WO2024099346A1 - Use of indazole compound in treatment of inflammasome activation-mediated diseases - Google Patents

Use of indazole compound in treatment of inflammasome activation-mediated diseases Download PDF

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WO2024099346A1
WO2024099346A1 PCT/CN2023/130416 CN2023130416W WO2024099346A1 WO 2024099346 A1 WO2024099346 A1 WO 2024099346A1 CN 2023130416 W CN2023130416 W CN 2023130416W WO 2024099346 A1 WO2024099346 A1 WO 2024099346A1
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methyl
asc
carboxylic acid
alkyl
indazole
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Chinese (zh)
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银巍
陈晨
黄奕俊
颜光美
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中山大学
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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Definitions

  • the present invention relates to the medical use of indazole compounds, and in particular to the use of lonidamine and its analogs in the treatment of diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • LND Lonidamine
  • indazole-3-carboxylic acid that inhibits hexokinase, the rate-limiting enzyme in the glycolytic pathway.
  • LND was first reported as a candidate for contraceptives and then used as an anti-tumor drug due to its anti-Warburg effect of inhibiting glycolysis.
  • Recently, the anti-inflammatory effects of LND have been observed in animal models of ischemic stroke and arthritis, but its exact mechanism of action has not been fully elucidated.
  • Inflammasomes are multi-molecular protein complexes in cells that respond to innate immune recognition caused by microbial infection and endogenous danger signals and are one of the key steps in the inflammatory response. Inflammasomes are composed of pattern recognition receptors (PRRs), adaptor proteins, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1.
  • PRRs pattern recognition receptors
  • ASC apoptosis-associated speck-like protein containing a CARD
  • caspase-1 caspase-1
  • PRRs include nucleotide-binding oligomerization domain (NOD) proteins, leucine-rich repeat (LRR) protein NLR (leucine-rich repeat (LRR)-containing protein) family members NLRP1 (NLR family, pyrin domain containing 1), NLRP3 (NLR family, pyrin domain containing 1) and NLRC4 (NLR Family CARD Domain Containing 4), as well as proteins absent in melanoma 2 (AIM2) and Pyrin.
  • NLRP3 can respond to a variety of stimuli, including bacteria, viruses, extracellular ATP, pollutants, metabolic disorders and tissue damage.
  • the senor binds to ASC and promotes ASC oligomerization to form ASC specks, which in turn recruits caspase-1 precursors and activates caspase-1.
  • Activated caspase-1 ultimately leads to the maturation and secretion of pro-inflammatory mediators IL-1 ⁇ and IL-18, and cleaves gasdermin D (GSDMD) to induce pyroptosis.
  • GDMD gasdermin D
  • ASC intracellular oligomerization to form ASC specks is necessary for the assembly and activation of a variety of inflammasomes.
  • extracellular ASC specks have been shown to have inflammatory signaling functions, including inducing neutrophil infiltration, accelerating A ⁇ deposition, and regulating adaptive immunity.
  • Increased ASC oligomers have been detected in the serum of patients with chronic respiratory diseases or autoinflammatory diseases, and ASC oligomers have also been detected in the cerebrospinal fluid of patients with subarachnoid hemorrhage and traumatic brain injury. Therefore, extracellular ASC specks can be used as potential biomarkers for these diseases.
  • inflammasome inhibitors In order to prevent and treat inflammasome-related diseases, inflammasome inhibitors with different targets have been designed and developed. Some compounds can directly bind to NLRP3 to block the activation of inflammasomes, including MCC950, CY-09, OLT1177, tranilast, and oridonin. Glyburide, BHB, and fenamate inhibit the activation of inflammasomes by inhibiting the upstream activation signals of NLRP3. However, since these upstream signals are involved in a variety of biological processes, these compounds can produce off-target effects and toxic side effects. For example, the highly selective and potent NLRP3 inhibitor MCC950 was terminated in a Phase II clinical trial for the treatment of rheumatoid arthritis due to liver toxicity. At present, these inflammasome inhibitors have shown therapeutic effects in experimental animal models, but have not been clinically used to treat various inflammatory-related diseases. In addition, inflammasome inhibitors with other non-NLRP3 targets are rarely reported.
  • ASC oligomerization is a key event in the assembly of inflammasomes, and the dysregulated activation of inflammasomes has been shown to be associated with the development and prognosis of a variety of diseases. Without being bound by theory, the inventors believe that blocking ASC oligomerization prevents inflammasome assembly events and ASC spot formation, thereby inhibiting the dysregulated activation of inflammasomes, and can be used to treat diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • One aspect of the present invention provides the use of a compound of formula Ia or Ib in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • R1 is selected from hydrogen, halogen, C1-3 alkyl, amino, C1-3 alkylamino and halogenated C1-3 alkyl
  • R2 is selected from carboxyl, C1-3 alkyl and hydrazide
  • R3 is H or C1-3 alkyl
  • R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl
  • R6 is H or C1-3 alkyl
  • Z is selected from C, N , O and S
  • p is 1, 2 or 3
  • the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • Another aspect of the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound represented by Formula Ia or Ib, wherein each substituent is defined as described above, and the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • Another aspect of the present invention provides a compound represented by formula Ia or Ib for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein each substituent is defined as described above, and the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • Another aspect of the present invention provides an ASC mutant protein, which comprises an amino acid residue mutation at at least one of I115, F163, W169 and L192, and the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2 (SEQ ID NO: 1).
  • Another aspect of the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, comprising identifying candidate drugs that interact with at least one of the amino acid residues I115, F163, W169 and L192 of the ASC protein.
  • FIG. 1 LND reduces inflammatory damage in EAE.
  • Body weight changes after EAE induction; n 12.
  • Representative H&E and Luxol Fast Blue (LFB) staining. Scale bar 100 ⁇ m.
  • LFD LND reduces inflammatory damage in EAE.
  • Body weight changes after EAE induction; n 10.
  • a, c, d, e, h, and i are unpaired t tests, and g and j are multiple unpaired t tests.
  • FIG. 1 LND inhibits inflammasome activation in vivo.
  • (c and d) ELISA analysis of IL-1 ⁇ (c) and IL-18 (d) in the serum of mice 4 hours after intraperitoneal injection of LPS (15 mg/kg), whether pretreated with LND or not, n 15. Data are expressed as mean ⁇ SEM. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
  • LND is a broad-spectrum inhibitor of the NLRP3 inflammasome.
  • (c and d) ELISA analysis of IL-1 ⁇ (c) and IL-18 (d) in the supernatant of BMDMs.
  • LPS-stimulated BMDMs were pretreated with or without LND (200 ⁇ M) for 30 min and then stimulated with nigericin, ATP, MSU, or imiquimod.
  • LND inhibits NLRP3 inflammasome activation independently of HK2.
  • Data are expressed as mean ⁇ SEM, *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
  • LND prevents the formation of ASC specks.
  • LPS-stimulated BMDMs were treated with LND (0.2 mM) for 0.5 h and then stimulated with inflammasome activators (ATP (5 mM, 30 min), poly(dA:dT) (1 ⁇ g/ml, 8 h), MDP (200 ng/ml, 8 h), or flagellin (200 ng/ml, 8 h)).
  • IL-1 ⁇ in the supernatant of BMDMs was analyzed by ELISA. Data are presented as mean ⁇ SEM. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
  • FIG. 6 LND directly binds to ASC to inhibit NLRP3 assembly.
  • the docking model of ASC and LND was established using Ligand scout software. LND is shown as a stick in green. The CARD domain of ASC is shown in cartoon form in pink, while the interaction site is in orange. The yellow dashed line represents the hydrogen bond.
  • the graph showing the binding of LND to recombinant ASC protein was obtained by SPR analysis using Biacore. Different concentrations of LND are presented as an overlay.
  • C LPS-stimulated BMDMs were lysed with protein lysis buffer and incubated with LND overnight at 4°C.
  • DARTS detection was performed using pronase enzyme (20 ng/ ⁇ g protein) and analyzed by Western blot.
  • pronase enzyme (20 ng/ ⁇ g protein) and analyzed by Western blot.
  • halogen refers to fluorine, chlorine, bromine or iodine or a group thereof.
  • number of halogens is not limited, it can be any suitable number, such as monohalogen, dihalogen, trihalogen; when the position of the halogen is not limited, it can be any suitable position, for example, the halogenated phenyl can be halogenated at the ortho position, para position, meta position or a combination thereof.
  • alkyl refers to a saturated straight or branched hydrocarbon chain.
  • alkyl group having a specific number of carbon atoms the term includes the corresponding normal alkyl group and its various isomeric forms (if any).
  • an alkyl group having 3 carbon atoms C3 alkyl
  • C1-3 alkyl groups include methyl, ethyl, propyl, isopropyl.
  • C 1-3 alkylamino refers to an amino group substituted by a C 1-3 alkyl group, and may be an amino group substituted by one or two C 1-3 alkyl groups, for example, methylamino, dimethylamino, diethylamino, methylethylamino and the like.
  • halogenated C 1-3 alkyl refers to a C 1-3 alkyl group substituted by halogen, and may be a C 1-3 alkyl group substituted by one or more halogens simultaneously, such as fluoromethyl, difluoromethyl, trifluoromethyl and the like.
  • carboxyphenyl refers to a phenyl group substituted by a carboxyl group, and may be a phenyl group substituted by one or more carboxyl groups.
  • hydrozide refers to -C(O)-NH- NH2 .
  • terapéuticaally effective amount refers to an amount of a conjugate of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, that participates in carrying or delivering the conjugate from one organ or part of the body to another organ or part of the body.
  • a pharmaceutically acceptable material such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, that participates in carrying or delivering the conjugate from one organ or part of the body to another organ or part of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.
  • treatment encompasses prevention, therapy, and cure.
  • the patient receiving such treatment is generally any animal in need, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.
  • diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway refers to diseases mediated by abnormal inflammasome activation, which is based on the oligomerization of ASC to form ASC specks, which in turn recruits caspase-1 precursors and activates caspase-1.
  • Activated caspase-1 ultimately leads to the maturation and secretion of pro-inflammatory mediators IL-1 ⁇ and IL-18, and cleaves gasdermin D (GSDMD) to induce pyroptosis.
  • GDMD gasdermin D
  • Forma I includes Formula Ia and Formula Ib
  • Formula II includes Formula IIa and IIb
  • Formula III includes Formula IIIa and IIIb
  • Formula IV includes Formula IVa, IVb, and IVc.
  • One aspect of the present invention provides the use of a compound represented by Formula Ia or Ib or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide
  • R 3 is H or C 1-3 alkyl
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl
  • R 6 is H or C 1-3 alkyl
  • Z is selected from C, N, O and S
  • p is 1, 2 or 3
  • the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • R 1 is selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is selected from C, N, O and S, preferably C or N, more preferably C;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is selected from C, N, O and S, preferably C or N, more preferably C;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is selected from C, N, O and S, preferably C or N, more preferably C;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is selected from C, N, O and S, preferably C or N, more preferably C;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H;
  • Z is selected from C, N, O and S, preferably C or N, more preferably C;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is C or N;
  • p is 1, 2 or 3, preferably 1.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl;
  • R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl;
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H or C 1-3 alkyl, preferably H;
  • Z is C or N;
  • p is 1.
  • R 1 is selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
  • R 2 is carboxyl;
  • R 3 is H or methyl;
  • R 4 and R 5 are independently selected from hydrogen, methyl, fluorine and chlorine;
  • R 6 is H;
  • Z is C;
  • p is 1.
  • a compound represented by Formula IIa or IIb or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from methyl, fluorine and chlorine
  • the disease mediated by inflammasome activation based on the ASC-Caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from methyl, fluorine and chlorine, more preferably fluorine or chlorine
  • the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic airway inflammation, gout, multiple sclerosis and se
  • a compound represented by Formula IVa, IVb or IVc or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
  • R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl
  • R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; more preferably selected from hydrogen, fluorine, chlorine and difluoromethyl
  • R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from fluorine and chlorine, more preferably both are chlorine
  • the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic air
  • the compound of Formula I is selected from any of the following (the numbers in brackets represent PubChem CID):
  • the compound of formula I is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid (ie, lonidamine, with a structural formula as shown in the following formula V) or a pharmaceutically acceptable salt or ester thereof.
  • the present invention provides the use of any compound of Formula I to Formula V as described above in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • the present invention provides use of any compound of Formula I to Formula V as described above in the preparation of a medicament for treating gout.
  • the present invention provides use of any one of the compounds of Formula I to Formula V as described above in the preparation of a medicament for treating multiple sclerosis.
  • the present invention provides use of any one of the compounds of Formula I to Formula V as described above in the preparation of a medicament for treating sepsis.
  • the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating gout.
  • the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating multiple sclerosis.
  • the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating sepsis.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any compound represented by any one of Formula I to Formula V or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable composition includes one or more of the above-mentioned compounds in a therapeutically effective amount, used alone or formulated with one or more pharmaceutically acceptable carriers (additives), excipients and/or diluents.
  • the compounds according to the invention may be formulated for administration in any convenient way by analogy from other drugs for use in human or veterinary medicine.
  • Another aspect of the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering to a subject in need thereof a therapeutically effective amount of any compound of any one of Formulas I to V described herein, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia, and AIDS.
  • the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis, and sepsis.
  • the disease is gout.
  • the disease is multiple sclerosis.
  • the disease is sepsis.
  • the compound of Formula I to Formula V is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof. Therefore, in some embodiments, the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof to a subject in need thereof, the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof to a subject in need thereof, the disease being selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • the present invention provides a method for treating multiple sclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
  • the present invention provides a method for treating sepsis, comprising administering to a subject in need thereof a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
  • Another aspect of the present invention provides any compound of any of Formula I to Formula V described herein for use in treating a disease mediated by inflammasome activation based on the ASC-Caspase-1 pathway, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • the disease is multiple sclerosis.
  • the disease is sepsis.
  • the compound of Formula I to Formula V is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof. Therefore, in some embodiments, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  • the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating gout.
  • the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating multiple sclerosis. In a preferred embodiment, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating sepsis.
  • Any compound of Formula I to Formula V provided by the present invention can be prepared by, for example, the method described in US20070015771A1 (Examples 8 to 39 thereof provide detailed preparation methods of indazole compounds), and the entirety of this patent document is incorporated herein by reference.
  • MS Multiple sclerosis
  • CNS central nervous system
  • the distribution, morphology, and signal performance of lesions on MRI are characteristic.
  • MS lesions are characterized by temporal multiple (DIT) and spatial multiple (DIS).
  • Clinically isolated syndrome refers to the first clinical attack, which often involves the unilateral optic nerve, spinal cord, brainstem, and can also involve the cerebral hemisphere. The lesions can be one or more. Not all patients progress to MS after the first attack, and CIS patients need close follow-up.
  • Remission-relapsing syndrome RRMS: In the early stages of the disease, patients often have repeated attacks, and most patients have complete relief of symptoms after the attack, which is called RRMS. 60-80% of patients will experience a stage of remission and relapse.
  • Secondary progressive syndrome After the patient experiences repeated attacks, the symptoms cannot be completely relieved, and the residual symptoms gradually accumulate and irreversible neurological disability occurs, that is, entering the progressive stage, so it is called secondary progressive syndrome.
  • Progressive relapsing syndrome PRMS
  • PRMS Progressive relapsing syndrome
  • PPMS Primary progressive syndrome
  • Acute MS is an acute variant of MS, which is aggressive, progresses rapidly in a short period of time, and often has multiple large lesions at the same time.
  • the present invention is intended to be used for any clinical classification of MS.
  • Sepsis also known as sepsis
  • sepsis-3 septic shock
  • SIRS systemic inflammatory response syndrome
  • Hyperuricemia causes gout, renal failure, etc.
  • the treatment of hyperuricemia mainly uses xanthine oxidase inhibitors such as allopurinol and febuxostat.
  • Gout attacks are caused by excessive uric acid in the blood and accumulation of uric acid crystals in the joints, which causes strong inflammation and onset of disease, accompanied by severe pain.
  • uric acid-lowering drugs There is a correlation between uric acid-lowering drugs and acute gout attacks. It is known that the sharp reduction of serum uric acid will produce short-term local precipitation of sodium urate crystals in cartilage and soft tissue, leading to acute gout attacks. That is, it is known that uric acid-lowering drugs in the past may induce gout attacks. Therefore, depending on the patient, it is sometimes necessary to stop treatment or change treatment guidelines due to gout attacks.
  • the present invention is expected to be used to treat gout.
  • Another aspect of the present invention provides an ASC mutant protein, which comprises an amino acid residue mutation at at least one of I115, F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising an amino acid residue mutation at I115, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising an amino acid residue mutation at F163, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising an amino acid residue mutation at W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising an amino acid residue mutation at L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and F163, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the ASC mutant protein further comprises amino acid residue mutations at one or more of T166, L178 and S195.
  • the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163, T166, W169, L178, L192 and S195, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • the present invention provides an ASC mutant protein comprising I115A, F163A, T166A, W169A, L178A, L192A and S195A mutations, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  • GenBank:BAA87339.2 The amino acid sequence of GenBank:BAA87339.2 is shown in SEQ ID NO:1, where the amino acid residues at the preferred mutation sites are shown in bold.
  • the present invention contemplates that the provided ASC mutant proteins can be used, for example, for disease treatment and drug screening.
  • a method for treating a disease mediated by activation of the ASC-caspase-1 inflammasome pathway is provided, the disease preferably being selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis, the method comprising administering a therapeutically effective amount of the ASC mutant protein or its encoding nucleic acid to a subject in need thereof.
  • the ASC mutant protein competes with the wild-type ASC protein to reduce the number of ASC oligomers and/or spots formed.
  • a method for treating a disease mediated by activation of the ASC-caspase-1 inflammasome pathway comprising administering a therapeutically effective amount of a base editor to a subject in need, the base editor causing a mutation in the ASC encoding gene, the mutation comprising at least one amino acid residue mutation at I115, F163, W169 and L192 to form any ASC mutant protein described in the present invention.
  • the base editor is, for example, an adenosine base editor (ABE) or a cytidine base editor (CBE).
  • the base editor is delivered to a cell via a vector such as a plasmid, a virus (eg, an adenovirus, an adeno-associated virus vector or a lentiviral vector).
  • a virus eg, an adenovirus, an adeno-associated virus vector or a lentiviral vector.
  • the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  • Another aspect of the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with at least one of the amino acid residues I115, F163, W169 and L192 of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the I115 amino acid residue of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the F163 amino acid residue of the ASC protein.
  • the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the W169 amino acid residue of the ASC protein.
  • the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the L192 amino acid residue of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and F163 amino acid residues of the ASC protein.
  • the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and W169 amino acid residues of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and L192 amino acid residues of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, and W169 amino acid residues of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, and L192 amino acid residues of the ASC protein.
  • the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the F163, W169, and L192 amino acid residues of the ASC protein.
  • the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, W169 and L192 amino acid residues of the ASC protein.
  • the method includes identifying a candidate drug that also interacts with at least one of the amino acid residues T166, L178 and S195 of the ASC protein.
  • LND lonidamine
  • MS multiple sclerosis
  • HK2 hexokinase 2
  • LND is a broad-spectrum inflammasome inhibitor that directly targets ASC, providing a new therapeutic approach for the clinical treatment of inflammasome-driven diseases.
  • LND blocks inflammasome activation in an HK2-independent manner by directly targeting ASC and shows therapeutic effects in animal models of multiple inflammasome-related diseases, including MS and sepsis.
