everolimus can enhance the degradation of IKZF3 and IKZF1 by pomalidomide
  • mTOR pathway inhibitors include everolimus. Unlike rapamycin, everolimus, a rapamycin derivative, has been approved by the U.S. Food and Drug Administration (FDA) for clinical treatment.
  • FDA Food and Drug Administration
  • Example 7 Combined use of rapamycin can enhance the degradation of IKZF3 by CC-92480 and improve the killing of myeloma cells by CC-92480
  • pomalidomide-resistant myeloma cell line OPM2-P5000 was obtained.
  • 0.5nM and 25nM pomalidomide and CC-92480 were administered in OPM2
  • 0.5nM and 3 ⁇ M pomalidomide and CC-92480 were administered in OPM2-P5000, respectively with a fixed concentration of 20nM rapamycin.
  • the pomalidomide-resistant myeloma cell line OPM2-P5000 is set up with a single administration group of mTOR signaling pathway inhibitor rapamycin (20 nM) and a single administration group of pomalidomide (3 ⁇ M).
  • rapamycin nM
  • pomalidomide 3 ⁇ M
  • combined administration group and control group were subjected to cell viability detection on days 0, 3 and 5 respectively (the detection method is the same as in Example 1).
  • the application of the mTOR inhibitor proposed in the present invention to enhance the efficacy of targeted protein degradation drugs expands the use of mTOR inhibitors and solves the problems of drug resistance and recurrence of various diseases such as tumors. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

Abstract

一种mTOR抑制剂增强靶向蛋白降解药物功效的应用。mTOR抑制剂能够显著增强分子胶水类和PROTAC等靶向蛋白降解类的小分子药物对底物蛋白的降解，以此达到治疗疾病的目的，尤其适用于对分子胶水类药物中的免疫调节药物IMiDs耐药而导致治疗效果下降以及复发的骨髓瘤病人。一种药物组合物，所述药物组合物包含分子胶水和PROTAC类蛋白降解剂和mTOR抑制剂。达到促进靶向蛋白降解药物降解底物蛋白的能力，从而提高该类药物治疗疾病的有效性。

Description

mTOR抑制剂增强靶向蛋白降解药物功效的应用 技术领域
本发明属于生物医药领域，公开了mTOR抑制剂增强靶向蛋白降解药物功效的应用。
背景技术
免疫调节类药物(Immunomodulatory Drug,IMiDs)主要包括第一代的沙利度胺(thalidomide)，以及沙利度胺的衍生物——来那度胺(lenalidomide)、泊马度胺(pomalidomide)，以及研发中尚未临床批准的CC-122，CC-220等，是一种分子胶水类药物。在上世纪90年代，一系列文献研究表明，沙利度胺具有较为明显的抗骨髓瘤的疗效，沙利度胺又重新回到人们的视野当中。为了改善沙利度胺对胎儿的致畸作用，人们又在沙利度胺的结构基础上开发出了来那度胺、泊马度胺等。沙利度胺及其衍生物因其对免疫***的调控作用被并称为免疫调节类药物。免疫调节类药物能够通过增强IFN-(interferon gamma)的分泌来诱导IL-2(interleukin-2)调节的T细胞的增殖，还能通过抑制TNF-，IL-1等来抑制肿瘤的生成。研究表明，其不仅能显著改善初期骨髓瘤患者的病症，亦能缓解难治复发性骨髓瘤患者的病情，是治疗骨髓瘤的明星药物和一线治疗药物，然而该类药物还无法完全根治骨髓瘤，对于复发性骨髓瘤的治疗效果也欠佳，一旦复发后，骨髓瘤患者缺少其他有效疗法。
2014年，哈佛医学院Ebert团队和诺贝尔生理学或医学奖获得者Kealin Jr团队在两篇《Science》同时发表研究结果，证明免疫调节类药物IMiDs治疗骨髓瘤的分子机制，其能够通过结合Cereblon(CRBN)，使得CRL4(CRBN)E3泛素连接酶识别新的底物蛋白，从而促进其下游两种转录因子IKZF1和IKZF3的降解。而IKZF1和IKZF3对于骨髓瘤细胞的生长增殖至关重要，这就导致了免疫调节类药物对肿瘤细胞的杀伤。该机制随后被《Nature》及其子刊进一步在结构基础上证实。IMiDs药物涉及的泛素化途径是机体内调控蛋白翻译后修饰的重要生理过程。通过向目标蛋白上共价连接一种由76个氨基酸构成的蛋白--泛素，机体能够达到降解不必要蛋白并维持稳态的目的。通常情况下，泛素活化酶(E1)能够激活并将泛素转移至泛素结合酶(E2)上，而泛素连接酶(E3ligase)能与目标蛋白结合，并将E2所携带的泛素转移至目标蛋白上。最终，26S蛋白酶体能够识别多聚泛素链，并降解目标蛋白。这其中，E3泛素连接酶在底物募集上起到重要作用。
