CN111194355A - 用于鉴定与stk24相互作用的治疗剂的方法 - Google Patents

用于鉴定与stk24相互作用的治疗剂的方法 Download PDF

Info

Publication number
CN111194355A
CN111194355A CN201880054044.9A CN201880054044A CN111194355A CN 111194355 A CN111194355 A CN 111194355A CN 201880054044 A CN201880054044 A CN 201880054044A CN 111194355 A CN111194355 A CN 111194355A
Authority
CN
China
Prior art keywords
mst3
lipid
modulator
leu
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201880054044.9A
Other languages
English (en)
Inventor
M.马拉普
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alexeira
Original Assignee
Alexeira
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alexeira filed Critical Alexeira
Publication of CN111194355A publication Critical patent/CN111194355A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Endocrinology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

本发明涉及用于鉴定化合物的方法，所述化合物可对脂质代谢有影响，并由此与几种人类疾病高度相关，所述人类疾病包括但不限于肥胖、2型糖尿病(T2D)、非酒精性脂肪肝疾病(NAFLD)、非酒精性脂肪性肝炎(NASH)、脂肪性胰腺疾病和脂肪性肾脏疾病。更具体地，本发明涉及用于鉴定人激酶哺乳动物无菌20样3(MST3)的表达或活性的调控剂的方法以及此类调控剂，特别是寡核苷酸在治疗代谢疾病中的用途。