  • Anti-IL-1 ⁇ antibody was from RD Systems
  • anti-GSDMD anti-GSDMD
  • anti-NEK7 antibodies anti-NEK7 antibodies
  • anti-caspase-1 AG-20B-0042B
  • anti-ASC antibodies AG-25B-0006
  • anti- ⁇ -tubulin antibody ARG65693
  • LPS Erichia coli O111:B4; #L2630
  • ATP A6419
  • nigericin 481990
  • R-837 were obtained from Selleck.
  • MSU crystals (tlrl-msu), muramyld dipeptide (tlrl-mdp), flagellin from S. typhimurium (tlrl-stfla), and poly(dA:dT)/LyoVecTM were from Invivogen.
  • Anti-hexokinase 2 antibody (NBP2-02272) was from Novus.
  • Anti-NLRP3 antibody (15101) was from Cell Signaling Technology.
  • Goat anti-rabbit IgG (HRP) (arg65351), Donkey anti-goat IgG (HRP) (arg65352), and Goat anti-Mouse IgG antibodies (HRP) (arg65350) were all from Arigo.
  • Disuccinimidyl was purchased from Thermo Scientific.
  • mice C57BL/6J mice were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China).
  • Conditional hexosidase II knockout mice B6.129P2(Cg)-Hk2tm1.1Uku/Kctt, HK2 CKO flox/flox
  • EMMA European Mouse Mutation Bank
  • B6J.B6n(Gg)-cx3cr1tm1.1(CRE)jung/j, Cx3cr1-Cre T) mice were obtained from Syy Biotech (Guangzhou) Co., Ltd. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee and the Laboratory Animal Ethics Committee of Sun Yat-sen University (No. SYSU-IACUC-2022-B0070).
  • BMDMs were isolated from the bone marrow of 6-8 week old mice and cultured in DMEM medium containing 10% FBS and 20% supernatant of L929 cells (ATCC) for 7 to 8 days.
  • ATCC supernatant of L929 cells
  • HK2 in the mononuclear-macrophage cell line was selectively knocked out using CX3CR1-cre ⁇ HK2flox/flox mice, and primary BMDMs were then isolated from these HK2 knockout mice.
  • J774A.1 cells were from ATCC and cultured in DMEM containing 10% FBS.
  • BMDMs (5 ⁇ 10 5 cells/mL) and J774A.1 cells (3 ⁇ 10 5 cells/mL) were plated in 6-well plates and cultured overnight, and the medium was replaced with fresh DMEM the next morning. Then, the cells were induced with LPS (500 ng/mL) for 3 hours. After that, LND was added to the culture and incubated for 30 minutes.
  • LPS 500 ng/mL
  • the cells were stimulated with ATP (5 mM) for 30 minutes, MSU (150 ⁇ g/mL), Iiquimod (100 ⁇ M) and nigericin (10 ⁇ M) for 4 hours, poly (dA: dT) (2 ⁇ g/mL, 6 hours), MDP (500 ng/mL, 6 hours) and flagellin (1 ⁇ g/mL, 6 hours).
  • ATP 5 mM
  • MSU 150 ⁇ g/mL
  • Iiquimod 100 ⁇ M
  • nigericin 10 ⁇ M
  • poly (dA: dT) (2 ⁇ g/mL, 6 hours)
  • MDP 500 ng/mL, 6 hours
  • flagellin 1 ⁇ g/mL, 6 hours
  • LPS-induced sepsis model 8-week-old male C57BL/6 mice were intraperitoneally injected with LND (40 mg/kg and 60 mg/kg) or solvent control ( ⁇ -cyclodextrin) 0.5 hours before intraperitoneal injection of LPS (15 mg/kg) (Sigma-Aldrich, L2630). Four hours later, the mice were euthanized, and the levels of IL-1 and IL-18 in the serum were measured using ELISA kits according to the manufacturer's instructions.
  • 0 no symptoms
  • 1 loss of tail rigidity
  • 2 unsteady gait
  • 3 hind limb paralysis
  • 4 forelimb paralysis
  • 5 moribund or dead. If the score was between two points, a grading of 0.5 points was used.
  • ASC oligomerization detection BMDMs were seeded in 6-well plates, washed with ice-cold PBS, and 500 ⁇ L of ice-cold buffer (20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 1% NP40, protease inhibitors) was added. The cells were lysed at 4°C for 30 min, and 50 ⁇ L of lysate was taken out for Western blot detection. Then, the remaining lysate was centrifuged at 2500 ⁇ g for 10 min at 4°C. The pellet was resuspended in 500 ⁇ L of ice-cold PBS.
  • succinimidyl ester (Thermo Fisher A39267) was added to the resuspended pellet and incubated with rotation at room temperature for 30 min. Then, centrifuged at 2500 ⁇ g for 10 min at 4°C. The supernatant was removed and the cross-linked pellet was resuspended in 30 ⁇ L of Laemmli buffer. The samples were boiled at 100°C for 10 min and analyzed by Western blot.
  • ELISA method Analyze mouse IL-1 ⁇ (LIANKE BIOTECH, EK201B/3), IL-18 (LIANKE BIOTECH, EK218) and TNF- ⁇ (LIANKE BIOTECH, EK282/3) in cell culture supernatant and serum according to the reagent manufacturer's instructions.
  • IP Immunoprecipitation
  • Immunohistochemical staining Brain and spinal cord samples were analyzed according to the manufacturer's instructions of the immunohistochemical (IHC) staining kit (Abcam, ab80436, Cambridge, MA, USA). Briefly, after deparaffinization and hydration, the sections were placed in 3% hydrogen peroxide for 15 minutes and antigen retrieval was performed for 20 minutes before cooling to room temperature. The sections were stained with H&E, Nissl, or LFB, respectively. The samples were incubated with the indicated primary antibodies overnight at 4°C in antibody diluent containing background reducing agent (DAKO, S3022, Santa Clara, CA, USA). Then, after washing three times with PBS, the sections were stained with diaminobenzidine (DAB) substrate-color-changing agent mixture and hematoxylin in sequence.
  • DAB diaminobenzidine
  • Immunofluorescence staining During tissue sectioning, the brain and spinal cord were routinely isolated and fixed. Then, the samples were embedded in paraffin and cut into 5 ⁇ m thick sections. After deparaffinization and hydration, the sections were subjected to antigen retrieval for 20 min and cooled to room temperature. The samples were incubated with the designated primary antibodies at 4 °C overnight in antibody diluent with background elimination reagent (DAKO, S3022, Santa Clara, CA, USA). The samples were washed three times with PBS and incubated with the designated fluorescence-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific, Rockford, IL, USA) for 1 h.
  • DAKO antibody diluent with background elimination reagent
  • Flow cytometry To analyze the infiltrating immune cells of the central nervous system, the brain tissue and spinal cord of MOG 35-55 immunized mice were ground with a tissue homogenizer to form a single cell suspension, which was filtered through a 300 mesh filter. After centrifugation, the single cell suspension was resuspended with 37% percoll and centrifuged for 30 minutes at 1000 ⁇ g, room temperature, minimum acceleration setting and no brake. Mononuclear cells were separated from the lowest layer. The cells were suspended in PBS containing 1% FBS. Washed three times and stained with cell surface marker antibodies for flow cytometric analysis. The following antibodies were used.
  • CD45-BV510 (BD, 563891), CD4-FITC (BioLegend, 100406), CD8-Alexa Fluor 700 (BD, 557959), and CD11b-BV421 (BioLegend, 101236).
  • Flow cytometric analysis was performed by flow cytometer (CytoFLEX, Beckman Coulter). Cell debris and dead cells were excluded based on forward and side scatter and Fixable Viability Stain 780 (BD, 565388).
  • siRNA-mediated gene interference in BMDMs Small interfering RNA was purchased from RiboBio (Guangzhou RiboBio Co., Ltd.). RNA interference was performed using Lipofectamine TM RNAiMAX (Invitrogen, 13778) according to the manufacturer's instructions. RNA interference was performed on cells that reached 60-70% confluence. Samples were collected 48 hours after RNA interference and Western blot or real-time PCR were performed as described above. The sequences of siRNA are as follows.
  • DARTS assay DARTS was performed according to published protocols. BMDMs (5 ⁇ 10 5 cells/mL) were plated in 10 cm dishes and cultured overnight, and the medium was replaced with fresh DMEM the next morning. Then, cells were induced with LPS for 4 h and lysed with M-PER (Thermo, 78501) lysis buffer. The lysate was centrifuged at 12,000 ⁇ g for 10 min at 4 °C, and the protein concentration was determined by BCA protein concentration assay. The lysate was incubated with LND overnight at 4 °C. 80 ⁇ g of protein lysate was used for each reaction.
  • M-PER Thermo, 78501
  • pronase enzyme Sigma, PRON-RO
  • 20 ng of pronase enzyme Sigma, PRON-RO
  • the digestion was terminated by adding 20 ⁇ protease inhibitor, and the samples were incubated on ice for 10 min.
  • 5 ⁇ SDS-PAGE loading buffer was added to the samples to reach a final concentration of 1 ⁇ SDS-PAGE buffer. Protein samples were analyzed by Western blot.
  • Transmission electron microscopy Purified expressed ASC protein samples (6 ⁇ M) were added with TEV enzyme (6 UI) and different concentrations of LND and incubated overnight at 4 °C. A drop of 10 ⁇ l of sample was placed on a clean Parafilm and a mesh copper pure carbon coated grid was floated on top for 10 min. Then, the grid was transferred and contrasted with 1% uranyl acetate for 5 min. Excess fluid was removed and allowed to dry before examination using a transmission electron microscope FEI Tecnai G2 Spirit (ThermoFisher Scientific Company, OR, USA). All images were acquired using Radius software and a Xarosa digital camera (EMSIS GmbH, Weg, Germany).
  • SPR Surface plasmon resonance analysis: The experiments were performed in a Biacore T100. Recombinant human ASC protein (Zeye Biotechnology) was immobilized on a CM5 sensor chip (BR100530, Cytiva). LND was dissolved in basic running buffer (PBS with 1% DMSO) and injected into the flow cell at a flow rate of 5 ⁇ L/min at 25 °C. The sensor chip was washed with basic running buffer between each concentration. ASC protein was immobilized in different channels of the same chip, and the response values obtained by injecting blank basic running buffer in the same way were used as controls. The kinetic parameters and affinity constants of the interaction were calculated using the Biacore T100 evaluation software.
  • Example 1 reduces inflammatory damage in experimental autoimmune encephalomyelitis (EAE).
  • Example 2 inhibits the activation of inflammasomes.
  • Inflammasomes are the main sensors of sterile inflammatory signals and a key trigger of inflammatory responses.
  • LND could inhibit inflammasome activation in vivo.
  • the spinal cord of EAE mice described above we found that the increased levels of GSDMD and caspase-1 were significantly reduced after LND treatment ( Figures 2a and 2b). This suggests that LND prevented inflammasome activation in the central nervous system tissues of EAE mice.
  • LPS lipopolysaccharide
  • Example 3 LND inhibited NLRP3 inflammasome activation induced by different agonists.
  • the NLRP3 inflammasome can be activated by other danger-associated molecular patterns, such as nigericin and monosodium urate (MSU), which are dependent on potassium ion efflux.
  • MSU monosodium urate
  • Treatment with LND inhibited caspase-1 cleavage and IL-1 ⁇ secretion triggered by nigericin and MSU ( Figures 3o and 3p).
  • imiquimod an NLRP3 inflammasome activator that is independent of potassium ion efflux, also inhibited imiquimod-induced caspase-1 and IL-1 ⁇ activation ( Figures 3o and 3p).
  • Example 4 inhibits NLRP3 inflammasome activation independently of HK2.
  • LND inhibits glycolysis by inhibiting the activity of HK2.
  • LND has other targets, including voltage-dependent anion channel (VDAC), mitochondrial pyruvate carrier (MPC) and monocarboxylate transporters (MCT) and succinate dehydrogenase (SDH).
  • VDAC voltage-dependent anion channel
  • MCT mitochondrial pyruvate carrier
  • MCT monocarboxylate transporters
  • SDH succinate dehydrogenase
  • siRNA-mediated interference with HK2 did not reduce caspase-1 cleavage and IL-1 ⁇ production in BMDMs ( Figures 4c and 4d) and J1774A.1 cells ( Figures 4e and 4f).
  • LPS- and ATP-induced NLRP3 inflammasome activation was comparable in BMDMs from wild-type (WT) mice and HK2 knockout mice ( Figure 4g), and LND still attenuated NLRP3 activation in BMDMs from HK2 knockout mice ( Figures 4h and 4i). This result suggests that LND inhibits NLRP3 inflammasome activation independent of HK2.
  • Example 5 selectively blocks oligomerization of ASC.
  • LND inhibits inflammasome activation.
  • a series of upstream events such as potassium ion efflux and the generation of reactive oxygen species (ROS)
  • ROS reactive oxygen species
  • LND can inhibit NLRP3 inflammasome activation induced by different activators that are dependent or independent of potassium ion efflux ( Figure 3).
  • LND did not inhibit the generation of ROS when inhibiting inflammasome activation ( Figure 5a).
  • NIMA-related kinase 7 (NEK7) has been identified as an essential component of the NLRP3 inflammasome, and the interaction between NEK7 and NLRP3 has been shown to be critical for NLRP3 oligomerization and inflammasome assembly.
  • LND had no effect on the binding between NEK7 and NLRP3 in BMDMs ( Figure 5b).
  • ASC-mediated ligation of NLRP3 and caspase-1 is another key step in inflammasome assembly.
  • LND did not block the interaction between NLRP3 and ASC ( Figure 5c), but inhibited the binding between ASC and caspase-1 ( Figure 5d).

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Abstract

The present invention provides use of a compound as represented by formula I or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating ASC-caspase-1 pathway-based inflammasome activation-mediated diseases. In particular, the compound of the present invention is preferably lonidamine, and is used for treating gout, multiple sclerosis or septicemia.

Description

吲唑类化合物在治疗炎症小体激活介导的疾病中的应用Application of indazole compounds in the treatment of diseases mediated by inflammasome activation 技术领域Technical Field
本发明涉及吲唑类化合物的医药用途，具体涉及氯尼达明及其类似物在在治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病中的应用。The present invention relates to the medical use of indazole compounds, and in particular to the use of lonidamine and its analogs in the treatment of diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway.
背景技术Background technique
氯尼达明(Lonidamine,LND)是吲唑-3-羧酸的衍生物，可以抑制糖酵解途径限速酶己糖激酶。LND首先被报道为避孕药的候选药物，然后由于其抑制糖酵解的反Warburg效应而被用作抗肿瘤药物。最近，在缺血性中风和关节炎的动物模型中观察到LND的抗炎作用，但其确切作用机制尚未完全阐明。Lonidamine (LND) is a derivative of indazole-3-carboxylic acid that inhibits hexokinase, the rate-limiting enzyme in the glycolytic pathway. LND was first reported as a candidate for contraceptives and then used as an anti-tumor drug due to its anti-Warburg effect of inhibiting glycolysis. Recently, the anti-inflammatory effects of LND have been observed in animal models of ischemic stroke and arthritis, but its exact mechanism of action has not been fully elucidated.
炎症小体是细胞内的多分子蛋白复合物，对微生物感染和内源性危险信号引起的先天免疫识别做出反应，是炎症反应的关键步骤之一。炎症小体由模式识别受体(pattern recognition receptor，PRR)、适配器蛋白即凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein containing a CARD，ASC)和caspase-1组成。目前已经确定了几种PRRs，包括核苷酸结合的寡聚结构域NOD(nucleotide-binding oligomerization domain)蛋白、含亮氨酸丰富的重复(LRR)蛋白NLR(leucine-rich repeat(LRR)-containing protein)家族成员NLRP1(NLR family,pyrin domain containing 1)、NLRP3(NLR family,pyrin domain containing 1)和NLRC4(NLR Family CARD Domain Containing 4)，以及黑色素瘤2缺失蛋白(proteins absent in melanoma 2,AIM2)和Pyrin。其中，NLRP3可以对各种各样的刺激作出反应，包括细菌、病毒、细胞外ATP、污染物、代谢失调和组织损伤。一旦被激活，传感器就会与ASC结合，并促进ASC低聚化形成ASC斑点(ASC specks)，进而招募caspase-1前体并激活caspase-1。被激活的caspase-1最终导致促炎症介质IL-1β和IL-18的成熟和分泌，并裂解gasdermin D(GSDMD)来诱导细胞焦亡。Inflammasomes are multi-molecular protein complexes in cells that respond to innate immune recognition caused by microbial infection and endogenous danger signals and are one of the key steps in the inflammatory response. Inflammasomes are composed of pattern recognition receptors (PRRs), adaptor proteins, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Several PRRs have been identified, including nucleotide-binding oligomerization domain (NOD) proteins, leucine-rich repeat (LRR) protein NLR (leucine-rich repeat (LRR)-containing protein) family members NLRP1 (NLR family, pyrin domain containing 1), NLRP3 (NLR family, pyrin domain containing 1) and NLRC4 (NLR Family CARD Domain Containing 4), as well as proteins absent in melanoma 2 (AIM2) and Pyrin. Among them, NLRP3 can respond to a variety of stimuli, including bacteria, viruses, extracellular ATP, pollutants, metabolic disorders and tissue damage. Once activated, the sensor binds to ASC and promotes ASC oligomerization to form ASC specks, which in turn recruits caspase-1 precursors and activates caspase-1. Activated caspase-1 ultimately leads to the maturation and secretion of pro-inflammatory mediators IL-1β and IL-18, and cleaves gasdermin D (GSDMD) to induce pyroptosis.
ASC细胞内寡聚化形成ASC斑点是多种炎症小体组装与激活所必需。此外，细胞外的ASC斑点已被证实具备炎症信号功能，包括可诱导中性粒细胞的浸润、加速Aβ沉积以及调节适应性免疫。在慢性呼吸道疾病或自体炎症疾病患者的血清中检测到ASC寡聚体增加，在蛛网膜下腔出血和创伤性脑损伤患者的脑脊液中同样检测到ASC寡聚体，因此细胞外的ASC斑点可作为这些疾病的潜在生物标志物。这些发现强烈表明，靶向ASC可能是治疗炎症性疾病的一个新策略。ASC intracellular oligomerization to form ASC specks is necessary for the assembly and activation of a variety of inflammasomes. In addition, extracellular ASC specks have been shown to have inflammatory signaling functions, including inducing neutrophil infiltration, accelerating Aβ deposition, and regulating adaptive immunity. Increased ASC oligomers have been detected in the serum of patients with chronic respiratory diseases or autoinflammatory diseases, and ASC oligomers have also been detected in the cerebrospinal fluid of patients with subarachnoid hemorrhage and traumatic brain injury. Therefore, extracellular ASC specks can be used as potential biomarkers for these diseases. These findings strongly suggest that targeting ASC may be a new strategy for treating inflammatory diseases.