免疫调节类药物IMiDs是人们第一次发现有小分子能够通过靶向E3泛素连接酶，来挟持该泛素连接酶特异性地降解新的底物蛋白靶点。IMiDs通过结合泛素连接酶的底物招募蛋白CRBN，形成了类似于一个接头结构，从而能够与新的底物蛋白结合，将其泛素化降解。传统的抑制剂药物依赖于通过化合物破坏酶活性口袋结构域，但大部分致病蛋白如癌蛋白并 不具备口袋结构，IMiDs的分子机制的揭示，使科学家们看到利用其靶向并降解传统的“不可成药”蛋白的潜力。2015年哈佛医学院Bradner团队在《Science》上报道成功地将治疗急性粒白血病(AML)的有效药物JQ-1与一种能紧密结合CRBN的邻苯二甲酰亚胺结构“偶联”，使得该“偶联物”(文中称为dBET1)能快速且广泛地降解BRD4，从而抑制相关突变肿瘤的生长基因表达。这掀起了PROTAC(proteolysis-targeting chimeras)药物的研发浪潮。人们逐渐意识到，传统的蛋白抑制剂只能阻断目的蛋白的单个结构域的功能，且通常情况下，癌症相关蛋白在机体内会行使多个功能。这就导致了在使用传统药物过后，机体通常会采取其它弥补措施来回复功能，也就是产生了所谓的耐药性。而特异性泛素化降解相关蛋白就可以避免这一类问题，人们也据此提出了PROTAC药物的研发策略。通过一端连接目的蛋白，一端连接泛素化降解***，目的蛋白可以不断地被彻底地降解，这大大降低了所需的药物浓度，且具有较高的选择性和速度。一系列PROTAC药物因此被研发出来，且相当一部分已进入临床试验。一些曾经复杂或难以成药的靶点蛋白，比如KRAS等亦有了针对性的PROTAC药物。
但PROTAC药物由于药物分子量过大，其代谢、吸收能力和安全性也受到颇多质疑。而IMiDs药物本身为代表的、或基于IMiDs结构开发的分子胶水降解剂在避免了分子量缺点后，同样具有PROTAC药物降解靶蛋白的优势和特点。由IMiDs引发的PROTAC药物和分子胶水类药物的思考与设计，正成为抗肿瘤药物生产中崭新而光明的发展方向。
mTOR信号通路关系到细胞与外界的物质交换过程，是细胞赖以正常生存和代谢的重要信号通路。mTOR信号通路参与很多基础生命过程，从蛋白质合成到细胞自噬，均有其参与调控的身影。mTOR信号通路中起到关键作用的主要是两个复合物，mTORC1和mTORC2。mTORC1能够磷酸化下游底物S6K1，从而进一步磷酸化CAD(氨甲酰磷酸酶)，促进嘧啶的合成。mTORC1还可以通过磷酸化底物4EBP使其与eIF4E解结合，从而促进mRNA的翻译。许多研究亦表明mTOR信号通路极有可能参与对许多肿瘤药物的耐药性的调控。而雷帕霉素及其衍生物依维莫司等可以对mTOR信号通路进行抑制。依维莫司的水溶性较雷帕霉素好，具有抑制血管内皮细胞增殖的作用，目前已被批准用于治疗黑色素瘤、直肠癌等。目前尚未见有mTOR抑制剂增强靶向蛋白降解药物功效的报道。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种mTOR抑制剂增强靶向蛋白降解药物功效的应用，用于解决现有技术中疾病耐药或复发等问题，本发明要以解决的技术问题是如何进一步提高临床一线靶向蛋白降解药物如免疫调节药物IMiDs降解底物蛋白的能 力，从而提高其药效的问题。本发明通过全基因组CRISPR文库筛选研究发现，mTOR信号通路抑制剂如雷帕霉素和依维莫司能够增强分子胶水类药物和PROTAC药物对底物蛋白的降解，联用mTOR信号通路抑制剂有望能在临床治疗中提高IMiDs为代表的分子胶水类药物和PROTAC药物等蛋白降解剂的治疗效果。
本发明的目的之一是提供一种mTOR抑制剂在制备增强靶向蛋白降解药物功效的产品中的应用。
本发明的另一目的是提供一种药物组合物，所述药物组合物包含分子胶水和PROTAC类蛋白降解剂和mTOR抑制剂，以及药学上可接受的辅料。
相对于现有技术，本发明的有益效果在于：
本发明提出的mTOR抑制剂能够显著增强分子胶水类和PROTAC等靶向蛋白降解的小分子药物对底物蛋白的降解，以此达到治疗疾病的目的。尤其是对于常见对分子胶水类药物中的免疫调节药物IMiDs耐药而导致治疗效果下降以及复发的现象的骨髓瘤病人，联用mTOR抑制剂能够帮助治疗此类耐药或复发型病人，提高现有临床药物的治疗效果。多种新一代靶向IKZF之外的分子胶水类和PROTAC类蛋白降解剂药物都在研发甚至临床试验中，被誉为打破传统小分子抑制剂的新一代药物，联用mTOR抑制剂将具有增强其疗效的潜力。
附图说明
图1.mTOR通路激活能够使得骨髓瘤细胞OPM2获得对免疫调节药物的耐药性。A为在骨髓瘤细胞系OPM2中构建的DEPDC5敲除细胞系情况；B-E分别为免疫调节药物泊马度胺(B)、来那度胺(C)、CC-220(D)、CC-122(E)分别对DEPDC5敲除的OPM2的杀伤曲线。
图2.泊马度胺与mTOR信号通路抑制剂雷帕霉素联用具有更强的杀伤骨髓瘤细胞效果。A-E为泊马度胺(Poma)、雷帕霉素(Rapa)以及两药联用分别在骨髓瘤细胞系MM1S(A)、H929(B)、OPM2(C)、U266(D)和RPMI-8226(E)中的杀伤曲线。
图3.联用雷帕霉素能增强泊马度胺对IKZF3、IKZF1的降解。A-C为蛋白质免疫印迹法下，在骨髓瘤细胞系OPM2(A)、RPMI-8226(B)和U266(C)中，泊马度胺(Pomalidomide)、雷帕霉素(Rapamycin)以及两药联用后底物蛋白IKZF3等的蛋白水平表达情况。
图4.联用雷帕霉素能增强CC90009和CC-885对其底物GSPT1的降解。A-B为HL60(A)和MOLT-4(B)中，CC90009、雷帕霉素(Rapamycin)以及两药联用后底物蛋白GSPT1等的蛋白水平表达情况。
图5.联用雷帕霉素能增强磺胺类药物和PROTAC类药物对其底物的降解。A-C为OPM2中磺胺类药物Indisulam(A)和PROTAC类药物ZNL-02-096(B)、ARV-771(C)在单用和联用雷 帕霉素后其底物蛋白的蛋白水平变化情况。
图6.联用mTOR信号通路抑制剂依维莫司能增强泊马度胺对IKZF3、IKZF1的降解。A为OPM2中泊马度胺(Pomalidomide)、依维莫司(Everolimus)以及两药联用后其底物蛋白IKZF3等的蛋白水平表达情况；B为KP4中CC90009、CC885分别与雷帕霉素(Rapamycine)、依维莫司(Everolimus)联用后底物蛋白GSPT1等的蛋白水平表达情况。