Description

用于鉴定与STK24相互作用的治疗剂的方法
发明领域
本发明涉及用于鉴定化合物的方法，所述化合物可对脂质代谢有影响，并由此与几种人类疾病高度相关，所述人类疾病包括但不限于肥胖、2型糖尿病(T2D)、非酒精性脂肪肝疾病(non-alcoholic fatty liver disease)(NAFLD)、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis)(NASH)、脂肪性胰腺疾病(fatty pancreas disease)和脂肪性肾脏疾病(fatty kidney disease)。更具体地，本发明涉及用于鉴定人激酶哺乳动物无菌20样3(MST3)的表达或活性的调控剂(modulator)的方法。
发明背景
人的激酶组(kinome)具有所谓“STE”激酶(其以酵母无菌20激酶命名)的大分支的特征。STE超家族包括几个亚家族，其中一个被命名为“哺乳动物无菌20样”(MST)家族(Creasy等人，1996)。哺乳动物中有五个MST激酶：MST1(也称为STK4)、MST2(也称为STK3)、MST3(也称为STK24)、MST4和YSK1(也称为STK25或SOK1)。五个哺乳动物MST激酶可根据其各自的调节、底物和功能大致分为三个亚组：MST1/2、MST3/4和YSK1(Thompson和Sahai，2015)。
已经鉴定MST1和MST2参与生长控制、增殖和迁移调节。已经鉴定MST3和MST4影响细胞迁移、细胞极性和凋亡。
最近已经显示YSK1是脂质和葡萄糖代谢的调节剂(regulator)(Nerstedt等人，2012)。
MST3在调节细胞极性和迁移中的作用
已经显示MST3在许多环境中调节肌动蛋白的动力学。在发育中的神经系统中，MST3是维持树突棘(dendritic spine)所需，并限制丝状伪足(filopodia)的形成(Ultanir等人，2014)。MST3还限制其他细胞类型中肌动蛋白依赖性突起(protrusion)。已经提示当MST3耗尽时，这导致2D表面上的迁移增加，但导致挤压通过3D矩阵(matrice)中间隙的缺陷(Lu等人，2006；Madsen等人，2015)。
哺乳动物细胞培养研究也暗示MST3调节细胞极性。MST3可能通过与Striatin蛋白相互作用而定位于高尔基体(Golgi apparatus)(Lu等人，2006)。与CCM3或Mo25的相互作用可触发MST3从高尔基体易位(translocation)到质膜。
MST3在调节细胞凋亡中的作用
MST3可以被胱天蛋白酶(caspase)切割(Lee等人，1998；Lee等人，2001)。切割发生在氨基酸313处，并将N末端激酶结构域与C末端调节序列分开。这导致活性激酶结构域的核积累，其可以促进细胞凋亡(Huang等人，2002；Lee等人，2004)。
MST3在疾病中的作用
与它在细胞迁移中的作用一致，已经暗示MST3与癌症有关。已经提示CCM3促进细胞皮层中MST3的活性，在所述细胞皮层中其协调ERM蛋白和MLC的磷酸化，使癌细胞挤压通过小间隙(Madsen等人2015；Tozluoglu等人2015)。
哺乳动物细胞中的蛋白质组学(proteomic)研究已经将MST3鉴定为称为STRIPAK复合物的大型PP2A复合物的组分(Glatter等人，2009；Kean等人，2011)。最近，癌症基因组测序已经暗示在癌症中的STRIPAK复合物。FAM40B以很高的频率突变，并且突变的数量和类型提示其具有致癌(oncogenic)功能(Davoli等人，2013)。对在肿瘤中发现的FAM40B截短突变体的分析表明，它们无法结合PP2A的催化亚基，并且可能在负调节MST3方面存在缺陷(Madsen等人，2015)。
MST3的调控缺陷也暗示与内皮畸形(endothelial malformation)的病理学有关(Stockton等人，2010；Zheng等人，2010)。
MST3在脂质代谢中的作用
现有技术未能鉴定MST3在脂质代谢中的作用。
发明描述
本发明人已经鉴定了MST3在哺乳动物脂质代谢中的作用。更具体地，本发明人已经认识到MST3在调控哺乳动物细胞系统中的脂质分配(partitioning)中具有作用，并且MST3在易于产生脂毒性(lipotoxicity)的外周组织中控制脂质利用相对于脂质存储的动态代谢平衡，并由此预期调控胰岛素敏感性，其与几种人类疾病具有高度相关性，所述疾病包括但不限于肥胖症、2型糖尿病(T2D)、非酒精性脂肪肝疾病(NAFLD)、非酒精性脂肪性肝炎(NASH)、脂肪性胰腺病和脂肪性肾脏疾病。
因此，一方面，本发明提供用于鉴定哺乳动物脂质代谢调控剂的方法。
该方法包括使用哺乳动物MST3，优选人MST3。
该方法可以包括确定MST3活性和/或MST3表达。
可以在表达MST3的哺乳动物细胞中确定MST3活性和/或MST3表达。哺乳动物细胞可以是具有组成型MST3表达的细胞或过表达重组MST3的细胞。
哺乳动物细胞可以是人或啮齿动物细胞。
在一个实施方案中，根据本发明的方法包括用于鉴定能够调控MST3的活性和/或表达的试剂的方法，该方法包括：
(i)使候选化合物调控剂与表达编码MST3多肽的多核苷酸序列的宿主细胞接触；
(ii)确定候选化合物调控剂对MST3的活性和/或表达的作用，由此将该化合物鉴定为MST3调控剂，并由此将该化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
可通过测量脂质沉积(deposition)、脂质分解代谢(catabolism)，例如脂肪酸氧化和/或脂质分泌和/或脂质合成代谢(anabolism)，例如脂质摄取和/或脂质合成来确定MST3活性。由于脂质沉积调节胰岛素敏感性，因此胰岛素敏感性参数(如胰岛素刺激的葡萄糖摄取(ISGU))也可以用作MST3活性的量度(measure)。
可以使用技术，如定量实时PCR(qRT-PCR)、蛋白质印迹(Western blot)或邻近连接(proximity ligation)测定法(PLA)确定MST3表达。
在另一个实施方案中，根据本发明的方法包括用于鉴定能够调控MST3活性的试剂的方法，该方法包括：
(i)使候选化合物调控剂与MST3多肽接触；
(ii)确定候选化合物调控剂对MST3活性的作用，由此将该化合物鉴定为MST3调控剂，并由此将该化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