为防治炎症小体相关疾病，不同作用靶点的炎症小体抑制剂被设计并研发。一些化合物可以直接结合NLRP3来阻断炎症小体的激活，包括MCC950、CY-09、OLT1177、tranilast和oridonin。而glyburide、BHB和fenamate通过抑制NLRP3的上游激活信号来抑制炎症小体的活化。然而，由于这些上游信号参与了多种多样的生物过程，这些化合物会产生脱靶效应与毒副作用。例如，高选择性和强效的NLRP3抑制剂MCC950由于肝脏毒性，在治疗类风湿性关节炎的II期临床试验中被终止。目前，这些炎症小体抑制剂在实验性动物模型上显示出治疗效果，但还没有在临床上应用于各种炎症相关疾病治疗。此外，以其他非NLRP3靶点的炎症小体抑制剂少见报道。In order to prevent and treat inflammasome-related diseases, inflammasome inhibitors with different targets have been designed and developed. Some compounds can directly bind to NLRP3 to block the activation of inflammasomes, including MCC950, CY-09, OLT1177, tranilast, and oridonin. Glyburide, BHB, and fenamate inhibit the activation of inflammasomes by inhibiting the upstream activation signals of NLRP3. However, since these upstream signals are involved in a variety of biological processes, these compounds can produce off-target effects and toxic side effects. For example, the highly selective and potent NLRP3 inhibitor MCC950 was terminated in a Phase II clinical trial for the treatment of rheumatoid arthritis due to liver toxicity. At present, these inflammasome inhibitors have shown therapeutic effects in experimental animal models, but have not been clinically used to treat various inflammatory-related diseases. In addition, inflammasome inhibitors with other non-NLRP3 targets are rarely reported.
发明内容Summary of the invention
ASC的寡聚化是炎症小体组装的关键事件，而炎症小体的失调激活已经被证明与多种疾病发生发展和预后有关。不愿受理论约束，发明人认为阻断ASC寡聚化阻止了炎症小体组装事件及ASC斑点形成，从而抑制炎症小体的失调激活，可用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病。ASC oligomerization is a key event in the assembly of inflammasomes, and the dysregulated activation of inflammasomes has been shown to be associated with the development and prognosis of a variety of diseases. Without being bound by theory, the inventors believe that blocking ASC oligomerization prevents inflammasome assembly events and ASC spot formation, thereby inhibiting the dysregulated activation of inflammasomes, and can be used to treat diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway.
本发明的一个方面提供式Ia或Ib的化合物在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病中的应用，
One aspect of the present invention provides the use of a compound of formula Ia or Ib in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
其中：R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1- 3烷基和酰肼基；R3为H或C1-3烷基；R4和R5独立地选自氢、卤素和C1-3烷基；R6为H或C1-3烷基；Z选自C、N、O和S；p为1、2或3；并且所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。Wherein: R1 is selected from hydrogen, halogen, C1-3 alkyl, amino, C1-3 alkylamino and halogenated C1-3 alkyl; R2 is selected from carboxyl, C1-3 alkyl and hydrazide; R3 is H or C1-3 alkyl; R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl; R6 is H or C1-3 alkyl; Z is selected from C, N , O and S; p is 1, 2 or 3; and the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
本发明的另一个方面提供一种治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的方法，所述方法包括向有此需要的对象施用治疗有效量的式Ia或Ib所示的化合物，其中各取代基定义如上所述，并且所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。 Another aspect of the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound represented by Formula Ia or Ib, wherein each substituent is defined as described above, and the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
本发明的另一个方面提供用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的式Ia或Ib所示的化合物，其中各取代基定义如上所述，并且所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。Another aspect of the present invention provides a compound represented by formula Ia or Ib for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein each substituent is defined as described above, and the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
本发明的另一个方面提供一种ASC突变蛋白，其包含在I115、F163、W169和L192中的至少一个处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定(SEQ ID NO:1)。Another aspect of the present invention provides an ASC mutant protein, which comprises an amino acid residue mutation at at least one of I115, F163, W169 and L192, and the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2 (SEQ ID NO: 1).
本发明的另一个方面提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，包括识别与ASC蛋白的I115、F163、W169和L192中的至少一个氨基酸残基相互作用的候选药物。Another aspect of the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, comprising identifying candidate drugs that interact with at least one of the amino acid residues I115, F163, W169 and L192 of the ASC protein.
本发明的其他方面将可从以下说明书中的详细描述中容易得到。Other aspects of the present invention will be readily apparent from the detailed description in the following specification.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1.LND减轻了EAE的炎症损伤。(a)如材料和方法中所述，用MOG35～55肽和百日咳毒素诱发小鼠EAE。EAE诱导后，每天给予溶剂或LND的小鼠的临床评分；n＝12。(b)EAE诱导后的体重变化；n＝12。(c)代表性的H&E和Luxol Fast Blue(LFB)染色。比例尺＝100μm。(d)CD45+在活细胞中的比例以及CD45+CD4+、CD45+CD8+和CD45+CD11b+细胞在中枢神经系统单核细胞群中的比例；这些细胞是在EAE诱导后第15天分离的。(e)如材料和方法中所述，用MOG35～55肽和百日咳毒素诱导小鼠的EAE。EAE发病后，每天给小鼠注射溶剂或LND的临床评分；n＝10。(f)EAE诱导后的体重变化；n＝10。(g)CD45+在活细胞中的比例和CD45+CD4+、CD45+CD8+和CD45+CD11b+细胞在中枢神经系统单核细胞群中的比例；这些细胞是在EAE诱导后第15天分离的。*P＜0.05；**P＜0.01；***P＜0.001；****P＜0.0001；NS，不显著。a、c、d、e、h和i为非配对t检验，g和j为多重非配对t检验。Figure 1. LND reduces inflammatory damage in EAE. (a) EAE was induced in mice with MOG35-55 peptide and pertussis toxin as described in Materials and Methods. Clinical scores of mice given solvent or LND daily after EAE induction; n = 12. (b) Body weight changes after EAE induction; n = 12. (c) Representative H&E and Luxol Fast Blue (LFB) staining. Scale bar = 100 μm. (d) The proportion of CD45 + in living cells and the proportion of CD45 + CD4 + , CD45 + CD8 + and CD45 + CD11b + cells in the central nervous system mononuclear cell population; these cells were isolated on day 15 after EAE induction. (e) EAE was induced in mice with MOG35-55 peptide and pertussis toxin as described in Materials and Methods. Clinical scores of mice given solvent or LND daily after EAE onset; n = 10. (f) Body weight changes after EAE induction; n = 10. (g) The proportion of CD45 + in living cells and the proportion of CD45 + CD4 + , CD45 + CD8 + , and CD45 + CD11b + cells in the CNS mononuclear cell population; these cells were isolated on day 15 after EAE induction. *P<0.05;**P<0.01;***P<0.001;****P<0.0001; NS, not significant. a, c, d, e, h, and i are unpaired t tests, and g and j are multiple unpaired t tests.
图2.LND抑制体内炎症小体的激活。(a and b)EAE诱导后脊髓组织中GSDMD和caspase-1的免疫组织化学染色(a)和定量分析(b)。(c and d)ELISA分析腹腔注射LPS(15mg/kg)4小时后小鼠血清中的IL-1β(c)和IL-18(d)，无论是否用LND预处理，n＝15。数据以平均值±SEM表示。*P<0.05；**P<0.01；***P<0.001；****P<0.0001；NS，不显著。数据通过单因素方差分析和Tukey's检验进行分析。Figure 2. LND inhibits inflammasome activation in vivo. (a and b) Immunohistochemical staining (a) and quantitative analysis (b) of GSDMD and caspase-1 in spinal cord tissue after EAE induction. (c and d) ELISA analysis of IL-1β (c) and IL-18 (d) in the serum of mice 4 hours after intraperitoneal injection of LPS (15 mg/kg), whether pretreated with LND or not, n = 15. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
图3.LND是NLRP3炎症小体的广谱抑制剂。LPS刺激的BMDMs(a)和J774A.1细胞(b)用LND处理0.5小时，然后用ATP(5mM)刺激0.5小时，其上清液(SN)中成熟的IL-1β和裂解的caspase-1(p20)的Western blot分析。BMDMs(a)和J774A.1细胞(b)裂解液中IL-1β前体和caspase-1前体的Western blot分析。(c and d)ELISA分析BMDMs上清液中的IL-1β(c)和IL-18(d)。(e and f)ELISA分析J774A.1细胞上清液中的IL-1β(e)和TNF-α(f)。(g)GSDMD的Western blot分析。(h)GSDMD-N灰度值的定量分析。(i和j)BMDMs(i)和J774A.1细胞(j)的上清液中LDH的释放。(k-m)用LND预处理30分钟，然后用LPS刺激3小时的J774A.1细胞中IL-1β、IL-18和TNF-α的mRNA水平。(o and p)LPS刺激的BMDMs用或不用LND(200μM)预处理30分钟，然后用尼日利亚菌素、ATP、MSU或imiquimod进行刺激。Western blot分析上清中成熟的IL-1β和裂解的caspase-1(p20)以及BMDMs裂解液中的IL-1β前体和caspase-1前体(o)。ELISA分析BMDMs上清液中的IL-1β(p)。数据以平均值±SEM表示。*P＜0.05；**P＜0.01；***P＜0.001；****P＜0.0001；NS，不显著。数据通过单因素方差分析和Tukey's检验进行分析。Figure 3. LND is a broad-spectrum inhibitor of the NLRP3 inflammasome. Western blot analysis of mature IL-1β and cleaved caspase-1 (p20) in the supernatant (SN) of LPS-stimulated BMDMs (a) and J774A.1 cells (b) treated with LND for 0.5 h and then stimulated with ATP (5 mM) for 0.5 h. Western blot analysis of IL-1β precursor and caspase-1 precursor in lysates of BMDMs (a) and J774A.1 cells (b). (c and d) ELISA analysis of IL-1β (c) and IL-18 (d) in the supernatant of BMDMs. (e and f) ELISA analysis of IL-1β (e) and TNF-α (f) in the supernatant of J774A.1 cells. (g) Western blot analysis of GSDMD. (h) Quantitative analysis of the gray value of GSDMD-N. (i and j) LDH release in the supernatants of BMDMs (i) and J774A.1 cells (j). (k–m) mRNA levels of IL-1β, IL-18, and TNF-α in J774A.1 cells pretreated with LND for 30 min and then stimulated with LPS for 3 h. (o and p) LPS-stimulated BMDMs were pretreated with or without LND (200 μM) for 30 min and then stimulated with nigericin, ATP, MSU, or imiquimod. Western blot analysis of mature IL-1β and cleaved caspase-1 (p20) in the supernatant and IL-1β precursor and caspase-1 precursor in the lysate of BMDMs (o). ELISA analysis of IL-1β in the supernatant of BMDMs (p). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
图4.LND抑制NLRP3炎症小体的激活不依赖于HK2。(a)通过qRT-PCR分析LPS刺激的BMDMs中LND靶向基因的mRNA水平，用LND处理30分钟，然后用ATP刺激3小时。(b)用不同剂量的ATP处理LPS刺激的BMDMs 30分钟后，用Western blot分析HK2的表达。(c)用对照siRNA或HK2特异性siRNA转染LPS刺激的BMDMs 24小时后，用ATP刺激，Western blot分析上清液(supernatant,SN)中的IL-1β和裂解的caspase-1(p20)以及裂解液中IL-1β前体、caspase-1前体和HK2的含量。(d)ELISA分析BMDMs上清液中的IL-1β。(e)对LPS刺激的J774A.1细胞转染对照siRNA或HK2特异性siRNA24小时后用ATP刺激，上清中的IL-1β和裂解的caspase-1(p20)以及原IL-1β、原caspase-1和HK2的裂解液进行Western blot分析。(f)ELISA分析J774A.1细胞上清液中的IL-1β。(g)对HK2敲除小鼠的LPS加ATP刺激的BMDMs的培养上清液(SN)中的IL-1β和裂解的caspase-1(p20)进行Western blot分析对这些细胞裂解液中的IL-1β前体、caspase-1前体和HK2进行Western blot分析。(h)用LND处理30分钟，然后用ATP刺激的HK2敲除小鼠的LPS刺激的BMDMs的培养上清液中IL-1β和裂解的caspase-1(p20)的Western blot分析。对这些细胞裂解液中的IL-1β前体、caspase-1前体和HK2进行Western blot分析。(i)ELISA分析BMDMs上清液中的IL-1β。数据以平均值±SEM表示，*P＜0.05；**P＜0.01；***P＜0.001；****P＜0.0001；NS，不显著。数据通过单因素方差分析和Tukey's检验进行分析。Figure 4. LND inhibits NLRP3 inflammasome activation independently of HK2. (a) qRT-PCR analysis of mRNA levels of LND-targeted genes in LPS-stimulated BMDMs treated with LND for 30 min and then stimulated with ATP for 3 h. (b) Western blot analysis of HK2 expression in LPS-stimulated BMDMs treated with different doses of ATP for 30 min. (c) 24 h after LPS-stimulated BMDMs were transfected with control siRNA or HK2-specific siRNA, and then stimulated with ATP, Western blot analysis of IL-1β and cleaved caspase-1 (p20) in the supernatant (SN) and IL-1β precursor, caspase-1 precursor, and HK2 in the lysate. (d) ELISA analysis of IL-1β in the supernatant of BMDMs. (e) Western blot analysis of IL-1β and cleaved caspase-1(p20) in the supernatant and lysates of pro-IL-1β, pro-caspase-1, and HK2 in J774A.1 cells stimulated with LPS and transfected with control siRNA or HK2-specific siRNA 24 h later with ATP. (f) ELISA analysis of IL-1β in the supernatant of J774A.1 cells. (g) Western blot analysis of IL-1β and cleaved caspase-1(p20) in the supernatant of cultured BMDMs (SN) stimulated with LPS plus ATP from HK2 knockout mice. Western blot analysis of IL-1β pro, pro-caspase-1, and HK2 in the lysates of these cells. (h) Western blot analysis of IL-1β and cleaved caspase-1(p20) in the supernatant of cultured BMDMs stimulated with LPS from HK2 knockout mice treated with LND for 30 min and then stimulated with ATP. Western blot analysis was performed on IL-1β precursor, caspase-1 precursor, and HK2 in these cell lysates. (i) ELISA analysis of IL-1β in the supernatant of BMDMs. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
图5.LND阻止ASC斑点的形成。(a)用FACS检测用或不用LND处理的LPS和ATP刺激的BMDMs中ROS+细胞的百分比。(b)在ATP处理30分钟前，用LND(0.2mM)处理LPS刺激的BMDMs 30分钟。对抗NEK7抗体免疫沉淀的细胞裂解液中的NLRP3蛋白的Western blot分析，以评估NLRP3-NEK7的相互作用。(c)在ATP处理前30分钟，用LND(0.2mM)处理LPS刺激的BMDMs，30分钟。对抗NLRP3抗体免疫沉淀的细胞裂解液中的ASC蛋白进行Western blot分析，以评估ASC-NLRP3的相互作用。(d)在ATP处理前30分钟，用LND(0.2mM)处理LPS刺激的BMDMs，30分钟。对抗ASC抗体免疫沉淀的细胞裂解液中的caspase-1蛋白的Western blot分析，以评估caspase-1和ASC的相互作用。(e和f)LPS刺激的BMDMs用LND(0.2mM)处理30分钟，然后用ATP刺激30分钟。用抗ASC抗体对NP-40不溶性沉淀中交联的ASC进行Western blot(e)和定量分析(f)。(g)用不同剂量的LND处理的LPS刺激的BMDMs中ASC斑点的免疫荧光分析。蓝色代表Hoechst 33342的核染色。箭头指征ASC斑点。比例尺＝10微米(h)用LND处理和ATP刺激后，含有ASC斑点的LPS刺激的BMDMs的百分比。显示的数据代表了所有测量的平均值±SEM。(i)在不同LND浓度存在下，人源重组ASC蛋白寡聚体的负染电子显微镜图像。(j)EAE小鼠脊髓中ASC的免疫荧光分析。白色箭头指征ASC斑点。比例尺＝25μm。(k)LPS刺激的BMDMs用LND(0.2mM)处理0.5小时，然后用炎症小体激活剂(ATP(5mM，30分钟)，poly(dA:dT)(1μg/ml，8小时)，MDP(200ng/ml，8小时)或鞭毛蛋白(200ng/ml，8小时))刺激。ELISA分析BMDMs上清液中的IL-1β。数据以平均值±SEM表示。*P＜0.05；**P＜0.01；***P＜0.001；****P＜0.0001；NS，不显著。数据通过单因素方差分析和Tukey's检验进行分析。Figure 5. LND prevents the formation of ASC specks. (a) The percentage of ROS + cells in LPS- and ATP-stimulated BMDMs treated with or without LND was measured by FACS. (b) LPS-stimulated BMDMs were treated with LND (0.2 mM) for 30 min before ATP treatment for 30 min. Western blot analysis of NLRP3 protein in cell lysates immunoprecipitated with anti-NEK7 antibody to assess NLRP3-NEK7 interaction. (c) LPS-stimulated BMDMs were treated with LND (0.2 mM) for 30 min before ATP treatment for 30 min. Western blot analysis of ASC protein in cell lysates immunoprecipitated with anti-NLRP3 antibody to assess ASC-NLRP3 interaction. (d) LPS-stimulated BMDMs were treated with LND (0.2 mM) for 30 min before ATP treatment for 30 min. Western blot analysis of caspase-1 protein in cell lysates immunoprecipitated with anti-ASC antibody to assess the interaction between caspase-1 and ASC. (e and f) LPS-stimulated BMDMs were treated with LND (0.2 mM) for 30 min and then stimulated with ATP for 30 min. Western blot analysis (e) and quantitative analysis (f) of cross-linked ASC in NP-40-insoluble precipitates using anti-ASC antibody. (g) Immunofluorescence analysis of ASC specks in LPS-stimulated BMDMs treated with different doses of LND. Blue represents nuclear staining with Hoechst 33342. Arrows indicate ASC specks. Scale bar = 10 μm. (h) Percentage of LPS-stimulated BMDMs containing ASC specks after treatment with LND and stimulation with ATP. Data shown represent the mean ± SEM for all measurements. (i) Negatively stained electron microscopy images of human recombinant ASC protein oligomers in the presence of different LND concentrations. (j) Immunofluorescence analysis of ASC in the spinal cord of EAE mice. White arrows indicate ASC specks. Scale bar = 25 μm. (k) LPS-stimulated BMDMs were treated with LND (0.2 mM) for 0.5 h and then stimulated with inflammasome activators (ATP (5 mM, 30 min), poly(dA:dT) (1 μg/ml, 8 h), MDP (200 ng/ml, 8 h), or flagellin (200 ng/ml, 8 h)). IL-1β in the supernatant of BMDMs was analyzed by ELISA. Data are presented as mean ± SEM. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001; NS, not significant. Data were analyzed by one-way ANOVA and Tukey's test.
图6.LND直接与ASC结合以抑制NLRP3的组装。(a)使用Ligand scout软件建立ASC与LND的对接模型。LND被显示为棍状，颜色为绿色。ASC的CARD结构域以卡通形式显示，颜色为粉红色，而相互作用的部位为橙色。黄色虚线代表氢键。(b)显示LND与重组ASC蛋白结合的图是用Biacore的SPR分析得到的。不同浓度的LND以叠加图的形式呈现。(C)LPS刺激的BMDMs用蛋白裂解液裂解，并与LND在4℃下孵育过夜。用pronase酶(20ng/μg蛋白)进行DARTS检测，并通过Western blot分析。(d)不同ASC突变体转染HEK293细胞中ASC斑点的分析。(e)用ASC突变体转染的HEK293细胞共聚焦成像。蓝色信号对应于Hoechst33342染色，红色信号对应于ASC。Figure 6. LND directly binds to ASC to inhibit NLRP3 assembly. (a) The docking model of ASC and LND was established using Ligand scout software. LND is shown as a stick in green. The CARD domain of ASC is shown in cartoon form in pink, while the interaction site is in orange. The yellow dashed line represents the hydrogen bond. (b) The graph showing the binding of LND to recombinant ASC protein was obtained by SPR analysis using Biacore. Different concentrations of LND are presented as an overlay. (C) LPS-stimulated BMDMs were lysed with protein lysis buffer and incubated with LND overnight at 4°C. DARTS detection was performed using pronase enzyme (20 ng/μg protein) and analyzed by Western blot. (d) Analysis of ASC spots in HEK293 cells transfected with different ASC mutants. (e) Confocal imaging of HEK293 cells transfected with ASC mutants. The blue signal corresponds to Hoechst33342 staining and the red signal corresponds to ASC.