图7.联用雷帕霉素能增强CC-92480对IKZF3的降解，并且提高CC-92480对骨髓瘤细胞的杀伤。A为在耐受泊马度胺的骨髓瘤细胞系OPM2-P5000中，CC-92480、雷帕霉素(Rapamycin)以及两药联用后底物蛋白IKZF3的蛋白表达水平情况；B为在对泊马度胺敏感的骨髓瘤细胞系OPM2中，CC-92480、雷帕霉素(Rapamycin)以及两药联用后底物蛋白IKZF3的蛋白表达水平情况；C为泊马度胺、CC-92480、雷帕霉素以及前述两种药分别与雷帕霉素联用在骨髓瘤细胞系OPM2-P5000中的杀伤曲线。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
本发明在多种人骨髓瘤细胞模型中展开试验。通过全基因组CRISPR体外文库筛选的方式，本发明发现mTOR通路的活性在骨髓瘤细胞对免疫调节药物IMiDs的耐药性中扮演至关重要的角色。本发明数据表明，联用mTOR通路抑制剂能够在血液瘤细胞系和实体瘤细胞系中，显著增强免疫调节药物及其他分子胶水类药物(泊马度胺、CC-90009、CC-885、CC-92480(Mezigdomide)和磺胺类药物等)和PROTAC类药物(ZNL-02-096、ARV-771等)等相应蛋白降解剂类的小分子药物的底物蛋白降解效果，提高其药效，提示临床上联用mTOR抑制剂(雷帕霉素、依维莫司等)能有效调高蛋白降解剂类药物的治疗效果。
本发明提供了mTOR抑制剂在制备增强靶向蛋白降解药物功效的产品中的应用。
所述mTOR抑制剂是指能够抑制mTOR信号通路的蛋白别构抑制剂或催化抑制剂；优选地，所述mTOR抑制剂包括雷帕霉素(Rapamycin,Sirolimus)、依维莫司(Everolimus)、坦西莫司(Temsirolimus)、地磷莫司(Ridaforolimus,deforolimus,MK-8669)、沙帕色替(Sapanisertib,MLN0128)、维妥色替(Vistusertib,AZD2014)、CC-115中的一种或几种。
在一些实施方式中，所述催化抑制剂是激酶抑制剂，例如为AKT抑制剂。
在一些实施方式中，所述mTOR抑制剂抑制mTORC1和mTORC2的活化。
在一些实施方式中，所述mTOR抑制剂抑制PI3K/Akt/mTOR信号通路中的PI3K蛋白、AKT蛋白、mTOR蛋白、S6K1蛋白、4EBP1蛋白中一种或几种的活化。
在一些实施方式中，所述的mTOR抑制剂为通过mTOR信号通路途径起作用的抗肿瘤药物、和/或治疗糖尿病药物、和/或治疗阿尔茨海默的药物、和/或延缓衰老的药物。
在一些实施方式中，所述的mTOR抑制剂为通过mTOR信号通路途径起作用的抗肿瘤药物，且至少抑制PI3K/Akt/mTOR信号通路中的PI3K蛋白、AKT蛋白、mTOR蛋白、S6K1蛋白、4EBP1蛋白中一种或几种的活化。
本发明所述应用中，所述靶向蛋白降解药物选自分子胶水类药物和PROTAC类药物中至少一种；优选地，所述靶向蛋白降解药物为分子胶水类药物；更优选地，所述靶向蛋白降解药物为免疫调节药物。
其中，所述分子胶水类药物包括沙利度胺、沙利度胺衍生物、CC-90009、CC-885、吲地磺胺或CC-92480中的一种或几种；优选地，所述沙利度胺衍生物选自来那度胺、泊马度胺、CC-122或CC-220中至少一种。
其中，所述PROTAC药物包括ZNL-02-096、ARV-771、ARV-110、ARV-471、KT-474、NX-2127中的一种或几种。
本发明所述应用中，在一些实施方式中，所述mTOR抑制剂用于增强靶向蛋白降解药物降解底物能力。
本发明所述应用中，在一些实施方式中，所述mTOR抑制剂用于增强底物蛋白对所述靶向蛋白降解药物的敏感性。
本发明所述应用中，在一些实施方式中，所述mTOR抑制剂用于降低所述靶向蛋白降解药物的耐药性。
本发明所述应用中，在一些实施方式中，所述mTOR抑制剂用于降低肿瘤细胞的活力。
本发明所述应用中，在一些实施方式中，所述mTOR抑制剂用于治疗靶向蛋白降解药物对症疾病。
本发明所述应用中，所述疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或几种。所述肿瘤选自淋巴瘤、血液瘤或实体瘤；优选地，选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾***状细胞癌、急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、*** 癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种；优选地，优选地，所述肿瘤为骨髓瘤、白血病、胰腺癌中的一种或多种。
所述神经性疾病选自帕金森病、亨廷顿病、多***萎缩、运动神经元病、阿尔茨海默病、脑部创伤、缺血性脑中风、脊髓疾病、肌萎缩性侧索硬化、多发性硬化或癫痫发作中的一种或多种。
所述自身免疫性疾病选自***性红斑狼疮、类风湿关节炎、***性硬化症、口眼干燥综合征、多发性肌炎中的一种或多种。
所述肿瘤为骨髓瘤；优选地，所述骨髓瘤细胞包括MM1S，NCI-H929，OPM2，U266和RPMI-8226。
在一些优选实施方式中，所述雷帕霉素能够提高泊马度胺对骨髓瘤细胞的杀伤作用；更优选地，所述骨髓瘤细胞选自对免疫调节药物泊马度胺敏感的骨髓瘤细胞系MM1S、H929、OPM2和对泊马度胺耐受的骨髓瘤细胞系U266、RPMI-8226之至少任一。
在一些优选实施方式中，所述雷帕霉素能够提高泊马度胺对底物蛋白IKZF3、IKZF1的降解作用；更优选地，所述雷帕霉素能够在骨髓瘤细胞系OPM2、RPMI-8226或U266中提高泊马度胺对底物蛋白IKZF3、IKZF1的降解作用。
在一些优选实施方式中，所述雷帕霉素能够提高CC-90009、CC-885对底物蛋白GSPT1的降解；更优选地，所述雷帕霉素能够在白血病细胞系HL60和MOLT-4中提高CC-90009、CC-885对底物蛋白GSPT1的降解。
在一些优选实施方式中，所述雷帕霉素能够提高磺胺类药物吲地磺胺对底物蛋白的降解；更优选地，所述雷帕霉素能够在骨髓瘤细胞系OPM2中提高磺胺类药物吲地磺胺对底物蛋白RBM39的降解。
在一些优选实施方式中，所述雷帕霉素能够提高PROTAC类药物ZNL-02-096、ARV-771对底物蛋白的降解；更优选地，所述雷帕霉素能够提高PROTAC类药物ZNL-02-096对底物蛋白Wee1的降解，提高PROTAC类药物ARV-771对底物BRD4的降解。
在一些优选实施方式中，所述依维莫司能够提高蛋白降解剂泊马度胺对底物蛋白的降解；更优选地，所述依维莫司能够在骨髓瘤细胞系OPM2中提高蛋白降解剂泊马度胺对底物蛋白IKZF3、IKZF1的降解。
在一些优选实施方式中，所述雷帕霉素能够提高蛋白降解剂CC-90009或CC-885对底物 蛋白的降解；更优选地，所述雷帕霉素能够在实体瘤细胞系人胰腺癌细胞系KP4中提高蛋白降解剂CC-90009或CC-885对底物蛋白GSPT1的降解。
在一些优选实施方式中，所述依维莫司能够提高蛋白降解剂CC-90009或CC-885对底物蛋白的降解；优选地，所述依维莫司能够在实体瘤细胞系人胰腺癌细胞系KP4中提高蛋白降解剂CC-90009或CC-885对底物蛋白GSPT1的降解。
在一些实施方式中，所述雷帕霉素能够提高CC-92480对底物蛋白的降解。在一些优选实施方式中，所述雷帕霉素能够提高CC-92480对底物蛋白IKZF3的降解。在一些实施方式中，所述雷帕霉素能够在骨髓瘤细胞系中提高CC-92480对底物蛋白IKZF3的降解。在一些优选实施方式中，所述雷帕霉素能够在对泊马度胺敏感或对泊马度胺耐受的骨髓瘤细胞系中提高CC-92480对底物蛋白IKZF3的降解。在一些更优选实施方式中，所述雷帕霉素能够在对泊马度胺敏感的骨髓瘤细胞系OPM2以及对泊马度胺耐受的OPM2-P5000中提高CC-92480对底物蛋白IKZF3的降解。
在一些实施方式中，所述雷帕霉素能够增强CC-92480对骨髓瘤细胞的杀伤作用。在一些优选实施方式中，所述雷帕霉素能够增强CC-92480对泊马度胺敏感或对泊马度胺耐受的骨髓瘤细胞的杀伤作用。在一些更优选实施方式中，所述雷帕霉素能够增强CC-92480对泊马度胺敏感的骨髓瘤细胞系OPM2或对泊马度胺耐受的骨髓瘤细胞系OPM2-P5000的杀伤作用。
本发明还提供了一种药物组合物，所述药物组合物包含靶向蛋白降解药物和mTOR抑制剂，以及药学上可接受的辅料。本发明所述药物组合物可用于治疗的疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或几种。
本发明药物组合物中，活性组分靶向蛋白降解药物和mTOR抑制剂的含量通常为安全有效量，所述安全有效量对于本领域技术人员来说应该是可以调整的，例如，所述活性组分的施用量通常依赖于患者的体重、应用的类型、疾病的病情和严重程度，例如，作为活性成分的施用量通常可以为1～1000mg/kg/day、20～200mg/kg/day、1～3mg/kg/day、3～5mg/kg/day、5～10mg/kg/day、10～25mg/kg/day、25～30mg/kg/day、30～40mg/kg/day、40～60mg/kg/day、60～80mg/kg/day、80～100mg/kg/day、100～150mg/kg/day、150～200mg/kg/day、200～300mg/kg/day、300～500mg/kg/day、或500～1000mg/kg/day。
本领域技术人员可以根据病症的严重程度和受试者的健康情况及年龄的不同考虑施用的有效量。有效量通常可在0.05ng/kg体重-约100mg/kg体重之间变动。
本发明中，所述药物组合物可以适应于任何形式的给药方式，可以是经口、鼻、直肠、 静脉、胃肠外给药等。药物组合物可以制成注射剂、注射用无菌粉末、片剂、丸剂、胶囊、锭剂、醑剂、散剂、颗粒剂、糖浆剂、溶液剂、酊剂、气雾剂、粉雾剂、或栓剂。
所述药物组合物中，所述靶向蛋白降解药物和mTOR抑制剂共同给予。“共同给予”表示在同一制剂中或在两种不同制剂中经由相同或不同途径同时给予，或通过相同或不同途径顺次给予。“顺次”给予表示在靶向蛋白降解药物和mTOR抑制剂之间具有以秒、分钟、小时或天计的时间差异。
本发明所述靶向蛋白降解药物和mTOR抑制剂、药物组合物还可与其他治疗手段结合使用，所述其他手段包括手术、放疗、化疗、靶向治疗。
本发明所述靶向蛋白降解药物和mTOR抑制剂、药物组合物主要针对的对象为哺乳动物或其离体癌细胞。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
本发明所述沙利度胺衍生物包括沙利度胺药学上可接受的盐、酯、异构体、前药、多晶型物或溶剂化物。所述药学上可接受的盐、酯包括所述沙利度胺、与如下酸形成的盐或酯：氢氯酸、氢溴酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、硫酸、柠檬酸、酒石酸、苯磺酸、或羟乙磺酸。
所述“前药”指当用适当的方法服用后，该“前药”在人体内进行代谢或化学反应而转变成所述具有活性的药物、或其药学上可接受的盐、酯、异构体、前药、多晶型物或溶剂化物。
本发明还提供了一种治疗疾病的方法，向有需要的个体或对象施用治疗有效量的所述靶向蛋白降解药物和mTOR抑制剂，或上述药物组合物。所述疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或几种。
本文所用的术语“治疗”是指给患有所述疾病、或者具有所述疾病的症状的受试者、例如哺乳动物、例如人施用一种或多种本文所述靶向蛋白降解药物、mTOR抑制剂或上述药物组合物，用以治愈、缓解、减轻或影响所述疾病或所述疾病的症状。在本发明具体的实施方案中，所述疾病是下文所定义的肿瘤或癌症。
本文所用的术语“癌症”或“肿瘤”是指异常的细胞生长和增殖，无论是恶性的还是良性的，和所有的癌前期细胞和癌细胞和组织。在一些实施方式中，所述肿瘤可以选自淋巴瘤、血液瘤或实体瘤；优选地，所述肿瘤选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾***状细胞癌、 急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、***癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种；更优选地，所述肿瘤为骨髓瘤、白血病、胰腺癌中的一种或多种。