具体实施方式Detailed ways
定义definition
术语“卤代”或“卤素”是指氟、氯、溴或碘或其基团。当未限定卤代数量时，可以是任何合适的数量，例如一卤代、二卤代、三卤代；当未限定卤代位置时，可以是任何合适的位置，例如卤代苯基可以是在邻位、对位、间位或其组合的卤代。 The term "halogen" or "halogen" refers to fluorine, chlorine, bromine or iodine or a group thereof. When the number of halogens is not limited, it can be any suitable number, such as monohalogen, dihalogen, trihalogen; when the position of the halogen is not limited, it can be any suitable position, for example, the halogenated phenyl can be halogenated at the ortho position, para position, meta position or a combination thereof.
术语“烷基”是指饱和的直链或支链烃链。对于具有特定数量的碳原子的烷基，该术语包含对应的正烷基及其各种异构体形式(如有)。例如，具有3个碳原子的烷基(C3烷基)包括正丙基(丙基)和异丙基。C1-3烷基包括甲基、乙基、丙基、异丙基。The term "alkyl" refers to a saturated straight or branched hydrocarbon chain. For an alkyl group having a specific number of carbon atoms, the term includes the corresponding normal alkyl group and its various isomeric forms (if any). For example, an alkyl group having 3 carbon atoms ( C3 alkyl) includes normal propyl (propyl) and isopropyl. C1-3 alkyl groups include methyl, ethyl, propyl, isopropyl.
术语“C1-3烷基氨基”是指被C1-3烷基取代的氨基，可以是被一个或两个C1-3烷基取代的氨基，例如甲基氨基、二甲基氨基、二乙基氨基、甲基乙基氨基等。The term "C 1-3 alkylamino" refers to an amino group substituted by a C 1-3 alkyl group, and may be an amino group substituted by one or two C 1-3 alkyl groups, for example, methylamino, dimethylamino, diethylamino, methylethylamino and the like.
术语“卤代C1-3烷基”是指被卤素取代的C1-3烷基，可以是被一个或多个卤素同时取代的C1-3烷基，例如氟甲基、二氟甲基、三氟甲基等。The term "halogenated C 1-3 alkyl" refers to a C 1-3 alkyl group substituted by halogen, and may be a C 1-3 alkyl group substituted by one or more halogens simultaneously, such as fluoromethyl, difluoromethyl, trifluoromethyl and the like.
术语“羧基苯基”是指被羧基取代的苯基，可以是被一个或多个羧基取代的苯基。The term "carboxyphenyl" refers to a phenyl group substituted by a carboxyl group, and may be a phenyl group substituted by one or more carboxyl groups.
术语“酰肼基”是指-C(O)-NH-NH2。The term "hydrazide" refers to -C(O)-NH- NH2 .
术语“治疗有效量”指一种本发明的共轭物或其组合物的在适用于任何医学治疗的合理效益/风险比下在动物中的细胞的至少一个亚群中有效产生某些所希望的治疗效果的量。The term "therapeutically effective amount" refers to an amount of a conjugate of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit/risk ratio applicable to any medical treatment.
术语“药学上可接受的”指位于正确医学判断的范围内、适于与人类和动物的组织接触而无过量毒性、刺激、过敏反应或其他问题或并发症、与一个合理效益/风险比相称的那些化合物、材料、组合物和/或剂型。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的载体”是指参与将所述共轭物从身体的一个器官或部位携载或运载到身体的另一个器官或部位的一种药学上可接受的材料、组合物或运载体，如一种液体或固体填充剂、稀释剂、赋形剂、制造助剂或溶剂包封材料。每种载体必须在与该组合物的其他成分相容并且对患者无害的意义上是“可接受的”。The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, that participates in carrying or delivering the conjugate from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.
术语“治疗”涵盖预防、治疗以及治愈。接受这一治疗的患者通常是任何有需要的动物，包括灵长类动物(特别是人类)以及其他哺乳动物，如马、牛、猪、羊、家禽和宠物。The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.
术语“基于ASC-caspase-1通路的炎症小体激活介导的疾病”是指由炎症小体异常激活介导的疾病，该炎症小体异常激活基于ASC低聚化形成ASC斑点(ASC specks)，进而招募caspase-1前体并激活caspase-1这一途径。被激活的caspase-1最终导致促炎症介质IL-1β和IL-18的成熟和分泌，并裂解gasdermin D(GSDMD)来诱导细胞焦亡。The term "diseases mediated by inflammasome activation based on the ASC-caspase-1 pathway" refers to diseases mediated by abnormal inflammasome activation, which is based on the oligomerization of ASC to form ASC specks, which in turn recruits caspase-1 precursors and activates caspase-1. Activated caspase-1 ultimately leads to the maturation and secretion of pro-inflammatory mediators IL-1β and IL-18, and cleaves gasdermin D (GSDMD) to induce pyroptosis.
术语“式I”包括式Ia和式Ib，“式II”包括式IIa和IIb，“式III”包括式IIIa和IIIb，“式IV”包括式IVa、IVb和式IVc。The term "Formula I" includes Formula Ia and Formula Ib, "Formula II" includes Formula IIa and IIb, "Formula III" includes Formula IIIa and IIIb, and "Formula IV" includes Formula IVa, IVb, and IVc.
吲唑类化合物及其类似物Indazole compounds and their analogs
本发明的一个方面提供式Ia或Ib所示的化合物或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，
One aspect of the present invention provides the use of a compound represented by Formula Ia or Ib or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
其中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1- 3烷基和酰肼基；R3为H或C1-3烷基；R4和R5独立地选自氢、卤素和C1-3烷基；R6为H或C1-3烷基；Z选自C、N、O和S；p为1、2或3；其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病，优选地所述疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。wherein R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide; R 3 is H or C 1-3 alkyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl; R 6 is H or C 1-3 alkyl; Z is selected from C, N, O and S; p is 1, 2 or 3; wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z选自C、N、O和S，优选为C或N，更优选为C；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is selected from C, N, O and S, preferably C or N, more preferably C; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z选自C、N、O和S，优选为C或N，更优选为C；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is selected from C, N, O and S, preferably C or N, more preferably C; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z选自C、N、O和S，优选为C或N，更优选为C；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is selected from C, N, O and S, preferably C or N, more preferably C; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、 C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z选自C、N、O和S，优选为C或N，更优选为C；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is selected from C, N, O and S, preferably C or N, more preferably C; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H；Z选自C、N、O和S，优选为C或N，更优选为C；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H; Z is selected from C, N, O and S, preferably C or N, more preferably C; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z是C或N；p为1、2或3，优选为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is C or N; p is 1, 2 or 3, preferably 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2选自羧基、C1-3烷基和酰肼基，优选为羧基；R3为H或C1-3烷基，优选为氢或甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选独立地选自氢、甲基、氟和氯；R6为H或C1-3烷基，优选为H；Z是C或N；p为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably carboxyl; R 3 is H or C 1-3 alkyl, preferably hydrogen or methyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H or C 1-3 alkyl, preferably H; Z is C or N; p is 1.
在一些实施方式中，在式Ia或Ib的结构式中，R1选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R2为羧基；R3为H或甲基；R4和R5独立地选自氢、甲基、氟和氯；R6为H；Z为C；p为1。In some embodiments, in the structural formula of Formula Ia or Ib, R 1 is selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 2 is carboxyl; R 3 is H or methyl; R 4 and R 5 are independently selected from hydrogen, methyl, fluorine and chlorine; R 6 is H; Z is C; p is 1.
在本发明的一些实施方式中，提供式IIa或IIb所示的化合物或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，
In some embodiments of the present invention, there is provided a use of a compound represented by Formula IIa or IIb or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
其中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1- 3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选选自甲基、氟和氯；并且其中所述基于ASC-Caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病，优选地所述疾病选自：哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。Wherein, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from methyl, fluorine and chlorine; and wherein the disease mediated by inflammasome activation based on the ASC-Caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
在本发明的一些实施方式中，提供式(IIIa)或(IIIb)所示的化合物或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，
In some embodiments of the present invention, there is provided a use of a compound represented by formula (IIIa) or (IIIb) or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
其中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1- 3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选选自甲基、氟和氯，更优选为氟或氯；并且其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病，优选地所述疾病选自：哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。 Wherein, R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from methyl, fluorine and chlorine, more preferably fluorine or chlorine; and wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
在本发明的一些实施方式中，提供式IVa、IVb或IVc所示的化合物或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，
In some embodiments of the present invention, there is provided a use of a compound represented by Formula IVa, IVb or IVc or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway.
其中，R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1- 3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；更优选选自氢、氟、氯和二氟甲基；R4和R5独立地选自氢、卤素和C1-3烷基，优选选自氟和氯，更优选均为氯；并且其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病，优选地所述疾病选自：哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。 wherein R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl; R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; more preferably selected from hydrogen, fluorine, chlorine and difluoromethyl; R 4 and R 5 are independently selected from hydrogen, halogen and C 1-3 alkyl, preferably selected from fluorine and chlorine, more preferably both are chlorine; and wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, maculopathy, benign prostatic hyperplasia and AIDS, preferably the disease is selected from: asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
在一些实施方式中，式I的化合物选自以下任何一种(括号内的数字表示PubChem CID)：In some embodiments, the compound of Formula I is selected from any of the following (the numbers in brackets represent PubChem CID):
6-氯-1-[(2,4-二氯苯基)甲基]吲唑-3-羧酸(11501294)；6-Chloro-1-[(2,4-dichlorophenyl)methyl]indazole-3-carboxylic acid (11501294);
5-(4-氯苯基)-1-[1-(2,4-二氯苯基)乙基]吡唑-3-羧酸(67565577)；5-(4-chlorophenyl)-1-[1-(2,4-dichlorophenyl)ethyl]pyrazole-3-carboxylic acid (67565577);
1-[(2-氯-4-氟苯基)甲基]-5-(二氟甲基)吲唑-3-羧酸(66888740)；1-[(2-chloro-4-fluorophenyl)methyl]-5-(difluoromethyl)indazole-3-carboxylic acid (66888740);
1-[(2,4-二氯苯基)甲基]-5-氟吲唑-3-羧酸(71221361)；1-[(2,4-Dichlorophenyl)methyl]-5-fluoroindazole-3-carboxylic acid (71221361);
1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸(39562)；1-[(2,4-Difluorophenyl)methyl]indazole-3-carboxylic acid (39562);
1-[(2,4-二氯苯基)甲基]-6-(氟甲基)吲唑-3-羧酸(57633631)；1-[(2,4-Dichlorophenyl)methyl]-6-(fluoromethyl)indazole-3-carboxylic acid (57633631);
1-[(2,4-二氯苯基)甲基]-6-(二氟甲基)吲唑-3-羧酸(11667849)；1-[(2,4-Dichlorophenyl)methyl]-6-(difluoromethyl)indazole-3-carboxylic acid (11667849);
1-[(2,4-二氯苯基)甲基]吲唑-3-羧酸盐(25271808)；1-[(2,4-Dichlorophenyl)methyl]indazole-3-carboxylate (25271808);
1-[(2,4-二氯苯基)甲基]-5-碘吲唑-3-羧酸(71216306)；1-[(2,4-Dichlorophenyl)methyl]-5-iodoindazole-3-carboxylic acid (71216306);
1-[(2,4-二氯苯基)甲基]-4-(二甲基氨基)吲唑-3-羧酸(66900636)；1-[(2,4-Dichlorophenyl)methyl]-4-(dimethylamino)indazole-3-carboxylic acid (66900636);
1-[(2,4-二氯苯基)甲基]-5-(氟甲基)吲唑-3-羧酸(66887973)；1-[(2,4-Dichlorophenyl)methyl]-5-(fluoromethyl)indazole-3-carboxylic acid (66887973);
1-(2,4-二氯苯基)-6-氟吲唑-3-羧酸(11652993)；1-(2,4-Dichlorophenyl)-6-fluoroindazole-3-carboxylic acid (11652993);
1-[(2,4-二氯苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(57633606)；1-[(2,4-Dichlorophenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (57633606);
1-[(2-氯-4-甲基苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(66888424)；1-[(2-chloro-4-methylphenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (66888424);
1-(2,4-二氯苯基)-3-(4-氯苯基)-1H-吡唑-5-羧酸(54148749)；1-(2,4-Dichlorophenyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxylic acid (54148749);
1-[1-(2,4-二氯苯基)乙基]-1H-吲唑-3-羧酸(58796770)；1-[1-(2,4-Dichlorophenyl)ethyl]-1H-indazole-3-carboxylic acid (58796770);
1-[(2,6-二氯-3-吡啶基)甲基]-1H-吲唑-3-羧酸(58796750)；1-[(2,6-Dichloro-3-pyridyl)methyl]-1H-indazole-3-carboxylic acid (58796750);
1-[(2-氯-4-氟苯基)甲基]-6-(二氟甲基)吲唑-3-羧酸(66888818)；1-[(2-chloro-4-fluorophenyl)methyl]-6-(difluoromethyl)indazole-3-carboxylic acid (66888818);
1-[(2-氯-4-甲基苯基)甲基]-6-甲基吲唑-3-羧酸(89564538)；1-[(2-Chloro-4-methylphenyl)methyl]-6-methylindazole-3-carboxylic acid (89564538);
1-[(2,4-二氯苯基)甲基]-5-(三氟甲基)吲唑-3-羧酸(11567264)；1-[(2,4-Dichlorophenyl)methyl]-5-(trifluoromethyl)indazole-3-carboxylic acid (11567264);
5-(4-氯苯基)-1-[(2,4-二氯苯基)甲基]-4-甲基吡唑-3-羧酸(54033453)；5-(4-chlorophenyl)-1-[(2,4-dichlorophenyl)methyl]-4-methylpyrazole-3-carboxylic acid (54033453);
1-[(2-氯-4-氟苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(57633637)；1-[(2-chloro-4-fluorophenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (57633637);
1-[(2,4-二氯苯基)甲基]-6-(二甲基氨基)吲唑-3-羧酸(11537863)；1-[(2,4-Dichlorophenyl)methyl]-6-(dimethylamino)indazole-3-carboxylic acid (11537863);
1-[(2,4-二氯苯基)甲基]-6-甲基吲唑-3-羧酸(11667156)。1-[(2,4-Dichlorophenyl)methyl]-6-methylindazole-3-carboxylic acid (11667156).
在优选的实施方式中，式I的化合物是1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸(即，氯尼达明，结构式如下式V所示)或其药学上可接受的盐或酯。
In a preferred embodiment, the compound of formula I is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid (ie, lonidamine, with a structural formula as shown in the following formula V) or a pharmaceutically acceptable salt or ester thereof.
基于ASC-Caspase-1通路的炎症小体激活介导的疾病Diseases mediated by inflammasome activation based on the ASC-Caspase-1 pathway
本发明提供如上所述的式I至式V的任一化合物在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。The present invention provides the use of any compound of Formula I to Formula V as described above in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
在优选的实施方式中，本发明提供如上所述的式I至式V的任一化合物在制备用于治疗痛风的药物中的应用。In a preferred embodiment, the present invention provides use of any compound of Formula I to Formula V as described above in the preparation of a medicament for treating gout.
在优选的实施方式中，本发明提供如上所述的式I至式V的任一化合物在制备用于治疗多发性硬化的药物中的应用。In a preferred embodiment, the present invention provides use of any one of the compounds of Formula I to Formula V as described above in the preparation of a medicament for treating multiple sclerosis.
在优选的实施方式中，本发明提供如上所述的式I至式V的任一化合物在制备用于治疗败血症的药物中的应用。In a preferred embodiment, the present invention provides use of any one of the compounds of Formula I to Formula V as described above in the preparation of a medicament for treating sepsis.
在优选的实施方式中，本发明提供1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。In a preferred embodiment, the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
在优选的实施方式中，本发明提供1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。In a preferred embodiment, the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
在优选的实施方式中，本发明提供1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯在制备用于治疗痛风的药物中的应用。In a preferred embodiment, the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating gout.
在优选的实施方式中，本发明提供1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯在制备用于治疗多发性硬化的药物中的应用。 In a preferred embodiment, the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating multiple sclerosis.
在优选的实施方式中，本发明提供1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯在制备用于治疗败血症的药物中的应用。In a preferred embodiment, the present invention provides the use of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating sepsis.
药物组合物Pharmaceutical composition
本发明的另一个方面提供一种药物组合物，其包含式I至式V的任一所示的任一化合物或其药学上可接受的盐或酯以及药学上可接受的载体。Another aspect of the present invention provides a pharmaceutical composition comprising any compound represented by any one of Formula I to Formula V or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable carrier.
本发明的式I至式V的任一所示的任一化合物或其药学上可接受的盐或酯可以被配制用于药物用途。药学上可接受的组合物包括治疗有效量的以上所述化合物中的一种或多种，单独使用或与一种或多种药学上可接受的载体(添加剂)、赋形剂和/或稀释剂配制在一起。Any compound shown in any of Formula I to Formula V of the present invention or a pharmaceutically acceptable salt or ester thereof can be formulated for pharmaceutical use. A pharmaceutically acceptable composition includes one or more of the above-mentioned compounds in a therapeutically effective amount, used alone or formulated with one or more pharmaceutically acceptable carriers (additives), excipients and/or diluents.
可以通过从其他药物类推将根据本发明的化合物配制为用于以任何便利方式给药以在人类或兽医学中使用。The compounds according to the invention may be formulated for administration in any convenient way by analogy from other drugs for use in human or veterinary medicine.
治疗方法及用途Treatment methods and uses
本发明的另一个方面提供一种治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本文所述的任一种式I至式V的任一化合物，所述疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。在优选的实施方式中，所述疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。在特别优选的实施方式中，所述疾病是痛风。在特别优选的实施方式中，所述疾病是多发性硬化。在另一特别优选的实施方式中，所述疾病是败血症。Another aspect of the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering to a subject in need thereof a therapeutically effective amount of any compound of any one of Formulas I to V described herein, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia, and AIDS. In a preferred embodiment, the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis, and sepsis. In a particularly preferred embodiment, the disease is gout. In a particularly preferred embodiment, the disease is multiple sclerosis. In another particularly preferred embodiment, the disease is sepsis.
优选地，所述式I至式V的化合物是1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。因此，在一些实施方式中，本发明提供一种治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的方法，所述方法包括向有需要的对象施用治疗有效量的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯，所述疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。在一些实施方式中，本发明提供一种治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的方法，所述方法包括向有需要的对象施用治疗有效量的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯，所述疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。在优选的实施方式中，本发明提供一种治疗多发性硬化的方法，所述方法包括向有需要的对象施用治疗有效量的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。在优选的实施方式中，本发明提供一种治疗败血症的方法，所述方法包括向有需要的对象施用治疗有效量的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。Preferably, the compound of Formula I to Formula V is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof. Therefore, in some embodiments, the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof to a subject in need thereof, the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS. In some embodiments, the present invention provides a method for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, the method comprising administering a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof to a subject in need thereof, the disease being selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis. In a preferred embodiment, the present invention provides a method for treating multiple sclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof. In a preferred embodiment, the present invention provides a method for treating sepsis, comprising administering to a subject in need thereof a therapeutically effective amount of 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
本发明的另一个方面提供了用于治疗基于ASC-Caspase-1通路的炎症小体激活介导的疾病的本文所述的任一种式I至式V的任一的化合物，所述疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。在优选的实施方式中，所述疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。在特别优选的实施方式中，所述疾病是多发性硬化。在另一特别优选的实施方式中，所述疾病是败血症。Another aspect of the present invention provides any compound of any of Formula I to Formula V described herein for use in treating a disease mediated by inflammasome activation based on the ASC-Caspase-1 pathway, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS. In a preferred embodiment, the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis. In a particularly preferred embodiment, the disease is multiple sclerosis. In another particularly preferred embodiment, the disease is sepsis.