本文所用的术语“神经性疾病”是指脑、脊髓或其他神经***的任何障碍等产生明显可识别的结构损伤。在一些实施方式中，所述神经性疾病可以选自帕金森病、亨廷顿病、多***萎缩、运动神经元病、阿尔茨海默病、脑部创伤、缺血性脑中风、脊髓疾病、肌萎缩性侧索硬化、多发性硬化或癫痫发作中的一种或多种。
在一些实施方式中，所述自身免疫性疾病可以选自***性红斑狼疮、类风湿关节炎、***性硬化症、口眼干燥综合征、多发性肌炎中的一种或多种。
在一些实施方式中，所述mTOR抑制剂是指能够抑制mTOR信号通路的蛋白别构抑制剂或催化抑制剂；优选地，所述mTOR抑制剂包括雷帕霉素(Rapamycin,Sirolimus)、依维莫司(Everolimus)、坦西莫司(Temsirolimus)、地磷莫司(Ridaforolimus,deforolimus,MK-8669)、沙帕色替(Sapanisertib,MLN0128)、维妥色替(Vistusertib,AZD2014)、CC-115中的一种或几种。
在一些实施方式中，所述靶向蛋白降解药物选自分子胶水类药物和PROTAC类药物中至少一种；优选地，所述靶向蛋白降解药物为分子胶水类药物；更优选地，所述靶向蛋白降解药物为免疫调节药物。
在一些实施方式中，所述分子胶水类药物可以包括沙利度胺、沙利度胺衍生物、CC-90009、CC-885、吲地磺胺或CC-92480中的一种或几种；优选地，所述沙利度胺衍生物选自来那度胺、泊马度胺、CC-220或CC-122中至少一种。
在一些实施方式中，所述PROTAC药物可以包括ZNL-02-096、ARV-771、ARV-110、ARV-471、KT-474、NX-2127中的一种或几种。
本文述及“包含”、“含有”等应理解为是包括性的意思，而没有排他性或穷尽的意思；即“包括但不限于”的意思。
本文述及“治疗有效量”通常指一用量在经过适当的给药期间后，能够达到治疗如上所列出的疾病的效果。
本文述及“治疗性”应理解为其最宽的意义。术语“治疗性”不一定暗示哺乳动物接受治疗直至完全恢复。因此，治疗包括缓解具体病症的症状。治疗也可降低已有病症的严重程度或急性发作的频率。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
本发明通过基因敲除DEPDC5基因使得mTOR通路激活后(下游S6K1被磷酸化激活为标志)，能够使得骨髓瘤细胞OPM2获得对免疫调节药物等分子胶水类蛋白降解剂的耐药性(见图1)。相反，本发明进一步发现，通过以临床药物雷帕霉素(Rapamycin)为代表的mTOR抑制剂，能够显著提高泊马度胺对IMiDs敏感性不同的多种骨髓瘤细胞(MM1S、NCI-H929，OPM2，U266和RPMI-8226)的杀伤(见图1)。
接着本发明对mTOR抑制剂提高骨髓瘤对免疫调节药物IMiDs的敏感性的分子机制展开研究，本发明设计实验探究mTOR抑制剂能否提高免疫调节药物IMiDs对底物蛋白的降解。实验结果表明，本发明在OPM2、RPMI-8226和U266等骨髓瘤细胞中联用雷帕霉素，能有效提高免疫调节药物IMiDs的底物蛋白IKZF的降解效率，该蛋白对于骨髓瘤细胞的存活至关重要(见图3)。
之后，本发明扩大了联用的靶向蛋白降解药物范围与其适应的疾病类型，探究mTOR抑制剂能否在多种疾病中广泛地提高由IMiDs药物发展而来的PROTAC和分子胶水类靶向蛋白降解药物药效。本发明在人白血病粒细胞系HL-60、人急性淋巴母细胞白血病细胞MOLT-4和人胰腺癌细胞系KP4中将雷帕霉素与分子胶水类药物CC-90009、CC-885联用，发现雷帕霉素仍然具有协助这一类靶向蛋白降解药物更多降解底物蛋白GSPT1的效果(见图4)。之 后，本发明测试了其他分子胶水类药物吲地磺胺以及PROTAC类药物ZNL-02-096(CRBN-based)、ARV771(VHL-based)分别与雷帕霉素的联用效果，观察到底物蛋白的降解增强(见图5)。
此外，本发明还检测了新一代的mTOR抑制剂依维莫司(Everolimus)对免疫调节药物IMiDs和分子胶水类药物的联用效果。结果显示新一代mTOR抑制剂依维莫司的联用也能增强IKZF和GSPT1等对细胞生存至关重要的蛋白降解(见图6)。
最后，本发明检测了雷帕霉素与新一代IKZF3降解剂CC-92480的药物联用效果。发现雷帕霉素能够提高CC-92480对底物蛋白的降解，并且增强CC-92480对骨髓瘤细胞的杀伤作用(见图7)。
实施例1 DEPDC5基因敲除骨髓瘤细胞系OPM2对免疫调节药物的耐药性
1、DEPDC5基因敲除骨髓瘤细胞系OPM2的建立
sgDEPDC5质粒的构建与包装病毒：
设计靶向DEPDC5基因的sgRNA，序列为：
正向：caccgGCTACATCAGTGAAGATACC(SEQ ID NO.1)；
反向：aaacGGTATCTTCACTGATGTAGCc(SEQ ID NO.2)。
合成上述sgRNA，退火后稀释100倍，将其连接到lentiCRISPR-V2质粒上。经转化、挑单克隆检验连接成功且无误后，对其进行质粒抽提。在15cm dish中包装病毒。将抽提成功的质粒(15ug)与慢病毒包装载体pVSV-G(7.5ug)、psPAX2(11.25ug)进行混合，在细胞转染液EZ Trans(李记生物)与质粒混合液3：1的比例下共孵育20分钟。20分钟后均匀滴加至处于对数生长期的悬浮状态的293FT细胞上，第二天换液。第四天吸取上清，在利用0.45μM滤网过滤上清后使用lenti-X-concentrator病毒浓缩液(Clontech)进行浓缩。过夜后收取浓缩病毒液(1ml/盘)。
悬浮骨髓瘤细胞系OPM2的病毒感染：
在12孔板中种下OPM2细胞，保持细胞密度为2M/孔，滴加浓缩病毒液300-400μl，并加入终浓度为10μg/μl的polybrene。配平后，在36℃，800g，120min的条件下离心。离好心后，将12孔板中的液体全部转移到T12.5中。第二天进行换液。第三天开始加Puromycin进行转导成功与否的检验。与control组相比，三天左右control组细胞因缺乏抗性，几乎能被Puromycin全部杀死，而病毒实验组中活细胞即为转导成功的细胞。
图1A为蛋白质免疫印迹法下检测在骨髓瘤细胞系OPM2中DEPDC5基因的敲除情况；由图1A可知，在构建的DEPDC5敲除细胞系中，与control组相比，DEPDC5的蛋白水平有 部分下降，mTOR下游通路p-S6K1的表达水平有所升高，提示了构建的DEPDC5细胞系中mTOR通路的激活，也意味着敲除是成功的。
2、免疫调节药物和分子胶水药物对细胞进行处理
(1)取对数生长期的OPM2细胞，以3000个/孔，90μl/孔的条件种在96孔板中。
(2)将1mM的来那度胺(Len)、泊马度胺(Pom)、CC-122和CC-220溶于培养基后加10μl至每孔中，使得每孔中药物的终浓度从1μM依次梯度递减，并设置不加药组，每种药物共7组，每组3个重复。
(3)将培养板放置在37℃，5％的CO2浓度的培养箱中培养5天。
(4)之后避光条件下在每孔中加入50μl的CTL溶液，避光放置在摇床上10min，在酶标仪上读数。
(5)取3个重复的平均吸光度值绘制药物处理下OPM2的细胞活力曲线
细胞活力检测，利用CellTiter-Lumi发光法细胞活力检测试剂盒(Beyotime)，借助细胞培养液中ATP催化的荧光素发光反应，由化学发光强度反映细胞活力。
测定结果：
mTOR通路激活能够使得骨髓瘤细胞OPM2获得对免疫调节药物的耐药性。DEPDC5是GATOR1复合物的一部分，而GATOR-RagGTPase通路能够抑制mTORC1的激活。本实施例图1B～E结果显示，在DEPDC5敲除后，可以使得mTOR通路激活。在骨髓瘤细胞系OPM2中敲除DEPDC5后，用免疫调节药物来那度胺(Pom)、泊马度胺(Len)、和新一代分子胶水药物CC-122、CC-220分别处理，可以看到与对照组相比，DEPDC5敲除的骨髓瘤细胞能够对免疫调节药物等分子胶水药物具有耐药性。
实施例2免疫调节药物泊马度胺与雷帕霉素联用对骨髓瘤细胞的杀伤作用
本实施例分别在对免疫调节药物泊马度胺敏感的骨髓瘤细胞系MM1S、H929、OPM2和对泊马度胺耐受的骨髓瘤细胞系U266、RPMI-8226中设置mTOR信号通路抑制剂雷帕霉素单独给药组、泊马度胺单独给药组、联合用药组和对照组，分别在第0、2、4、5天进行细胞活力检测(检测方法同实施例1)。
结果如图2所示，在此五种细胞系中，泊马度胺和雷帕霉素联合用药与泊马度胺单独给药相比P＜0.001(two-wayANOVA)，说明泊马度胺(Pom)与mTOR信号通路抑制剂雷帕霉素(RaPa)联用具有更强的杀伤骨髓瘤细胞的效果(图2中数据代表两次独立实验结果，n＝3，mean±s.e.m.)。
实施例3联用雷帕霉素增强泊马度胺对IKZF3、IKZF1的降解作用
分别向骨髓瘤细胞系OPM2、RPMI-8226和U266中加入25nM的泊马度胺与不同浓度梯度的雷帕霉素，共处理24h后收样，通过RIPA裂解细胞样品后，将取得的蛋白样利用免疫印迹法进行蛋白水平检测。
实验结果如图3所示，单独给予雷帕霉素时，泊马度胺的底物作用蛋白IKZF3水平不变；而联用雷帕霉素后，IKZF3的水平明显降低，说明联用雷帕霉素能增强泊马度胺对IKZF3、IKZF1的降解。
实施例4联用雷帕霉素能增强CC90009和CC-885对其底物GSPT1的降解
在白血病细胞系HL60和MOLT-4中分别加入10nM的CC-90009与不同浓度梯度的雷帕霉素，共处理12h后收样，通过与实施例3相同的方法进行蛋白水平检测。
实验结果如图4所示，单独给予雷帕霉素时，CC-90009的底物作用蛋白GSPT1的水平不变；而联用雷帕霉素后，GSPT1的水平明显降低，说明联用雷帕霉素能增强CC90009和CC-885对其底物GSPT1的降解。
实施例5联用雷帕霉素能增强磺胺类药物和PROTAC类药物对其底物的降解
在骨髓瘤细胞系OPM2中加入不同浓度(0、0.1、1μM)的吲地磺胺(Indisulam)和固定20nM浓度的雷帕霉素(Rapamycin)，24h后采用与实施例3相同的方法进行蛋白水平检测，结果如图5a所示，在1μM的吲地磺胺工作浓度下，联用雷帕霉素有更强的降解吲地磺胺底物RBM39的效果(Vinculin为内参)。
在骨髓瘤细胞系OPM2中加入不同浓度(0、1、10nM)的ZNL-02-096和固定20nM浓度的雷帕霉素(Rapamycin)，24h后采用与实施例3相同的方法进行蛋白水平检测，结果如图5b所示，能观察到在10nM的ZNL-02-096工作浓度下，联用雷帕霉素有更强的降解ZNL-02-096底物Wee1的效果(Vinculin为内参)。
在骨髓瘤细胞系OPM2中加入不同浓度(0、0.5、1nM)的ARV-771和固定20nM浓度的雷帕霉素(Rapamycin)，24h后采用与实施例3相同的方法进行蛋白水平检测，结果如图5c所示，能观察到在0.5nM或1nM的ARV-771工作浓度下，联用雷帕霉素有更强的降解ARV-771底物BRD4的效果(Vinculin为内参)。结果说明，联用雷帕霉素能增强磺胺类药物和PROTAC类药物对其底物的降解。
本实施例中使用的吲地磺胺是磺胺类药物的一种，亦是一种分子胶水类药物，其能够挟持dcaf15令其降解RBM39。ZNL-02-096是PROTAC类药物的一种，底物蛋白为Wee1(2020年于Dana-Farber癌症研究所合成的，结构已知，参考文献如下Li et al(2020)Development and characterization of a Wee1 kinase degrader.Cell Chem.Biol.27 57 PMID:31735695。ARV-771是 PROTAC类药物的一种，能够挟持VHL令其降解BET蛋白，如BRD4等，结构已知，参考文献如下Raina et al(2016)PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer.Proc Natl Acad SciU SA.1137124PMID:27274052。
实施例6联用mTOR信号通路抑制剂依维莫司能增强泊马度胺对IKZF3、IKZF1的降解
除了雷帕霉素，mTOR通路抑制剂还有依维莫司(Everolimus)等。与雷帕霉素不同的是，作为雷帕霉素衍生物，依维莫司得到了美国食品药品监督管理局(FDA)的批准用于临床治疗。
在骨髓瘤细胞系OPM2中，加入不同浓度(0、1、10、102、103nM)的ZNL-02-096和固定25nM浓度的泊马度胺(Pomalidomide)，24h后，采用与实施例3相同的方法进行蛋白水平检测，结果如图6a所示，结果显示在骨髓瘤细胞系OPM2中，浓度梯度的依维莫司能够增强泊马度胺对底物蛋白IKZF3、IKZF1的降解。