优选地，所述式I至式V的化合物是1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。因此，在一些实施方式中，本发明提供用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯，所述疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。在一些实施方式中，本发明提供用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯，所述疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。在优选的实施方式中，本发明提供用于治疗痛风的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。在优选的实施方式中，本发明提供用于治疗多发性硬化的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。在优选的实施方式中，本发明提供用于治疗败血症的1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。Preferably, the compound of Formula I to Formula V is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof. Therefore, in some embodiments, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS. In some embodiments, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway, wherein the disease is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis. In a preferred embodiment, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating gout. In a preferred embodiment, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating multiple sclerosis. In a preferred embodiment, the present invention provides 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof for use in treating sepsis.
本发明提供的式I至式V的任一化合物可通过例如US20070015771A1描述的方法制备(其实施例8至39提供了吲唑类化合物的详细制备方法)，通过引用将该专利文献整体合并至本文中。Any compound of Formula I to Formula V provided by the present invention can be prepared by, for example, the method described in US20070015771A1 (Examples 8 to 39 thereof provide detailed preparation methods of indazole compounds), and the entirety of this patent document is incorporated herein by reference.
多发性硬化(multiple sclerosis，MS)是一种以中枢神经系统(CNS)炎性脱髓鞘病变为主要特点的免疫介导性疾病，病变主要累及白质。其病因尚不明确，可能与遗传、环境、病毒感染等多种因素相关。MS病理上表现为CNS多发髓鞘脱失，可伴有神经细胞及其轴索损伤，MRI上病灶分布、形态及信号表现具有一定特征性。MS病变具有时间多发(DIT)和空间多发(DIS)的特点。Multiple sclerosis (MS) is an immune-mediated disease characterized by inflammatory demyelinating lesions in the central nervous system (CNS), with the lesions mainly affecting the white matter. The etiology is still unclear and may be related to multiple factors such as genetics, environment, and viral infection. MS pathologically manifests as multiple demyelination in the CNS, which may be accompanied by damage to nerve cells and their axons. The distribution, morphology, and signal performance of lesions on MRI are characteristic. MS lesions are characterized by temporal multiple (DIT) and spatial multiple (DIS).
MS的临床分类主要有：临床孤立综合征(CIS)是指首次的临床发作，常常累及单侧视神经、脊髓、脑干，也可以累及大脑半球，病灶可以1个也可以多个。患者经历首次发作后并非全部进展为MS，CIS患者需要严密随访。缓解复发型(RRMS)：在疾病早期阶段，患者常常有反复的发作，多数病人发作后症状完全缓解，称为RRMS。60-80％的患者会经历缓解复发的阶段。继发进展型(SPMS)：患者经历反复的发作后，症状不能完全缓解，残留的症状逐渐累积出现不可逆的神经功能残疾，即进入进展期，故称为继发进展型。进展复发型(PRMS)：部分患者一开始症状就逐步累积，同时在病程中出现典型的发作症状，称为进展复发型。原发进展型(PPMS)：患者从疾病开始就表现为隐匿的慢性的神经功能残疾的进展，病程超过1年，常常表现为慢性进展性脊髓病变，故称为原发进展型。急性MS是MS的急性变异性，来势凶猛，短时间内快速进展，常常同时出现多发的大病灶。本发明预期用于任何一种临床分型的MS。The clinical classification of MS is mainly as follows: Clinically isolated syndrome (CIS) refers to the first clinical attack, which often involves the unilateral optic nerve, spinal cord, brainstem, and can also involve the cerebral hemisphere. The lesions can be one or more. Not all patients progress to MS after the first attack, and CIS patients need close follow-up. Remission-relapsing syndrome (RRMS): In the early stages of the disease, patients often have repeated attacks, and most patients have complete relief of symptoms after the attack, which is called RRMS. 60-80% of patients will experience a stage of remission and relapse. Secondary progressive syndrome (SPMS): After the patient experiences repeated attacks, the symptoms cannot be completely relieved, and the residual symptoms gradually accumulate and irreversible neurological disability occurs, that is, entering the progressive stage, so it is called secondary progressive syndrome. Progressive relapsing syndrome (PRMS): Some patients gradually accumulate symptoms from the beginning, and typical symptoms of attacks appear during the course of the disease, which is called progressive relapsing syndrome. Primary progressive syndrome (PPMS): Patients show hidden and chronic progression of neurological disability from the beginning of the disease, and the course of the disease exceeds 1 year, often showing chronic progressive spinal cord lesions, so it is called primary progressive syndrome. Acute MS is an acute variant of MS, which is aggressive, progresses rapidly in a short period of time, and often has multiple large lesions at the same time. The present invention is intended to be used for any clinical classification of MS.
败血症(也称为脓毒症)是可能变得危及生命的对感染的全身炎症反应。当身体对感染的反应失去平衡时，就会出现败血症，而释放到血流中以对抗感染的化学物质会导致炎症并严重伤害身体自身的组织和器官。在最严重的情况下，这会导致败血性休克(此时也称为“败血症-3”)，其中血液循环和细胞损伤极大地增加了死亡率。Sepsis (also known as sepsis) is a systemic inflammatory response to infection that can become life-threatening. Sepsis occurs when the body's response to infection becomes out of balance, and chemicals released into the bloodstream to fight the infection cause inflammation and severe damage to the body's own tissues and organs. In the most severe cases, this can lead to septic shock (also known as "sepsis-3"), in which blood circulation and cellular damage greatly increase mortality.
败血症是复杂的病症并且不断被重新定义。在2016年，败血症和针对败血症的第三次国际共识定义被重新定义。由于全身炎症反应综合症(SIRS)的特异性和敏感性较差，因此SIRS被序贯器官衰竭评估(SOFA)取代，SOFA由于其大量性而最初通过qSOFA评分进行评估。qSOFA的评分范围为0至3分，针对每个测试阳性的生命体征为1分。这些qSOFA生命体征标准是呼吸频率≥22次呼吸/分钟，动脉收缩压小于或等于100/mmHg，以及精神状态改变(格拉斯哥昏迷量表评分低于15)。已经确定qSOFA评分至少为2的患者的院内死亡率为24％，以及qSOFA评分低于2的患者的院内死亡率为3％。通常，如果qSOFA评分至少为2，那么患者将通过完整的SOFA测试进行评估。SOFA测试的评分范围为0至24，并且涉及评估特定器官系统(呼吸系统、心血管系统、肝脏系统、肾脏系统、凝血系统和中枢神经系统)。如果SOFA评分也大于或等于2，则认为患者为败血症。Sepsis is a complex condition and is continually being redefined. In 2016, sepsis and the third international consensus definition for sepsis were redefined. Due to the poor specificity and sensitivity of the systemic inflammatory response syndrome (SIRS), SIRS was replaced by the sequential organ failure assessment (SOFA), which was initially assessed by the qSOFA score due to its high volume. The qSOFA score ranges from 0 to 3 points, with 1 point for each vital sign tested positive. These qSOFA vital sign criteria are respiratory rate ≥ 22 breaths/min, systolic arterial blood pressure less than or equal to 100/mmHg, and altered mental status (Glasgow Coma Scale score less than 15). It has been determined that patients with a qSOFA score of at least 2 have an in-hospital mortality of 24%, and patients with a qSOFA score less than 2 have an in-hospital mortality of 3%. Typically, if the qSOFA score is at least 2, then the patient will be evaluated with a full SOFA test. The SOFA test has a score range of 0 to 24 and involves the assessment of specific organ systems (respiratory, cardiovascular, hepatic, renal, coagulation, and central nervous systems). If the SOFA score is also greater than or equal to 2, the patient is considered to have sepsis.
高尿酸血症引起痛风、肾衰竭等。目前，高尿酸血症的治疗主要使用了别嘌呤醇、非布司他等黄嘌呤氧化酶抑制剂。痛风发作是由于血液中的尿酸变得过量、尿酸的晶体在关节堆积，由此引起强烈的炎症而发病，并伴有剧烈的疼痛。降尿酸药和急性痛风发作存在关联性，已知血清尿酸的急剧降低会产生软骨和软组织的尿酸钠晶体的短暂的局部沉淀，导致急性痛风发作。即，已知以往的降尿酸药可能诱发痛风发作。因此，根据患者的不同，有时也会因痛风发作而需要中止治疗或变更治疗方针。本发明预期可用于治疗痛风。Hyperuricemia causes gout, renal failure, etc. At present, the treatment of hyperuricemia mainly uses xanthine oxidase inhibitors such as allopurinol and febuxostat. Gout attacks are caused by excessive uric acid in the blood and accumulation of uric acid crystals in the joints, which causes strong inflammation and onset of disease, accompanied by severe pain. There is a correlation between uric acid-lowering drugs and acute gout attacks. It is known that the sharp reduction of serum uric acid will produce short-term local precipitation of sodium urate crystals in cartilage and soft tissue, leading to acute gout attacks. That is, it is known that uric acid-lowering drugs in the past may induce gout attacks. Therefore, depending on the patient, it is sometimes necessary to stop treatment or change treatment guidelines due to gout attacks. The present invention is expected to be used to treat gout.
突变蛋白及其用途Mutant proteins and their uses
本发明的另一个方面提供一种ASC突变蛋白，其包含在I115、F163、W169和L192中的至少一个处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。 Another aspect of the present invention provides an ASC mutant protein, which comprises an amino acid residue mutation at at least one of I115, F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising an amino acid residue mutation at I115, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在F163处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising an amino acid residue mutation at F163, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在W169处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising an amino acid residue mutation at W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising an amino acid residue mutation at L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115和F163处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and F163, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115和W169处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在F163和W169处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在F163和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在W169和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115、F163和W169处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163 and W169, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115、F163和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在F163、W169和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115、F163、W169和L192处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在以上任一ASC突变蛋白的实施方式中，ASC突变蛋白还包含在T166，L178和S195的一个或多个处的氨基酸残基突变。In any of the above ASC mutant protein embodiments, the ASC mutant protein further comprises amino acid residue mutations at one or more of T166, L178 and S195.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含在I115、F163、T166、W169、L178、L192和S195处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising amino acid residue mutations at I115, F163, T166, W169, L178, L192 and S195, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
在一些实施方式中，本发明提供一种ASC突变蛋白，其包含I115A、F163A、T166A、W169A、L178A、L192A和S195A突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。In some embodiments, the present invention provides an ASC mutant protein comprising I115A, F163A, T166A, W169A, L178A, L192A and S195A mutations, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
GenBank:BAA87339.2的氨基酸序列如SEQ ID NO:1所示，其中优选的突变位点所在的氨基酸残基以粗体显示。The amino acid sequence of GenBank:BAA87339.2 is shown in SEQ ID NO:1, where the amino acid residues at the preferred mutation sites are shown in bold.
SEQ ID NO:1
SEQ ID NO:1
本发明预期，所提供的ASC突变蛋白可用于例如疾病治疗和药物筛选。例如，在一些实施方式中，提供一种治疗由ASC-caspase-1炎症小体通路激活介导的疾病的方法，所述疾病优选选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症，所述方法包括向有需要的对象施用治疗有效量的所述ASC突变蛋白或其编码核酸。该ASC突变蛋白与野生型ASC蛋白竞争以减少形成ASC寡聚体和/或斑点的数量。The present invention contemplates that the provided ASC mutant proteins can be used, for example, for disease treatment and drug screening. For example, in some embodiments, a method for treating a disease mediated by activation of the ASC-caspase-1 inflammasome pathway is provided, the disease preferably being selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis, the method comprising administering a therapeutically effective amount of the ASC mutant protein or its encoding nucleic acid to a subject in need thereof. The ASC mutant protein competes with the wild-type ASC protein to reduce the number of ASC oligomers and/or spots formed.
在一些实施方式中，提供一种治疗由ASC-caspase-1炎症小体通路激活介导的疾病的方法，所述方法包括向有需要的对象施用治疗有效量的碱基编辑器，所述碱基编辑器导致ASC编码基因发生突变，所述突变包含在I115、F163、W169和L192处的至少一个氨基酸残基突变，以形成本发明所述的任一ASC突变蛋白。所述碱基编辑器例如是腺苷碱基编辑器(ABE)或胞苷碱基编辑器(CBE)。在优选的实施方式中，所述碱基编辑器通过质粒、病毒(例如腺病毒、腺相关病毒载体或慢病毒载体)等载体递送至细胞。在优选的实施方式中，所述由ASC-caspase-1炎症小体通路激活介导的疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。In some embodiments, a method for treating a disease mediated by activation of the ASC-caspase-1 inflammasome pathway is provided, the method comprising administering a therapeutically effective amount of a base editor to a subject in need, the base editor causing a mutation in the ASC encoding gene, the mutation comprising at least one amino acid residue mutation at I115, F163, W169 and L192 to form any ASC mutant protein described in the present invention. The base editor is, for example, an adenosine base editor (ABE) or a cytidine base editor (CBE). In a preferred embodiment, the base editor is delivered to a cell via a vector such as a plasmid, a virus (eg, an adenovirus, an adeno-associated virus vector or a lentiviral vector). In a preferred embodiment, the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
基于计算机的药物筛选方法Computer-based drug screening methods
本发明的再一个方面提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115、F163、W169和L192中的至少一个氨基酸残基相互作用的候选药物。Another aspect of the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with at least one of the amino acid residues I115, F163, W169 and L192 of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the I115 amino acid residue of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的F163氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the F163 amino acid residue of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的W169氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the W169 amino acid residue of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的L192氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying a candidate drug that interacts with the L192 amino acid residue of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115和F163氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and F163 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115和W169氨基酸残基相互作用的候选药物。 In some embodiments, the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and W169 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115和L192氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115 and L192 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115、F163和W169氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, and W169 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115、F163和L192氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, and L192 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的F163、W169和L192氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the F163, W169, and L192 amino acid residues of the ASC protein.
在一些实施方式中，本发明提供一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，所述方法包括识别与ASC蛋白的I115、F163、W169和L192氨基酸残基相互作用的候选药物。In some embodiments, the present invention provides a computer-based method for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method comprising identifying candidate drugs that interact with the I115, F163, W169 and L192 amino acid residues of the ASC protein.
在以上的任一基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法中，所述方法包括识别还与ASC蛋白的T166、L178和S195中的至少一个氨基酸残基相互作用的候选药物。In any of the above computer-based methods for screening drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway, the method includes identifying a candidate drug that also interacts with at least one of the amino acid residues T166, L178 and S195 of the ASC protein.
实施例Example
许多NLRP3的炎症小体抑制剂已被设计和开发用于治疗炎症小体相关疾病，而靶向ASC可能是一种新的有前景的策略。在这里，我们发现一种用作抗肿瘤药物的小分子糖酵解抑制剂氯尼达明(Lonidamine,LND)显著减弱多发性硬化症(multiple sclerosis,MS)和败血症这两个炎症小体相关疾病损伤。此外，LND阻断了多种类型的炎症小体激活，而且这种阻断作用不依赖于包括己糖激酶2(HK2)在内的LND已知靶点。我们的结果进一步揭示，LND直接与炎症小体配体ASC结合并抑制其寡聚化。我们的研究结果表明，LND是一种直接靶向ASC的广谱炎症小体抑制剂，为临床治疗炎症小体驱动的疾病提供了一种新的治疗方法。我们证明LND通过直接靶向ASC，以不依赖于HK2的方式阻断炎症小体的激活，并在多种炎症小体相关疾病的动物模型中显示出治疗效果，包括多发性硬化症和脓毒症。Many NLRP3 inflammasome inhibitors have been designed and developed for the treatment of inflammasome-related diseases, and targeting ASC may be a new and promising strategy. Here, we found that lonidamine (LND), a small molecule glycolysis inhibitor used as an antitumor drug, significantly attenuated damage in two inflammasome-related diseases, multiple sclerosis (MS) and sepsis. In addition, LND blocked multiple types of inflammasome activation, and this blocking effect was independent of LND's known targets, including hexokinase 2 (HK2). Our results further revealed that LND directly binds to the inflammasome ligand ASC and inhibits its oligomerization. Our results indicate that LND is a broad-spectrum inflammasome inhibitor that directly targets ASC, providing a new therapeutic approach for the clinical treatment of inflammasome-driven diseases. We demonstrated that LND blocks inflammasome activation in an HK2-independent manner by directly targeting ASC and shows therapeutic effects in animal models of multiple inflammasome-related diseases, including MS and sepsis.
试剂和抗体：抗IL-1β抗体(AF-401-NA)来自RD Systems，抗GSDMD(ab209845)和抗NEK7抗体(ab133514)来自Abcam，抗caspase-1(AG-20B-0042B)和抗ASC抗体(AG-25B-0006)来自Adipogen，抗α-tubulin抗体(ARG65693)来自Arigo。LPS(大肠杆菌O111:B4；#L2630)、ATP(A6419)和nigericin(481990)均来自Sigma。LND(AF-1890)和咪喹莫特(R-837)从Selleck获得。MSU晶体(tlrl-msu)、muramyld二肽(tlrl-mdp)、来自S.typhimurium的鞭毛蛋白(tlrl-stfla)和poly(dA:dT)/LyoVecTM均来自Invivogen。抗hexokinase 2抗体(NBP2-02272)来自Novus。抗NLRP3抗体(15101)来自Cell Signaling Technology。Goat抗rabbit IgG(HRP)(arg65351)，Donkey抗goat IgG(HRP)(arg65352)和Goat抗Mouse IgG抗体(HRP)(arg65350)均来自Arigo。双琥珀酰亚胺(DSS)购自Thermo Scientific。Reagents and antibodies: Anti-IL-1β antibody (AF-401-NA) was from RD Systems, anti-GSDMD (ab209845) and anti-NEK7 antibodies (ab133514) were from Abcam, anti-caspase-1 (AG-20B-0042B) and anti-ASC antibodies (AG-25B-0006) were from Adipogen, and anti-α-tubulin antibody (ARG65693) was from Arigo. LPS (Escherichia coli O111:B4; #L2630), ATP (A6419), and nigericin (481990) were from Sigma. LND (AF-1890) and imiquimod (R-837) were obtained from Selleck. MSU crystals (tlrl-msu), muramyld dipeptide (tlrl-mdp), flagellin from S. typhimurium (tlrl-stfla), and poly(dA:dT)/LyoVecTM were from Invivogen. Anti-hexokinase 2 antibody (NBP2-02272) was from Novus. Anti-NLRP3 antibody (15101) was from Cell Signaling Technology. Goat anti-rabbit IgG (HRP) (arg65351), Donkey anti-goat IgG (HRP) (arg65352), and Goat anti-Mouse IgG antibodies (HRP) (arg65350) were all from Arigo. Disuccinimidyl (DSS) was purchased from Thermo Scientific.
小鼠：C57BL/6J小鼠来自广东省医学实验动物中心(广州，中国)。条件性敲除己糖酶II小鼠(B6.129P2(Cg)-Hk2tm1.1Uku/Kctt,HK2 CKO flox/flox)从欧洲小鼠突变库(EMMA，项目编号：02074)引进，由广东药明生物技术有限公司培育。(B6J.B6n(Gg)-cx3cr1tm1.1(CRE)jung/j,Cx3cr1-Cre T)小鼠来自赛业(广州)生物科技有限公司。本研究中的所有动物实验都得到了中山大学机构动物护理与使用委员会和实验动物伦理委员会的批准(编号：SYSU-IACUC-2022-B0070)。Mice: C57BL/6J mice were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Conditional hexosidase II knockout mice (B6.129P2(Cg)-Hk2tm1.1Uku/Kctt, HK2 CKO flox/flox) were introduced from the European Mouse Mutation Bank (EMMA, Project No. 02074) and bred by Guangdong WuXi Biological Technology Co., Ltd. (B6J.B6n(Gg)-cx3cr1tm1.1(CRE)jung/j, Cx3cr1-Cre T) mice were obtained from Syy Biotech (Guangzhou) Co., Ltd. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee and the Laboratory Animal Ethics Committee of Sun Yat-sen University (No. SYSU-IACUC-2022-B0070).
细胞培养和刺激：从6-8周龄的小鼠骨髓中分离出BMDMs，在含有10％FBS和20％L929细胞(ATCC)上清液的DMEM培养基中培养7至8天。用CX3CR1-cre×HK2flox/flox小鼠选择性地敲除单核-巨噬细胞系中的HK2，然后从这HK2敲除小鼠中分离出原代BMDMs。J774A.1细胞来自ATCC，在含有10％FBS的DMEM中培养。Cell culture and stimulation: BMDMs were isolated from the bone marrow of 6-8 week old mice and cultured in DMEM medium containing 10% FBS and 20% supernatant of L929 cells (ATCC) for 7 to 8 days. HK2 in the mononuclear-macrophage cell line was selectively knocked out using CX3CR1-cre×HK2flox/flox mice, and primary BMDMs were then isolated from these HK2 knockout mice. J774A.1 cells were from ATCC and cultured in DMEM containing 10% FBS.
为了诱导炎症小体的激活，将BMDMs(5×105个细胞/mL)和J774A.1细胞(3×105个细胞/mL)置于6孔板中并培养过夜，第二天早上将培养基换成新鲜的DMEM。然后，用LPS(500ng/mL)诱导细胞3小时。之后，向培养物中加入LND孵育30分钟，然后，用ATP(5mM)刺激细胞30分钟，用MSU(150μg/mL)、Iiquimod(100μM)和nigericin(10μM)刺激4小时，用poly(dA:dT)(2μg/mL，6小时)、MDP(500ng/mL，6小时)和鞭毛蛋白(1μg/mL，6小时)。细胞提取物和收集的上清液通过Western blot进行分析。To induce inflammasome activation, BMDMs (5 × 10 5 cells/mL) and J774A.1 cells (3 × 10 5 cells/mL) were plated in 6-well plates and cultured overnight, and the medium was replaced with fresh DMEM the next morning. Then, the cells were induced with LPS (500 ng/mL) for 3 hours. After that, LND was added to the culture and incubated for 30 minutes. Then, the cells were stimulated with ATP (5 mM) for 30 minutes, MSU (150 μg/mL), Iiquimod (100 μM) and nigericin (10 μM) for 4 hours, poly (dA: dT) (2 μg/mL, 6 hours), MDP (500 ng/mL, 6 hours) and flagellin (1 μg/mL, 6 hours). Cell extracts and collected supernatants were analyzed by Western blot.
LPS诱导的脓毒症模型：在腹腔注射LPS(15mg/kg)(Sigma-Aldrich，L2630)前0.5小时，对8周龄雄性C57BL/6小鼠腹腔注射LND(40mg/kg和60mg/kg)或溶剂对照(β-环糊精)。4小时后，小鼠被安乐死，根据试剂商的说明，用ELISA试剂盒测量血清中IL-1和IL-18的水平。LPS-induced sepsis model: 8-week-old male C57BL/6 mice were intraperitoneally injected with LND (40 mg/kg and 60 mg/kg) or solvent control (β-cyclodextrin) 0.5 hours before intraperitoneal injection of LPS (15 mg/kg) (Sigma-Aldrich, L2630). Four hours later, the mice were euthanized, and the levels of IL-1 and IL-18 in the serum were measured using ELISA kits according to the manufacturer's instructions.
EAE的诱导和评估：这种建模方法是基于以前的研究。在8周大的雌性C57BL/6小鼠背侧皮下注射150μg乳化在CFA(5μg/mL)中的MOG35-55肽(RS合成公司)。24小时后腹腔注射百日咳毒素(每只小鼠500ng，List labs)。7天后进行第二次MOG和百日咳注射。从疾病诱发开始，每天腹腔注射LND(60mg/kg)。在相同的时间点，对溶剂组的小鼠注射β-CD。每天对动物进行监测和评分，直到它们被安乐死。临床评分的评估如下。0＝无症状，1＝尾巴强直性丧失，2＝步态不稳，3＝后肢瘫痪，4＝前肢瘫痪，5＝奄奄一息或死亡。如果分数介于两点之间，则使用0.5分的分级。Induction and evaluation of EAE: This modeling approach was based on previous studies. Female C57BL/6 mice aged 8 weeks were injected subcutaneously on the dorsal flank with 150 μg of MOG 35-55 peptide (RS Synthetics) emulsified in CFA (5 μg/mL). Pertussis toxin (500 ng per mouse, List labs) was injected intraperitoneally 24 hours later. A second MOG and pertussis injection was performed 7 days later. From the onset of disease induction, LND (60 mg/kg) was injected intraperitoneally daily. At the same time points, mice in the vehicle group were injected with β-CD. Animals were monitored and scored daily until they were euthanized. Clinical scores were evaluated as follows. 0 = no symptoms, 1 = loss of tail rigidity, 2 = unsteady gait, 3 = hind limb paralysis, 4 = forelimb paralysis, 5 = moribund or dead. If the score was between two points, a grading of 0.5 points was used.
ASC寡聚化检测：BMDMs种6孔板，用冰PBS清洗，并加入500μL冰缓冲液(20mM HEPES-KOH，pH 7.5，150mM KCl 1％NP40，蛋白酶抑制剂)。细胞在4℃下裂解30分钟，并取出50μL裂解液用于Westernblot检测。然后，剩余的裂解液在4℃下2500×g离心10分钟。将颗粒重悬在500微升冰PBS中。然后，将2mM的琥珀酰亚胺酯(Thermo Fisher A39267)加入重悬的颗粒中，并在室温下旋转孵化30分钟。然后在4℃下以2500×g离心10分钟。去除上清液，并将交联的沉淀重新悬浮在30μL Laemmli缓冲液中。将样品在100℃下煮沸10分钟，并通过Westernblot法进行分析。ASC oligomerization detection: BMDMs were seeded in 6-well plates, washed with ice-cold PBS, and 500 μL of ice-cold buffer (20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 1% NP40, protease inhibitors) was added. The cells were lysed at 4°C for 30 min, and 50 μL of lysate was taken out for Western blot detection. Then, the remaining lysate was centrifuged at 2500 × g for 10 min at 4°C. The pellet was resuspended in 500 μL of ice-cold PBS. Then, 2 mM succinimidyl ester (Thermo Fisher A39267) was added to the resuspended pellet and incubated with rotation at room temperature for 30 min. Then, centrifuged at 2500 × g for 10 min at 4°C. The supernatant was removed and the cross-linked pellet was resuspended in 30 μL of Laemmli buffer. The samples were boiled at 100°C for 10 min and analyzed by Western blot.
ELISA法：根据试剂商的说明分析细胞培养上清液和血清中的小鼠IL-1β(LIANKE BIOTECH,EK201B/3)、IL-18(LIANKE BIOTECH,EK218)和TNF-α(LIANKE BIOTECH,EK282/3)。ELISA method: Analyze mouse IL-1β (LIANKE BIOTECH, EK201B/3), IL-18 (LIANKE BIOTECH, EK218) and TNF-α (LIANKE BIOTECH, EK282/3) in cell culture supernatant and serum according to the reagent manufacturer's instructions.
免疫沉淀(IP)：LPS(500ng/ml)刺激BMDMs 30分钟之后，用LND处理，再用ATP刺激30分钟，将细胞重悬在含有蛋白酶抑制剂的IP裂解缓冲液(Beyotime，P0013J)中。然后，在4℃下以12,000g离心10分钟。将上清液与一抗或正常的兔/鼠IgG在4℃下孵育过夜。第二天，用蛋白G珠在4℃下沉淀抗体-蛋白复合物2小时，并进行免疫印迹分析。Immunoprecipitation (IP): After BMDMs were stimulated with LPS (500 ng/ml) for 30 min, treated with LND, and stimulated with ATP for 30 min, the cells were resuspended in IP lysis buffer (Beyotime, P0013J) containing protease inhibitors. Then, centrifuged at 12,000 g for 10 min at 4°C. The supernatant was incubated with primary antibody or normal rabbit/mouse IgG at 4°C overnight. The next day, the antibody-protein complex was precipitated with protein G beads at 4°C for 2 h and subjected to immunoblot analysis.
免疫组化染色：根据免疫组化(IHC)染色试剂盒(Abcam,ab80436,Cambridge,MA,USA)制造商的说明，对大脑和脊髓样本进行分析。简而言之，在去石蜡和水化后，将切片放在3％的过氧化氢中15分钟，并进行20分钟的抗原修复，之后冷却到室温。分别用H&E、Nissl或LFB对切片进行染色。在含有背景还原剂的抗体稀释液(DAKO,S3022,Santa Clara,CA,USA)中，将样品与指定的一抗在4℃下孵育过夜。然后，在用PBS清洗3次后，切片依次用二氨基联苯胺(DAB)底物-变色剂混合物和苏木精进行染色。Immunohistochemical staining: Brain and spinal cord samples were analyzed according to the manufacturer's instructions of the immunohistochemical (IHC) staining kit (Abcam, ab80436, Cambridge, MA, USA). Briefly, after deparaffinization and hydration, the sections were placed in 3% hydrogen peroxide for 15 minutes and antigen retrieval was performed for 20 minutes before cooling to room temperature. The sections were stained with H&E, Nissl, or LFB, respectively. The samples were incubated with the indicated primary antibodies overnight at 4°C in antibody diluent containing background reducing agent (DAKO, S3022, Santa Clara, CA, USA). Then, after washing three times with PBS, the sections were stained with diaminobenzidine (DAB) substrate-color-changing agent mixture and hematoxylin in sequence.
使用倒置显微镜(Eclipse Ti-U，日本尼康)进行拍摄。使用Image-Pro Plus软件(6.0版，Media Cybernetics,Inc.,Silver Springs,MD,USA)进行图像分析。An inverted microscope (Eclipse Ti-U, Nikon, Japan) was used for photography and Image-Pro Plus software (version 6.0, Media Cybernetics, Inc., Silver Springs, MD, USA) was used for image analysis.
免疫荧光染色：在组织切片过程中，大脑和脊髓被常规地分离和固定。然后，将样本嵌入石蜡中并切成5μm厚的切片。去石蜡和水化后，将切片进行抗原修复20分钟并冷却至室温。在带有背景消除试剂的抗体稀释液(DAKO，S3022，Santa Clara，CA，USA)中，将样品与指定的一抗在4℃下孵育过夜。用PBS清洗样品3次，并与指定的荧光结合的二抗(Molecular Probes,Thermo Fisher Scientific,Rockford,IL,USA)孵育1小时。然后，清洗样品并用Hoechst 33342(5μg/mL,Sigma-Aldrich)染色15分钟，用PBS清洗三次后，用水溶性封片剂封片。使用尼康A1光谱共聚焦显微镜(尼康，日本)对切片进行拍照。Immunofluorescence staining: During tissue sectioning, the brain and spinal cord were routinely isolated and fixed. Then, the samples were embedded in paraffin and cut into 5 μm thick sections. After deparaffinization and hydration, the sections were subjected to antigen retrieval for 20 min and cooled to room temperature. The samples were incubated with the designated primary antibodies at 4 °C overnight in antibody diluent with background elimination reagent (DAKO, S3022, Santa Clara, CA, USA). The samples were washed three times with PBS and incubated with the designated fluorescence-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific, Rockford, IL, USA) for 1 h. Then, the samples were washed and stained with Hoechst 33342 (5 μg/mL, Sigma-Aldrich) for 15 min, washed three times with PBS, and then mounted with water-soluble mounting medium. The sections were photographed using a Nikon A1 spectral confocal microscope (Nikon, Japan).
实时逆转录PCR：使用试剂(Invitrogen，15596)提取细胞的总RNA，然后使用oligo(dT)和RevertAid逆转录酶(Thermo Fisher Scientific，EP0442)进行逆转录。使用SuperReal PreMix SYBR Green(Tiangen，FP205)和7500快速实时PCR系统(Applied Biosystems，美国)进行实时PCR。通过比较Ct法(RQ＝2-ΔCt)计算和分析相对mRNA表达水平。引物的序列如下。
Real-time reverse transcription PCR: Use Reagent (Invitrogen, 15596) was used to extract total RNA of cells, and then reverse transcription was performed using oligo (dT) and RevertAid reverse transcriptase (Thermo Fisher Scientific, EP0442). Real-time PCR was performed using SuperReal PreMix SYBR Green (Tiangen, FP205) and 7500 fast real-time PCR system (Applied Biosystems, USA). Relative mRNA expression levels were calculated and analyzed by comparative Ct method (RQ = 2-ΔCt). The sequences of primers are as follows.
流式细胞仪：为了分析中枢神经系统的浸润性免疫细胞，用组织匀浆仪研磨MOG35- 55免疫小鼠的大脑组织和脊髓，形成单细胞悬浮液，通过300目滤膜过滤。离心后，用37％的percoll重新悬浮单细胞悬液，并在1000×g、室温、最低加速度设置和无制动的情况下离心30分钟。从最低层分离出单核细胞。将细胞悬浮在含有1％FBS的PBS中。清洗三次，并用细胞表面标志物抗体染色，进行流式细胞分析。使用了以下抗体。CD45-BV510(BD，563891)，CD4-FITC(BioLegend，100406)，CD8-Alexa Fluor 700(BD，557959)，以及CD11b-BV421(BioLegend，101236)。通过流式细胞仪(CytoFLEX,Beckman Coulter)进行流式细胞分析。根据正向和侧向散射以及Fixable Viability Stain 780(BD，565388)，排除细胞碎片和死细胞。Flow cytometry: To analyze the infiltrating immune cells of the central nervous system, the brain tissue and spinal cord of MOG 35-55 immunized mice were ground with a tissue homogenizer to form a single cell suspension, which was filtered through a 300 mesh filter. After centrifugation, the single cell suspension was resuspended with 37% percoll and centrifuged for 30 minutes at 1000×g, room temperature, minimum acceleration setting and no brake. Mononuclear cells were separated from the lowest layer. The cells were suspended in PBS containing 1% FBS. Washed three times and stained with cell surface marker antibodies for flow cytometric analysis. The following antibodies were used. CD45-BV510 (BD, 563891), CD4-FITC (BioLegend, 100406), CD8-Alexa Fluor 700 (BD, 557959), and CD11b-BV421 (BioLegend, 101236). Flow cytometric analysis was performed by flow cytometer (CytoFLEX, Beckman Coulter). Cell debris and dead cells were excluded based on forward and side scatter and Fixable Viability Stain 780 (BD, 565388).
细胞死亡检测：细胞处理后，收集培养基，根据试剂商的说明，使用CytoTox96非放射性细胞毒性试验(Promega,G1780)评估LDH的释放。Cell death detection: After cell treatment, the culture medium was collected and the release of LDH was evaluated using CytoTox96 non-radioactive cytotoxicity assay (Promega, G1780) according to the manufacturer's instructions.
siRNA介导的BMDMs基因干扰：小干扰RNA购自RiboBio(Guangzhou RiboBio Co.,Ltd.)。根据试剂商的说明，用LipofectamineTMRNAiMAX(Invitrogen,13778)进行RNA干扰。对达到60～70％汇合度的细胞进行RNA干扰。RNA干扰后48小时收集样品，并按上述方法进行Western blot或实时PCR检测。siRNA的序列如下。
siRNA-mediated gene interference in BMDMs: Small interfering RNA was purchased from RiboBio (Guangzhou RiboBio Co., Ltd.). RNA interference was performed using Lipofectamine TM RNAiMAX (Invitrogen, 13778) according to the manufacturer's instructions. RNA interference was performed on cells that reached 60-70% confluence. Samples were collected 48 hours after RNA interference and Western blot or real-time PCR were performed as described above. The sequences of siRNA are as follows.
DARTS检测：DARTS是根据已发表的实验步骤进行的。将BMDMs(5×105个细胞/mL)铺在10cm的大皿中，培养过夜，第二天早上将培养基换成新鲜的DMEM。然后，用LPS诱导细胞4小时，用M-PER(Thermo，78501)裂解缓冲液裂解。裂解液在4℃下以12,000×g离心10分钟，并进行BCA蛋白浓度检测以确定蛋白浓度。将裂解物与LND在4℃下孵育过夜。每个反应使用80μg的蛋白裂解液。然后，每μg蛋白质加入20ng的pronase酶(Sigma，PRON-RO)并在室温下孵育30分钟。加入20×蛋白酶抑制剂终止消化，并将样品在冰上孵育10分钟。接下来，向样品中加入5×SDS-PAGE加载缓冲液，以达到最终浓度为1×SDS-PAGE缓冲液。蛋白质样品通过Western blot进行分析。DARTS assay: DARTS was performed according to published protocols. BMDMs (5 × 10 5 cells/mL) were plated in 10 cm dishes and cultured overnight, and the medium was replaced with fresh DMEM the next morning. Then, cells were induced with LPS for 4 h and lysed with M-PER (Thermo, 78501) lysis buffer. The lysate was centrifuged at 12,000 × g for 10 min at 4 °C, and the protein concentration was determined by BCA protein concentration assay. The lysate was incubated with LND overnight at 4 °C. 80 μg of protein lysate was used for each reaction. Then, 20 ng of pronase enzyme (Sigma, PRON-RO) was added per μg of protein and incubated at room temperature for 30 min. The digestion was terminated by adding 20× protease inhibitor, and the samples were incubated on ice for 10 min. Next, 5× SDS-PAGE loading buffer was added to the samples to reach a final concentration of 1× SDS-PAGE buffer. Protein samples were analyzed by Western blot.
用LigandScout进行分子模拟对接：从PDB数据库中下载了全长的人类ASC(PDB代码2KN6)的结构，并使用Ligandscout 4.4.7软件进行分析。使用默认设置将LND与ASC的CARD结构域对接。分子图形由PyMOL制备。Molecular modeling and docking with LigandScout: The structure of full-length human ASC (PDB code 2KN6) was downloaded from the PDB database and analyzed using Ligandscout 4.4.7 software. LND was docked to the CARD domain of ASC using default settings. Molecular graphics were prepared by PyMOL.