在实体瘤细胞系—人胰腺癌细胞系KP4中，加入不同浓度(0、0.01、0.1μM)雷帕霉素(Rapamycine)、依维莫司(Everolimus)，以及和固定1μM浓度的CC-90009和0.1μM浓度的CC-885，24h后，采用与实施例3相同的方法进行蛋白水平检测。结果如图6b所示，结果显示在人胰腺癌细胞系KP4中，不同浓度的雷帕霉素或依维莫司能够增强CC-90009或CC-885对底物GSPT1的降解。
实施例7联用雷帕霉素能增强CC-92480对IKZF3的降解，并且提高CC-92480对骨髓瘤细胞的杀伤
本实施例在对免疫调节药物泊马度胺敏感的骨髓瘤细胞系OPM2进行长时间高浓度的泊马度胺给药后，获得了对泊马度胺耐药的骨髓瘤细胞系OPM2-P5000。在OPM2中给药0.5nM、25nM的泊马度胺、CC-92480，在OPM2-P5000中给药0.5nM、3μM的泊马度胺、CC-92480,并分别与固定浓度20nM的雷帕霉素联用。
结果如图7a、图7b所示，在对泊马度按耐药的骨髓瘤细胞系中，联用雷帕霉素能够增强泊马度胺和CC-92480对底物蛋白IKZF3的降解。
同时，本实施例对泊马度胺耐受的骨髓瘤细胞系OPM2-P5000中设置mTOR信号通路抑制剂雷帕霉素单独给药组(20nM)、泊马度胺单独给药组(3μM)、CC-92480单独给药组(3μM)、联合用药组和对照组，分别在第0、3、5天进行细胞活力检测(检测方法同实施例1)。
结果如图7c所示，联用雷帕霉素后CC-92480具有更强的杀伤骨髓瘤细胞的效果(n＝3，mean±s.e.m.)。
综上所述，本发明提出的mTOR抑制剂增强靶向蛋白降解药物功效的应用，扩大了mTOR抑制剂的用途，解决了多种疾病如肿瘤的耐药和复发问题。所以，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (33)

  1. mTOR抑制剂在制备增强靶向蛋白降解药物功效的产品中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述mTOR抑制剂是指能够抑制mTOR信号通路的蛋白别构抑制剂或催化抑制剂。
  3. 如权利要求1所述的应用，其特征在于，所述mTOR抑制剂包括雷帕霉素(Rapamycin,Sirolimus)、依维莫司(Everolimus)、坦西莫司(Temsirolimus)、地磷莫司(Ridaforolimus,deforolimus,MK-8669)、沙帕色替(Sapanisertib,MLN0128)、维妥色替(Vistusertib,AZD2014)、CC-115中的一种或几种。
  4. 如权利要求1所述的应用，其特征在于，所述靶向蛋白降解药物选自分子胶水类药物和PROTAC类药物中至少一种。
  5. 如权利要求1所述的应用，其特征在于，所述靶向蛋白降解药物为免疫调节药物。
  6. 如权利要求4所述的应用，其特征在于，包括以下至少任一：
    1)所述分子胶水类药物包括沙利度胺、沙利度胺衍生物、CC-90009、CC-885、吲地磺胺或CC-92480中的一种或几种；
    2)所述PROTAC药物包括ZNL-02-096、ARV-771、ARV-110、ARV-471、KT-474、NX-2127中的一种或几种。
  7. 如权利要求6所述的应用，其特征在于，所述沙利度胺衍生物选自来那度胺、泊马度胺、CC-220或CC-122中至少一种。
  8. 如权利要求1所述的应用，其特征在于，所述增强靶向蛋白降解药物功效包括以下至少任一项：
    1)增强靶向蛋白降解药物降解底物蛋白的能力；
    2)增强底物蛋白对所述靶向蛋白降解药物的敏感性；
    3)降低所述靶向蛋白降解药物的耐药性；
    4)增强靶向蛋白降解药物对肿瘤细胞活力的抑制作用；
    5)增强靶向蛋白降解药物治疗疾病的效果。
  9. 如权利要求8所述的应用，其特征在于，包括以下至少任一：
    1)增强泊马度胺对骨髓瘤细胞的杀伤作用；
    2)提高泊马度胺对底物蛋白的降解作用；
    3)提高CC-90009、CC-885对底物蛋白的降解；
    4)提高吲地磺胺对底物蛋白的降解；
    5)提高PROTAC类药物对底物蛋白的降解；
    6)提高CC-92480对底物蛋白的降解；
    7)增强CC-92480对骨髓瘤细胞的杀伤作用。
  10. 如权利要求9所述的应用，其特征在于，包括以下至少任一：
    1)所述骨髓瘤细胞对泊马度胺敏感或对泊马度胺耐受；
    2)提高泊马度胺对底物蛋白IKZF3、IKZF1的降解作用；
    3)提高CC-90009、CC-885对底物蛋白GSPT1的降解；
    4)提高吲地磺胺对底物蛋白RBM39的降解；
    5)提高PROTAC类药物ZNL-02-096、ARV-771对底物蛋白Wee1的降解；
    6)提高CC-92480对底物蛋白IKZF3的降解；
    7)增强CC-92480对泊马度胺敏感或对泊马度胺耐受的骨髓瘤细胞的杀伤作用。
  11. 如权利要求9所述的应用，其特征在于，增强CC-92480对泊马度胺敏感的骨髓瘤细胞系OPM2或对泊马度胺耐受的骨髓瘤细胞系OPM2-P5000的杀伤作用。
  12. 如权利要求1所述的应用，其特征在于，所述靶向蛋白降解药物用于治疗的疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或几种。
  13. 如权利要求12所述的应用，其特征在于，所述肿瘤选自淋巴瘤、血液瘤或实体瘤；
    和/或，所述神经性疾病选自帕金森病、亨廷顿病、多***萎缩、运动神经元病、阿尔茨海默病、脑部创伤、缺血性脑中风、脊髓疾病、肌萎缩性侧索硬化、多发性硬化或癫痫发作中的一种或多种；
    和/或，所述自身免疫性疾病选自***性红斑狼疮、类风湿关节炎、***性硬化症、口眼干燥综合征、多发性肌炎中的一种或多种。