LND结构类似物分子模拟对接：我们从NCBI数据库中搜索并下载了与LND结构类似的化合物111个，对上述化合物进行了构象优化(MMF94力场，Ligandscout)，获得LND结构类似化合物库。我们对ASC的结构数据(PDB：2kn6)进行了可能的结合口袋搜寻(Calculate Pocket Ligandscout)，参数为：Buriedness 0.18,Threshold 0.30。在CARD结构域构建了结合口袋。最后，我们把LND结构类似化合物库中的化合物对接(docking)进结合口袋(AutoDock Vina 1.1)，并打分(Binding affinity score)。对接参数：Exaustiveness 40,Max.Number of modes 10,Max.energy difference 3，对接结果按照亲和力大小进行排序。Molecular simulation docking of LND structural analogs: We searched and downloaded 111 compounds with similar structures to LND from the NCBI database, optimized the conformations of the above compounds (MMF94 force field, Ligandscout), and obtained a library of compounds with similar structures to LND. We searched for possible binding pockets (Calculate Pocket Ligandscout) on the structural data of ASC (PDB: 2kn6) with the parameters of Buriedness 0.18 and Threshold 0.30. A binding pocket was constructed in the CARD domain. Finally, we docked the compounds in the library of LND structural analogs into the binding pocket (AutoDock Vina 1.1) and scored them (Binding affinity score). Docking parameters: Exaustiveness 40, Max. Number of modes 10, Max. energy difference 3. The docking results were sorted according to affinity.
透射电镜：纯化表达的ASC蛋白样品(6μM)，分别加入TEV酶(6UI)，同时加入不同浓度的LND，在4℃孵育过夜。将一滴10μl样品放置在干净的Parafilm上，并将一个网孔铜纯碳涂层网格浮在顶部10分钟。然后，转移网格并与1％乙酸铀酰进行对比5分钟。在使用透射电子显微镜FEI Tecnai G2Spirit(ThermoFisher Scientific Company，OR，USA)检查之前，去除多余的流体并使其干燥。所有图像均使用Radius软件和Xarosa数码相机(EMSIS GmbH，Münster，Germany)采集。Transmission electron microscopy: Purified expressed ASC protein samples (6 μM) were added with TEV enzyme (6 UI) and different concentrations of LND and incubated overnight at 4 °C. A drop of 10 μl of sample was placed on a clean Parafilm and a mesh copper pure carbon coated grid was floated on top for 10 min. Then, the grid was transferred and contrasted with 1% uranyl acetate for 5 min. Excess fluid was removed and allowed to dry before examination using a transmission electron microscope FEI Tecnai G2 Spirit (ThermoFisher Scientific Company, OR, USA). All images were acquired using Radius software and a Xarosa digital camera (EMSIS GmbH, Münster, Germany).
表面等离子体共振(SPR)分析：实验在Biacore T100中进行。重组的人ASC蛋白(Zeye Biotechnology)被固定在CM5传感器芯片(BR100530，Cytiva)上。将LND溶于基本运行缓冲液(含1％DMSO的PBS)，在25℃下以5μL/分钟的流速注入流动池。在每个浓度之间用基本运行缓冲液清洗传感器芯片。ASC蛋白被固定在同一芯片的不同通道中，以同样的方式注入空白的基本运行缓冲液得到的反应值被用作对照。用Biacore T100评估软件计算相互作用的动力学参数和亲和力常数。Surface plasmon resonance (SPR) analysis: The experiments were performed in a Biacore T100. Recombinant human ASC protein (Zeye Biotechnology) was immobilized on a CM5 sensor chip (BR100530, Cytiva). LND was dissolved in basic running buffer (PBS with 1% DMSO) and injected into the flow cell at a flow rate of 5 μL/min at 25 °C. The sensor chip was washed with basic running buffer between each concentration. ASC protein was immobilized in different channels of the same chip, and the response values obtained by injecting blank basic running buffer in the same way were used as controls. The kinetic parameters and affinity constants of the interaction were calculated using the Biacore T100 evaluation software.
实施例1.LND减轻了实验性自身免疫性脑脊髓炎(EAE)的炎性损伤。Example 1. LND reduces inflammatory damage in experimental autoimmune encephalomyelitis (EAE).
应用EAE模型证明了LND的抗炎作用。从第一次免疫注射髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)35～55肽之日起，用对EAE小鼠进行LND给药，直到实验的终点。我们发现在EAE疾病的高峰期，LND明显改善了EAE小鼠的神经系统缺陷(图1a)，并抑制了体重下降(图1b)。此外，组织学分析显示LND减少了单核细胞在脊髓中的浸润，减少了神经元的脱髓鞘(图1c)。The anti-inflammatory effect of LND was demonstrated using the EAE model. From the date of the first immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, EAE mice were administered LND until the end of the experiment. We found that at the peak of EAE disease, LND significantly improved the neurological deficits of EAE mice (Figure 1a) and inhibited weight loss (Figure 1b). In addition, histological analysis showed that LND reduced the infiltration of monocytes in the spinal cord and reduced the demyelination of neurons (Figure 1c).
外周免疫细胞对中枢神经系统的大量浸润是EAE的主要病理特征之一，通过流式细胞仪分析各组重的脊髓和脑组织中外周免疫细胞的比例来分析LND是否能减少这种EAE损伤。流式细胞仪分析显示，用LND治疗的小鼠中枢神经系统的CD4+T细胞、CD8+T细胞和髓系细胞(CD45+CD11b+)的浸润明显减少(图1d)。为了进一步评估LND对EAE的治疗效果，在EAE小鼠发病后再给予LND，发现LND明显降低了EAE的严重程度(图1e)以及减轻了体重下降(图1f)。同样，LND给药治疗后，小鼠中枢神经系统中来自外周循环的CD4+和CD8+T细胞的浸润显著减少，并减少了浸润中枢神经组织的髓系细胞(图1g)。这些结果显示，LND治疗有效抑制炎症并明显减弱了EAE所致的神经损伤。The massive infiltration of peripheral immune cells into the central nervous system is one of the main pathological characteristics of EAE. The proportion of peripheral immune cells in the spinal cord and brain tissues of each group was analyzed by flow cytometry to analyze whether LND can reduce this EAE damage. Flow cytometric analysis showed that the infiltration of CD4 + T cells, CD8 + T cells and myeloid cells (CD45 + CD11b + ) in the central nervous system of mice treated with LND was significantly reduced (Figure 1d). To further evaluate the therapeutic effect of LND on EAE, LND was given to EAE mice after the onset of the disease, and it was found that LND significantly reduced the severity of EAE (Figure 1e) and reduced weight loss (Figure 1f). Similarly, after LND administration, the infiltration of CD4 + and CD8 + T cells from the peripheral circulation in the central nervous system of mice was significantly reduced, and the myeloid cells infiltrating the central nervous tissue were reduced (Figure 1g). These results show that LND treatment effectively inhibits inflammation and significantly attenuates nerve damage caused by EAE.
实施例2.LND抑制炎症小体的激活。Example 2. LND inhibits the activation of inflammasomes.
炎症小体是无菌性炎症信号的主要传感器，是炎症反应的一个关键触发因素，我们进一步研究了LND在体内抑制炎症小体激活的可能性。在上述EAE小鼠脊髓中，我们发现在LND处理后增加的GSDMD和caspase-1水平明显降低(图2a和2b)。这表明，LND阻止了EAE小鼠中枢神经系统组织的炎症小体激活。除了中枢神经系统炎症疾病，我们进一步研究了LND介导的对脂多糖(lipopolysaccharide,LPS)诱导的败血症模型中炎症小体的抑制作用。腹腔注射LPS可诱发小鼠NLRP3依赖性炎症和脓毒症休克。如图2c和2d所示，LND处理明显缓解了LPS诱导的IL-1β和IL-18的血清水平的增加。Inflammasomes are the main sensors of sterile inflammatory signals and a key trigger of inflammatory responses. We further investigated the possibility that LND could inhibit inflammasome activation in vivo. In the spinal cord of EAE mice described above, we found that the increased levels of GSDMD and caspase-1 were significantly reduced after LND treatment (Figures 2a and 2b). This suggests that LND prevented inflammasome activation in the central nervous system tissues of EAE mice. In addition to central nervous system inflammatory diseases, we further investigated the LND-mediated inhibition of inflammasomes in a lipopolysaccharide (LPS)-induced sepsis model. Intraperitoneal injection of LPS can induce NLRP3-dependent inflammation and septic shock in mice. As shown in Figures 2c and 2d, LND treatment significantly alleviated the LPS-induced increase in serum levels of IL-1β and IL-18.
上述结果表明，LND抑制了体内炎症小体的激活。The above results indicate that LND inhibits the activation of inflammasomes in vivo.
实施例3.LND抑制了不同激动剂诱导的NLRP3炎症小体激活。Example 3. LND inhibited NLRP3 inflammasome activation induced by different agonists.
应用LPS和ATP诱导小鼠骨髓来源巨噬细胞(Bone marrow derived macrophages,BMDMs)的NLRP3炎症小体激活的经典模型，在加入ATP之前用LND预处理0.5小时，来探究LND对炎症小体激活的抑制作用。免疫印迹结果显示，在BMDMs(图3a)和J1774A.1细胞(图3b)中，LND剂量依赖性地抑制了ATP诱导的caspase-1前体裂解为其生物活性形式P20以及IL-1β前体裂解成其生物活性形式P17。此外，酶联免疫吸附试验(ELISA)结果还表明，LND抑制了BMDMs和J1774A.1细胞中IL-1β(图3c)和IL-18(图3d)的细胞外分泌(图3e)，但对不依赖于炎症小体产生的细胞因子TNF-α释放没有影响(图3f)。LND还抑制了细胞焦亡执行蛋白GSDMD的裂解(图3g和3h)和随后的乳酸脱氢酶(lactate dehydrogenase,LDH)释放(图3i和图3j)。这些结果表明，LND显著抑制LPS和ATP诱导的NLRP3炎症小体的激活和细胞焦亡。接下来，我们探究了LND是否影响了LPS诱导的炎症小体激活的启动阶段。当BMDMs在LPS刺激3小时前用LND预处理时，LND既不影响pro-IL-1β、NLRP3、pro-IL-18或TNF-α的mRNA水平的增加(图3k-3m)，也不抑制pro-IL-1β和NLRP3的蛋白丰度增加(图3n)，说明LND对炎症小体激活的启动阶段没有影响。The classic model of NLRP3 inflammasome activation in mouse bone marrow-derived macrophages (BMDMs) induced by LPS and ATP was used to investigate the inhibitory effect of LND on inflammasome activation by pre-treating with LND for 0.5 h before adding ATP. Immunoblotting results showed that LND dose-dependently inhibited ATP-induced cleavage of caspase-1 precursor to its bioactive form P20 and cleavage of IL-1β precursor to its bioactive form P17 in BMDMs (Figure 3a) and J1774A.1 cells (Figure 3b). In addition, enzyme-linked immunosorbent assay (ELISA) results also showed that LND inhibited the extracellular secretion of IL-1β (Figure 3c) and IL-18 (Figure 3d) in BMDMs and J1774A.1 cells (Figure 3e), but had no effect on the release of cytokine TNF-α, which is independent of inflammasome production (Figure 3f). LND also inhibited the cleavage of the pyroptosis executioner protein GSDMD (Figures 3g and 3h) and the subsequent release of lactate dehydrogenase (LDH) (Figures 3i and 3j). These results indicate that LND significantly inhibited LPS- and ATP-induced NLRP3 inflammasome activation and pyroptosis. Next, we investigated whether LND affected the initiation phase of LPS-induced inflammasome activation. When BMDMs were pretreated with LND 3 hours before LPS stimulation, LND neither affected the increase in mRNA levels of pro-IL-1β, NLRP3, pro-IL-18, or TNF-α (Figures 3k-3m) nor inhibited the increase in protein abundance of pro-IL-1β and NLRP3 (Figure 3n), indicating that LND had no effect on the initiation phase of inflammasome activation.
除了ATP和LPS刺激以外，NLRP3炎症小体还可以被其他危险相关分子模式激活，如尼日利亚菌素和尿酸结晶(monosodium urate,MSU)，这些激活剂依赖于钾离子外流。用LND处理可抑制由尼日利亚菌素和MSU引发的caspase-1裂解和IL-1β的分泌(图3o和3p)。此外，咪喹莫特是一种不依赖于钾离子外流的NLRP3炎症小体激活剂，LND还抑制了咪喹莫特诱导的caspase-1和IL-1β的激活(图3o和3p)。In addition to ATP and LPS stimulation, the NLRP3 inflammasome can be activated by other danger-associated molecular patterns, such as nigericin and monosodium urate (MSU), which are dependent on potassium ion efflux. Treatment with LND inhibited caspase-1 cleavage and IL-1β secretion triggered by nigericin and MSU (Figures 3o and 3p). In addition, imiquimod, an NLRP3 inflammasome activator that is independent of potassium ion efflux, also inhibited imiquimod-induced caspase-1 and IL-1β activation (Figures 3o and 3p).
总之，这些结果表明LND可以抑制各种不同激动剂诱导的NLRP3炎症小体激活。Together, these results suggest that LND can inhibit NLRP3 inflammasome activation induced by a variety of different agonists.
实施例4.LND不依赖于HK2来抑制NLRP3炎症小体的激活。Example 4. LND inhibits NLRP3 inflammasome activation independently of HK2.
LND通过抑制HK2的活性来抑制糖酵解，另外LND还有其他作用靶点，包括电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)、线粒体丙酮酸载体(mitochondrial pyruvate carrier,MPC)和单羧酸转运体(monocarboxylate transporters,MCT)以及琥珀酸脱氢酶(succinate dehydrogenase,SDH)。为了研究LND通过哪个靶点来抑制NLRP3炎症小体激活，我们进一步测试LPS和ATP诱导的NLRP3炎症小体激活模型中这些已知的LND靶点的mRNA水平。结果显示，只有HK2的mRNA水平(图4a)增加，而LND的其他靶点没有变化(图4a)。免疫印迹试验也证实，LPS和ATP处理诱导了HK2的蛋白水平(图4b)，提示HK2可能在NLRP3炎症小体的激活中发挥作用。LND inhibits glycolysis by inhibiting the activity of HK2. In addition, LND has other targets, including voltage-dependent anion channel (VDAC), mitochondrial pyruvate carrier (MPC) and monocarboxylate transporters (MCT) and succinate dehydrogenase (SDH). To investigate which targets LND inhibits NLRP3 inflammasome activation, we further tested the mRNA levels of these known LND targets in the LPS- and ATP-induced NLRP3 inflammasome activation model. The results showed that only the mRNA level of HK2 (Figure 4a) increased, while other targets of LND did not change (Figure 4a). Immunoblotting also confirmed that LPS and ATP treatment induced the protein level of HK2 (Figure 4b), suggesting that HK2 may play a role in the activation of NLRP3 inflammasome.
意外的是，在BMDMs(图4c和4d)和J1774A.1细胞(图4e和4f)中，由siRNA介导的对HK2的干扰并没有减少caspase-1的裂解和IL-1β的产生。此外在来自野生型(WT)小鼠和HK2基因敲除小鼠的BMDMs中，LPS和ATP诱导的NLRP3炎症小体的激活程度相当(图4g)，且LND仍然减弱了HK2基因敲除小鼠的BMDMs中NLRP3的激活(图4h和4i)。这个结果表明，LND抑制NLRP3炎症小体的激活不依赖于HK2。Unexpectedly, siRNA-mediated interference with HK2 did not reduce caspase-1 cleavage and IL-1β production in BMDMs (Figures 4c and 4d) and J1774A.1 cells (Figures 4e and 4f). In addition, LPS- and ATP-induced NLRP3 inflammasome activation was comparable in BMDMs from wild-type (WT) mice and HK2 knockout mice (Figure 4g), and LND still attenuated NLRP3 activation in BMDMs from HK2 knockout mice (Figures 4h and 4i). This result suggests that LND inhibits NLRP3 inflammasome activation independent of HK2.
实施例5.LND选择性地阻断了ASC的寡聚化。Example 5. LND selectively blocks oligomerization of ASC.
然后我们进一步探讨了LND抑制炎症小体激活的可能机制。钾离子外流和活性氧(reactive oxygen species,ROS)的产生等一系列的上游事件可以触发NLRP3炎症小体的激活。LND可以抑制依赖或独立于钾离子外流的不同激活剂诱导的NLRP3炎症小体激活(图3)。此外，LND在抑制炎症小体激活时并没有抑制ROS的产生(图5a)。这些结果表明，LND不作用于NLRP3炎症小体的这些上游激活事件。We then further explored the possible mechanism by which LND inhibits inflammasome activation. A series of upstream events, such as potassium ion efflux and the generation of reactive oxygen species (ROS), can trigger the activation of NLRP3 inflammasome. LND can inhibit NLRP3 inflammasome activation induced by different activators that are dependent or independent of potassium ion efflux (Figure 3). In addition, LND did not inhibit the generation of ROS when inhibiting inflammasome activation (Figure 5a). These results indicate that LND does not act on these upstream activation events of NLRP3 inflammasome.
然后，我们分析了LND直接作用于炎症小体组装的可能性。最近，NIMA相关激酶7(NIMA-related kinase 7,NEK7)被认为是NLRP3炎症小体的一个重要组成部分，NEK7与NLRP3的相互作用被证明是NLRP3寡聚化和炎症小体组装的关键。然而，LND对BMDMs中NEK7和NLRP3之间的结合没有影响(图5b)。ASC介导的NLRP3和caspase-1的连接是炎症小体组装的另一个关键步骤。我们发现，LND没有阻断NLRP3和ASC之间的相互作用(图5c)，但抑制了ASC和caspase-1之间的结合(图5d)。然后我们进一步检查了LND对炎症小体激活必要步骤ASC寡聚化的影响，发现LND剂量依赖性的抑制ATP诱导的ASC寡聚化(图5e和5f)。此外，在BMDMs的炎症小体激活过程中，ASC寡聚化在核周围形成一个大斑点(即ASC斑点(ASC specks)，图5g和5h)，而用LND预处理明显抑制了这种ASC斑点的形成(图5g和5h)。为了探索LND阻断ASC寡聚化的能力，我们加入不同浓度LND的情况下，观察纯化的人重组ASC蛋白中体外形成ASC寡聚体的能力。电子显微镜分析表明，LND显著抑制ASC寡聚体的形成。此外，在EAE小鼠的脊髓中LND治疗下降了小胶质细胞中ASC斑点增加(图5i)。除了NLRP3炎症小体，ASC的寡聚化还参与了AIM2、NLRP1和NLRC4等其他类型的炎症小体激活。因此，我们也测试了LND是否能抑制其他炎症小体的激活，结果表明LND抑制了由MDP(一种NLRP1炎症小体激活剂)、鞭毛蛋白(一种NLRC4炎症小体激活剂)或poly(dA:dT)(一种AIM2炎症小体激活剂)诱发的IL-1β的分泌(图5j)。综上所述，这些结果证实LND可以阻断ASC的寡聚化，是广谱炎症小体抑制剂，并且提示LND有可能直接与ASC结合。We then analyzed the possibility that LND directly acts on inflammasome assembly. Recently, NIMA-related kinase 7 (NEK7) has been identified as an essential component of the NLRP3 inflammasome, and the interaction between NEK7 and NLRP3 has been shown to be critical for NLRP3 oligomerization and inflammasome assembly. However, LND had no effect on the binding between NEK7 and NLRP3 in BMDMs (Figure 5b). ASC-mediated ligation of NLRP3 and caspase-1 is another key step in inflammasome assembly. We found that LND did not block the interaction between NLRP3 and ASC (Figure 5c), but inhibited the binding between ASC and caspase-1 (Figure 5d). We then further examined the effect of LND on ASC oligomerization, an essential step for inflammasome activation, and found that LND dose-dependently inhibited ATP-induced ASC oligomerization (Figures 5e and 5f). In addition, during inflammasome activation in BMDMs, ASC oligomerizes to form a large spot around the nucleus (i.e., ASC specks, Figures 5g and 5h), while pretreatment with LND significantly inhibited the formation of such ASC specks (Figures 5g and 5h). To explore the ability of LND to block ASC oligomerization, we observed the ability of purified human recombinant ASC protein to form ASC oligomers in vitro when different concentrations of LND were added. Electron microscopy analysis showed that LND significantly inhibited the formation of ASC oligomers. In addition, LND treatment reduced the increase of ASC specks in microglia in the spinal cord of EAE mice (Figure 5i). In addition to the NLRP3 inflammasome, ASC oligomerization is also involved in the activation of other types of inflammasomes such as AIM2, NLRP1, and NLRC4. Therefore, we also tested whether LND could inhibit the activation of other inflammasomes, and the results showed that LND inhibited the secretion of IL-1β induced by MDP (an NLRP1 inflammasome activator), flagellin (an NLRC4 inflammasome activator), or poly(dA:dT) (an AIM2 inflammasome activator) (Figure 5j). Taken together, these results confirm that LND can block the oligomerization of ASC and is a broad-spectrum inflammasome inhibitor, and suggest that LND may directly bind to ASC.