  14. 如权利要求12所述的应用，其特征在于，所述肿瘤选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾***状细胞癌、急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、***癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种。
  15. 如权利要求12所述的应用，其特征在于，所述肿瘤为骨髓瘤、白血病、胰腺癌中的一种或多种。
  16. 一种药物组合物，其特征在于，所述药物组合物包含靶向蛋白降解药物和mTOR抑制剂，以及药学上可接受的辅料。
  17. 如权利要求16所述的药物组合物，其特征在于，所述mTOR抑制剂是指能够抑制mTOR信号通路的蛋白别构抑制剂或催化抑制剂。
  18. 如权利要求16所述的药物组合物，其特征在于，所述mTOR抑制剂包括雷帕霉素(Rapamycin,Sirolimus)、依维莫司(Everolimus)、坦西莫司(Temsirolimus)、地磷莫司(Ridaforolimus,deforolimus,MK-8669)、沙帕色替(Sapanisertib,MLN0128)、维妥色替(Vistusertib,AZD2014)、CC-115中的一种或几种。
  19. 如权利要求16所述的药物组合物，其特征在于，所述靶向蛋白降解药物选自分子胶水类药物和PROTAC类药物中至少一种。
  20. 如权利要求16所述的药物组合物，其特征在于，所述靶向蛋白降解药物为免疫调节药物。
  21. 如权利要求16所述的药物组合物，其特征在于，所述靶向蛋白降解药物用于治疗的疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或几种。
  22. 如权利要求21所述的药物组合物，其特征在于，所述肿瘤选自淋巴瘤、血液瘤或实体瘤；
    和/或，所述神经性疾病选自帕金森病、亨廷顿病、多***萎缩、运动神经元病、阿尔茨海默病、脑部创伤、缺血性脑中风、脊髓疾病、肌萎缩性侧索硬化、多发性硬化或癫痫发作中的一种或多种；
    和/或，所述自身免疫性疾病选自***性红斑狼疮、类风湿关节炎、***性硬化症、口眼干燥综合征、多发性肌炎中的一种或多种。
  23. 如权利要求21所述的药物组合物，其特征在于，所述肿瘤选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾***状细胞癌、急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、***癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种。
  24. 如权利要求21所述的药物组合物，其特征在于，所述肿瘤为骨髓瘤、白血病、胰腺 癌中的一种或多种。
  25. 一种治疗疾病的方法，向有需要的个体或对象施用治疗有效量的靶向蛋白降解药物、mTOR抑制剂或如权利要求16-24任一所述的药物组合物，所述疾病选自肿瘤、神经性疾病、自身免疫性疾病、传染性疾病或炎性疾病中的一种或多种。
  26. 如权利要求25所述的方法，其特征在于，所述肿瘤选自淋巴瘤、血液瘤或实体瘤；
    和/或，所述神经性疾病选自帕金森病、亨廷顿病、多***萎缩、运动神经元病、阿尔茨海默病、脑部创伤、缺血性脑中风、脊髓疾病、肌萎缩性侧索硬化、多发性硬化或癫痫发作中的一种或多种；
    和/或，所述自身免疫性疾病选自***性红斑狼疮、类风湿关节炎、***性硬化症、口眼干燥综合征、多发性肌炎中的一种或多种。
  27. 如权利要求25所述的方法，其特征在于，所述肿瘤选自肾上腺皮质癌、膀胱尿路上皮癌、乳腺癌、宫颈鳞状细胞癌、宫颈内腺癌、胆管癌、结肠腺癌、淋巴样肿瘤、弥散性大B细胞淋巴瘤、食管癌、多形性成胶质细胞瘤、头颈部鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾***状细胞癌、急性髓性白血病、脑低度胶质瘤、肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮细胞癌、卵巢癌、胰腺癌、嗜铬细胞瘤和副神经节瘤、***癌、直肠癌、恶性肉瘤、黑色素瘤、胃癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺癌、子宫内膜癌、子宫肉瘤、葡萄膜黑色素瘤、多发性骨髓瘤、急性淋系白血病、慢性淋系白血病、慢性髓性白血病、T细胞淋巴瘤、B细胞淋巴瘤肿瘤细胞中的一种或多种。
  28. 如权利要求25所述的方法，其特征在于，所述肿瘤为骨髓瘤、白血病、胰腺癌中的一种或多种。
  29. 如权利要求25所述的方法，其特征在于，所述mTOR抑制剂是指能够抑制mTOR信号通路的蛋白别构抑制剂或催化抑制剂；
    和/或，所述靶向蛋白降解药物选自分子胶水类药物和PROTAC类药物中至少一种。
  30. 如权利要求25所述的方法，其特征在于，所述mTOR抑制剂包括雷帕霉素(Rapamycin,Sirolimus)、依维莫司(Everolimus)、坦西莫司(Temsirolimus)、地磷莫司(Ridaforolimus,deforolimus,MK-8669)、沙帕色替(Sapanisertib,MLN0128)、维妥色替(Vistusertib,AZD2014)、CC-115中的一种或几种；
    和/或，所述靶向蛋白降解药物为分子胶水类药物和PROTAC药物。
  31. 如权利要求25所述的方法，其特征在于，所述靶向蛋白降解药物为免疫调节药物。
  32. 如权利要求29所述的方法，其特征在于，所述分子胶水类药物包括沙利度胺、沙利 度胺衍生物、CC-90009、CC-885、吲地磺胺、或CC-92480中的一种或几种；
    所述PROTAC药物包括ZNL-02-096、ARV-771、ARV-110、ARV-471、KT-474、NX-2127中的一种或几种。
  33. 如权利要求32所述的方法，其特征在于，所述沙利度胺衍生物选自来那度胺、泊马度胺、CC-122或CC-220中至少一种。
PCT/CN2023/089697 2022-05-06 2023-04-21 mTOR抑制剂增强靶向蛋白降解药物功效的应用 WO2023213197A1 (zh)

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