实施例6.LND直接结合ASC蛋白Example 6. LND directly binds to ASC protein
为了研究LND是否直接与ASC结合，我们应用ASC的晶体结构(PDB 2KN6)进行分子建模分析。结果显示，LND很容易与CARD口袋对接，并与ASC蛋白的I115、F163、T166、W169、L178、L192和S195等多个氨基酸残基位点相互作用(图6a)。其中，已经有报道表明W169、L178和L192对ASC的寡聚化至关重要。因此，LND可能通过与这些氨基酸的相互作用来抑制ASC的寡聚化和随后的ASC对caspase-1前体的招募。应用表面等离子体共振(surface plasmon resonance,SPR)实验来分析LND和重组人ASC蛋白的直接相互作用，结果显示LND以剂量依赖的方式直接与ASC结合(图6b)。然后应用药物亲和力反应靶标稳定性(drug affinity responsive target stability,DARTS)方法来确认LND和ASC的直接相互作用。我们发现，LND保护ASC免受pronase蛋白酶介导的蛋白质降解，但不保护NLRP3和pro-caspase-1(图6c)。这些结果显示，LND通过选择性与ASC而不是NLRP3和pro-caspase-1结合，来抑制炎症小体激活。为了进一步探究模拟对接到的氨基酸残基对于ASC聚合体的重要性，我们在ASC-EGFP质粒中通过定点诱变突变这些位置，将位点突变质粒转染进HEK293T细胞中以研究ASC寡聚体的形成。除了T166，L178和S195以外，I115，F163，W169和L192位点突变都显著减少了ASC寡聚体和斑点的数量(图6d，e)，而不影响蛋白表达(图3d)，因此表明LND结合位点对ASC斑点形成至关重要，并发现了抑制ASC聚合的新位点。To investigate whether LND directly binds to ASC, we used the crystal structure of ASC (PDB 2KN6) for molecular modeling analysis. The results showed that LND easily docked with the CARD pocket and interacted with multiple amino acid residues of ASC protein, including I115, F163, T166, W169, L178, L192, and S195 (Figure 6a). Among them, W169, L178, and L192 have been reported to be essential for the oligomerization of ASC. Therefore, LND may inhibit the oligomerization of ASC and the subsequent recruitment of ASC to caspase-1 precursors by interacting with these amino acids. Surface plasmon resonance (SPR) experiments were applied to analyze the direct interaction between LND and recombinant human ASC protein, and the results showed that LND directly bound to ASC in a dose-dependent manner (Figure 6b). Then the drug affinity responsive target stability (DARTS) method was applied to confirm the direct interaction between LND and ASC. We found that LND protected ASC from pronase-mediated protein degradation, but not NLRP3 and pro-caspase-1 (Fig. 6c). These results suggest that LND inhibits inflammasome activation by selectively binding to ASC rather than NLRP3 and pro-caspase-1. To further explore the importance of the amino acid residues to which the mimic docking is linked for ASC aggregation, we mutated these positions by site-directed mutagenesis in the ASC-EGFP plasmid and transfected the site-mutated plasmid into HEK293T cells to study the formation of ASC oligomers. In addition to T166, L178 and S195, mutations in sites I115, F163, W169 and L192 all significantly reduced the number of ASC oligomers and specks (Fig. 6d, e) without affecting protein expression (Fig. 3d), thus indicating that the LND binding site is essential for ASC speck formation and discovering a new site for inhibiting ASC aggregation.
实施例7.LND及其类似物与ASC蛋白的亲和力分析Example 7. Affinity analysis of LND and its analogs with ASC protein
对LND的结构类似物与ASC CARD结构域进行分子模拟对接分析，部分化合物的对接结果按照亲和力排序如下。



Molecular simulation docking analysis was performed on the structural analogs of LND and the ASC CARD domain, and the docking results of some compounds are ranked according to affinity as follows.



Claims (20)

  1. 式Ia或Ib所示的化合物或其药学上可接受的盐或酯在制备用于治疗基于ASC-caspase-1通路的炎症小体激活介导的疾病的药物中的应用，
    Use of a compound represented by formula Ia or Ib or a pharmaceutically acceptable salt or ester thereof in the preparation of a drug for treating a disease mediated by inflammasome activation based on the ASC-caspase-1 pathway,
    其中：in:
    R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R2选自羧基、C1-3烷基和酰肼基；R3为H或C1-3烷基；R4和R5独立地选自氢、卤素和C1-3烷基；R6为H或C1-3烷基；Z选自C、N、O和S；p为1、2或3；并且 R1 is selected from hydrogen, halogen, C1-3 alkyl, amino, C1-3 alkylamino and halogenated C1-3 alkyl; R2 is selected from carboxyl, C1-3 alkyl and hydrazide; R3 is H or C1-3 alkyl; R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl; R6 is H or C1-3 alkyl; Z is selected from C, N, O and S; p is 1, 2 or 3; and
    其中所述基于ASC-caspase-1通路的炎症小体激活介导的疾病不是以下任一种疾病：癌症、关节炎、急性中枢神经系统损伤、阿尔兹海默症、不孕不育症、黄斑病变、良性前列腺增生和艾滋病。The disease mediated by inflammasome activation based on the ASC-caspase-1 pathway is not any of the following diseases: cancer, arthritis, acute central nervous system injury, Alzheimer's disease, infertility, macular degeneration, benign prostatic hyperplasia and AIDS.
  2. 根据权利要求1所述的应用，其中R1选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基。The use according to claim 1, wherein R 1 is selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl.
  3. 根据权利要求1或2所述的应用，其中R2为羧基或酰肼基。The use according to claim 1 or 2, wherein R2 is a carboxyl group or a hydrazide group.
  4. 根据权利要求1-3任一项所述的应用，其中R3为氢或甲基。The use according to any one of claims 1 to 3, wherein R 3 is hydrogen or methyl.
  5. 根据权利要求1-4任一项所述的应用，其中R4和R5独立地选自氢、甲基、氟和氯。The use according to any one of claims 1 to 4, wherein R 4 and R 5 are independently selected from hydrogen, methyl, fluorine and chlorine.
  6. 根据权利要求1-5任一项所述的应用，其中Z选自C和N。The use according to any one of claims 1 to 5, wherein Z is selected from C and N.
  7. 根据权利要求1-6任一项所述的应用，其中p为1。The use according to any one of claims 1 to 6, wherein p is 1.
  8. 根据权利要求1所述的应用，其中所述化合物为式IIa或IIb所示的化合物或其药学上可接受的盐或酯，
    The use according to claim 1, wherein the compound is a compound represented by Formula IIa or IIb or a pharmaceutically acceptable salt or ester thereof,
    其中，in,
    R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl;
    R2选自羧基、C1-3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
    R4和R5独立地选自氢、卤素和C1-3烷基，优选选自甲基、氟和氯。 R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl, preferably selected from methyl, fluorine and chlorine.
  9. 根据权利要求1所述的应用，其中所述化合物为式(IIIa)或(IIIb)所示的化合物或其药学上可接受的盐或酯，
    The use according to claim 1, wherein the compound is a compound represented by formula (IIIa) or (IIIb) or a pharmaceutically acceptable salt or ester thereof,
    其中，in,
    R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl;
    R2选自羧基、C1-3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl;
    R4和R5独立地选自氢、卤素和C1-3烷基，优选选自甲基、氟和氯，更优选为氟或氯。 R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl, preferably selected from methyl, fluorine and chlorine, more preferably fluorine or chlorine.
  10. 根据权利要求1所述的应用，其中所述化合物为式IVa、IVb或IVc所示的化合物或其药学上可接受的盐或酯，

    The use according to claim 1, wherein the compound is a compound represented by formula IVa, IVb or IVc or a pharmaceutically acceptable salt or ester thereof,

    其中，in,
    R1选自氢、卤素、C1-3烷基、氨基、C1-3烷基氨基和卤代C1-3烷基；R 1 is selected from hydrogen, halogen, C 1-3 alkyl, amino, C 1-3 alkylamino and halogenated C 1-3 alkyl;
    R2选自羧基、C1-3烷基和酰肼基，优选选自氢、氟、氯、碘、甲基、乙基、氨基、二甲基氨基、氟代甲基、三氟甲基和二氟甲基；更优选选自氢、氟、氯和二氟甲基；R 2 is selected from carboxyl, C 1-3 alkyl and hydrazide, preferably selected from hydrogen, fluorine, chlorine, iodine, methyl, ethyl, amino, dimethylamino, fluoromethyl, trifluoromethyl and difluoromethyl; more preferably selected from hydrogen, fluorine, chlorine and difluoromethyl;
    R4和R5独立地选自氢、卤素和C1-3烷基，优选选自氟和氯，更优选均为氯。 R4 and R5 are independently selected from hydrogen, halogen and C1-3 alkyl, preferably selected from fluorine and chlorine, more preferably both are chlorine.
  11. 根据权利要求1-10任一项所述的应用，其中所述化合物选自：The use according to any one of claims 1 to 10, wherein the compound is selected from:
    6-氯-1-[(2,4-二氯苯基)甲基]吲唑-3-羧酸(11501294)；6-Chloro-1-[(2,4-dichlorophenyl)methyl]indazole-3-carboxylic acid (11501294);
    5-(4-氯苯基)-1-[1-(2,4-二氯苯基)乙基]吡唑-3-羧酸(67565577)；5-(4-chlorophenyl)-1-[1-(2,4-dichlorophenyl)ethyl]pyrazole-3-carboxylic acid (67565577);
    1-[(2-氯-4-氟苯基)甲基]-5-(二氟甲基)吲唑-3-羧酸(66888740)；1-[(2-chloro-4-fluorophenyl)methyl]-5-(difluoromethyl)indazole-3-carboxylic acid (66888740);
    1-[(2,4-二氯苯基)甲基]-5-氟吲唑-3-羧酸(71221361)；1-[(2,4-Dichlorophenyl)methyl]-5-fluoroindazole-3-carboxylic acid (71221361);
    1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸(39562)；1-[(2,4-Difluorophenyl)methyl]indazole-3-carboxylic acid (39562);
    1-[(2,4-二氯苯基)甲基]-6-(氟甲基)吲唑-3-羧酸(57633631)；1-[(2,4-Dichlorophenyl)methyl]-6-(fluoromethyl)indazole-3-carboxylic acid (57633631);
    1-[(2,4-二氯苯基)甲基]-6-(二氟甲基)吲唑-3-羧酸(11667849)；1-[(2,4-Dichlorophenyl)methyl]-6-(difluoromethyl)indazole-3-carboxylic acid (11667849);
    1-[(2,4-二氯苯基)甲基]吲唑-3-羧酸盐(25271808)；1-[(2,4-Dichlorophenyl)methyl]indazole-3-carboxylate (25271808);
    1-[(2,4-二氯苯基)甲基]-5-碘吲唑-3-羧酸(71216306)；1-[(2,4-Dichlorophenyl)methyl]-5-iodoindazole-3-carboxylic acid (71216306);
    1-[(2,4-二氯苯基)甲基]-4-(二甲基氨基)吲唑-3-羧酸(66900636)；1-[(2,4-Dichlorophenyl)methyl]-4-(dimethylamino)indazole-3-carboxylic acid (66900636);
    1-[(2,4-二氯苯基)甲基]-5-(氟甲基)吲唑-3-羧酸(66887973)；1-[(2,4-Dichlorophenyl)methyl]-5-(fluoromethyl)indazole-3-carboxylic acid (66887973);
    1-(2,4-二氯苯基)-6-氟吲唑-3-羧酸(11652993)； 1-(2,4-Dichlorophenyl)-6-fluoroindazole-3-carboxylic acid (11652993);
    1-[(2,4-二氯苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(57633606)；1-[(2,4-Dichlorophenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (57633606);
    1-[(2-氯-4-甲基苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(66888424)；1-[(2-chloro-4-methylphenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (66888424);
    1-(2,4-二氯苯基)-3-(4-氯苯基)-1H-吡唑-5-羧酸(54148749)；1-(2,4-Dichlorophenyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxylic acid (54148749);
    1-[1-(2,4-二氯苯基)乙基]-1H-吲唑-3-羧酸(58796770)；1-[1-(2,4-Dichlorophenyl)ethyl]-1H-indazole-3-carboxylic acid (58796770);
    1-[(2,6-二氯-3-吡啶基)甲基]-1H-吲唑-3-羧酸(58796750)；1-[(2,6-Dichloro-3-pyridyl)methyl]-1H-indazole-3-carboxylic acid (58796750);
    1-[(2-氯-4-氟苯基)甲基]-6-(二氟甲基)吲唑-3-羧酸(66888818)；1-[(2-chloro-4-fluorophenyl)methyl]-6-(difluoromethyl)indazole-3-carboxylic acid (66888818);
    1-[(2-氯-4-甲基苯基)甲基]-6-甲基吲唑-3-羧酸(89564538)；1-[(2-Chloro-4-methylphenyl)methyl]-6-methylindazole-3-carboxylic acid (89564538);
    1-[(2,4-二氯苯基)甲基]-5-(三氟甲基)吲唑-3-羧酸(11567264)；1-[(2,4-Dichlorophenyl)methyl]-5-(trifluoromethyl)indazole-3-carboxylic acid (11567264);
    5-(4-氯苯基)-1-[(2,4-二氯苯基)甲基]-4-甲基吡唑-3-羧酸(54033453)；5-(4-chlorophenyl)-1-[(2,4-dichlorophenyl)methyl]-4-methylpyrazole-3-carboxylic acid (54033453);
    1-[(2-氯-4-氟苯基)甲基]-6-(三氟甲基)吲唑-3-羧酸(57633637)；1-[(2-chloro-4-fluorophenyl)methyl]-6-(trifluoromethyl)indazole-3-carboxylic acid (57633637);
    1-[(2,4-二氯苯基)甲基]-6-(二甲基氨基)吲唑-3-羧酸(11537863)；1-[(2,4-Dichlorophenyl)methyl]-6-(dimethylamino)indazole-3-carboxylic acid (11537863);
    1-[(2,4-二氯苯基)甲基]-6-甲基吲唑-3-羧酸(11667156)；以及1-[(2,4-Dichlorophenyl)methyl]-6-methylindazole-3-carboxylic acid (11667156); and
    它们各自的药学上可接受的盐或酯。Their respective pharmaceutically acceptable salts or esters.
  12. 根据权利要求11所述的应用，其中所述化合物选自：The use according to claim 11, wherein the compound is selected from:
    6-氯-1-[(2,4-二氯苯基)甲基]吲唑-3-羧酸(11501294)；6-Chloro-1-[(2,4-dichlorophenyl)methyl]indazole-3-carboxylic acid (11501294);
    5-(4-氯苯基)-1-[1-(2,4-二氯苯基)乙基]吡唑-3-羧酸(67565577)；5-(4-chlorophenyl)-1-[1-(2,4-dichlorophenyl)ethyl]pyrazole-3-carboxylic acid (67565577);
    1-[(2-氯-4-氟苯基)甲基]-5-(二氟甲基)吲唑-3-羧酸(66888740)；1-[(2-chloro-4-fluorophenyl)methyl]-5-(difluoromethyl)indazole-3-carboxylic acid (66888740);
    1-[(2,4-二氯苯基)甲基]-5-氟吲唑-3-羧酸(71221361)；1-[(2,4-Dichlorophenyl)methyl]-5-fluoroindazole-3-carboxylic acid (71221361);
    1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸(39562)；以及1-[(2,4-Difluorophenyl)methyl]indazole-3-carboxylic acid (39562); and
    它们各自的药学上可接受的盐或酯。Their respective pharmaceutically acceptable salts or esters.
  13. 根据权利要求11所述的应用，其中所述化合物是1-[(2,4-二氟苯基)甲基]吲唑-3-羧酸或其药学上可接受的盐或酯。The use according to claim 11, wherein the compound is 1-[(2,4-difluorophenyl)methyl]indazole-3-carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
  14. 根据权利要求1、8、9或10所述的应用，其中所述由ASC-caspase-1炎症小体通路激活介导的疾病选自哮喘、过敏性气道炎症、痛风、多发性硬化和败血症。The use according to claim 1, 8, 9 or 10, wherein the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is selected from asthma, allergic airway inflammation, gout, multiple sclerosis and sepsis.
  15. 根据权利要求1、8、9或10所述的应用，其中所述由ASC-caspase-1炎症小体通路激活介导的疾病选自痛风、多发性硬化和败血症。The use according to claim 1, 8, 9 or 10, wherein the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is selected from gout, multiple sclerosis and sepsis.
  16. 根据权利要求1、8、9或10所述的应用，其中所述由ASC-caspase-1炎症小体通路激活介导的疾病是痛风。 The use according to claim 1, 8, 9 or 10, wherein the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is gout.
  17. 根据权利要求1、8、9或10所述的应用，其中所述由ASC-caspase-1炎症小体通路激活介导的疾病是多发性硬化。The use according to claim 1, 8, 9 or 10, wherein the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is multiple sclerosis.
  18. 根据权利要求1、8、9或10所述的应用，其中所述由ASC-caspase-1炎症小体通路激活介导的疾病是败血症。The use according to claim 1, 8, 9 or 10, wherein the disease mediated by activation of the ASC-caspase-1 inflammasome pathway is sepsis.
  19. 一种ASC突变蛋白，其包含在I115、F163、W169和L192中的至少一个处的氨基酸残基突变，所述ASC突变蛋白相对于野生型蛋白具有减少的ASC寡聚体和/或斑点的数量，其中所述氨基酸残基编号根据GenBank:BAA87339.2确定。An ASC mutant protein comprising an amino acid residue mutation at at least one of I115, F163, W169 and L192, wherein the ASC mutant protein has a reduced number of ASC oligomers and/or spots relative to the wild-type protein, wherein the amino acid residue numbering is determined according to GenBank: BAA87339.2.
  20. 一种基于计算机的筛选用于治疗由ASC-caspase-1炎症小体通路激活介导的疾病的药物的方法，包括识别与ASC蛋白的I115、F163、W169和L192中的至少一个氨基酸残基相互作用的候选药物。 A method for computer-based screening of drugs for treating diseases mediated by activation of the ASC-caspase-1 inflammasome pathway comprises identifying a candidate drug that interacts with at least one of the amino acid residues I115, F163, W169, and L192 of the ASC protein.
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