可以使用MST3多肽，优选经重组产生和纯化的MST3多肽，以及MST3肽底物，并确定肽底物的磷酸化水平作为MST3活性的量度，来测定MST3活性。
在又一个实施方案中，根据本发明的方法包括用于鉴定能够调控MST3活性的试剂的方法，该方法包括：
(i)使候选化合物调控剂与MST3多肽接触；
(ii)确定候选化合物调控剂与MST3的结合，由此将该化合物鉴定为MST3结合剂(binder)，并由此将该化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
可以使用MST3多肽，优选经重组产生和纯化的MST3多肽，和候选化合物，并如Olesen等人2016所述测定该化合物对MST3解链温度的影响，来确定MST3结合。
MST3多肽可以是人MST3。人MST3的氨基酸序列可以例如见于UniProtKB数据库登录号nr-Q9Y6E0(STK24_HUMAN)和NCBI GenBank数据库登录号nr NP_003567，在本文中鉴定为SEQ ID NO：2。
根据本发明的MST3多肽可以包含根据SEQ ID NO：2的氨基酸序列或其变体，所述变体与根据SEQ ID NO：2的氨基酸序列或其功能活性片段具有至少80％的序列同一性。
将MST3的功能活性片段定义为与具有根据SEQ ID NO：2的氨基酸序列的MST3多肽的MST3激酶活性相比，保留至少90％的MST3激酶活性，如至少80％、70％、60％或50％MST3激酶活性的MST3多肽。
用于筛选MST3的调控剂的方法可以基于经重组产生和纯化的MST3多肽和MST3肽底物的使用。酶促测定，如TR-FRET或ADP猎人测定(hunter assay)可适用于MST3测定，并用于确定肽底物的磷酸化水平。
可以使用标准的辐射滤板(radiometric filter plate)测定法针对一组激酶评估鉴定的调控剂的选择性。表面等离子体共振(SPR)可用于评估候选化合物与MST3的结合。
可以在根据本发明的方法中测试的候选化合物包括通常称为“小分子”的简单有机分子，例如具有分子量小于2000道尔顿的那些。该方法还可用于筛选化合物文库，如肽文库，包括合成肽文库和肽噬菌体文库。
一旦鉴定出MST3活性的调控剂，即抑制剂或刺激剂，那么就可以应用药物化学技术进一步改善其性质，例如增强功效和/或减少副作用。
其他合适的候选分子包括寡核苷酸和多核苷酸，如dsRNA、siRNA、shRNA、miRNA和反义RNA或DNA，以及潜在地可以调控MST3的活性和/或表达的任何其他分子。
编码人MST的cDNA序列可以例如见于NCBI GenBank数据库登录号NM_003576，在本文中鉴定为SEQ ID NO：1。
优选的寡核苷酸和多核苷酸在长度上由8-80个碱基组成，其包含可与核酸序列SEQ ID NO：1杂交的序列。
应当理解，本领域中已知有许多规程，其可以用来执行本发明。可用于鉴定MST3调控剂的合适规程的实例包括平衡结合混合物的快速过滤、酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)和荧光共振能量转移测定(FRET)、闪烁亲近(scintillation proximity)测定(SPA)、电泳迁移率变动(electrophoretic mobility shift)分析(EMSA)、染色质免疫沉淀分析(ChIP)、表面等离子体共振(SPR)、qRT-PCR、蛋白质印迹和PLA测定。
在第二方面，本发明提供了用于治疗代谢疾病的化合物、药物组合物和方法。
在一个实施方案中，本发明提供了寡核苷酸和多核苷酸，用于治疗代谢疾病，所述寡核苷酸和多核苷酸在长度上由8-80个碱基组成，包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列。
在另一个实施方案中，本发明提供了包含寡核苷酸或多核苷酸的药物组合物，用于治疗代谢疾病，所述寡核苷酸或多核苷酸在长度上由8-80个碱基组成，包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列。
在另一个实施方案中，本发明提供了在长度上由8-80个碱基组成的寡核苷酸和多核苷酸，其包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列，用于制造用于治疗代谢疾病的药物。
在又一个实施方案中，本发明提供了用于治疗代谢疾病的方法，该方法包括向需要此种治疗的受试者施用药物有效量的寡核苷酸或多核苷酸，所述寡核苷酸或多核苷酸在长度上由8-80个碱基组成，包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列。
所述代谢疾病可以选自但不限于肥胖症、2型糖尿病(T2D)、非酒精性脂肪肝疾病(NAFLD)、非酒精性脂肪性肝炎(NASH)、脂肪性胰腺疾病和脂肪性肾脏疾病。
优选的寡核苷酸和多核苷酸是dsRNA、siRNA、shRNA、miRNA和反义RNA或DNA。
寡核苷酸和多核苷酸的长度可以是8-80个碱基，优选长度是8-70、8-60、8-50、8-40或8-30个碱基。
反义寡核苷酸或多核苷酸中的核苷酸可以是RNA残基、DNA残基或核苷酸类似物。
反义寡核苷酸和多核苷酸可选自下组：肽核酸(PNA)、磷酸二酰胺吗啉低聚物(phosphorodimidate mopholino oligomer)(PMO)和硫代磷酸酯(phosphorothioate)寡核苷酸(PS-ODN)。
图注
图1.人肝细胞中MST3的消耗通过增加的β-氧化和三酰基甘油(triacylglycerol) (TAG)分泌与抑制的脂质摄取和合成组合来抑制脂质积累。用MST3或非靶向对照(NTC)小干扰RNA(siRNA)转染永生化(immortalised)的人肝细胞(IHH)；与油酸(OA)温育48小时后和在基础条件下进行评估。
(A)利用抗MST3抗体的代表性蛋白质印迹；肌动蛋白用作上样对照。(B)用于中性脂质的油红O(Oil Red O)染色；显示了代表性的细胞图像(比例尺，100μm)。(C)脂质组学分析。
Figure BDA0002387899710000061
siRNA基础，
Figure BDA0002387899710000062
siRNA基础，□NTC siRNA+OA，
■MST3 siRNA+OA
(D)放射性标记的棕榈酸酯的氧化。
(E)[3H]TAG分泌到培养基中。-□-NTC siRNA,-■-MST3 siRNA
(F)OA的摄取。-△-NTC siRNA基础，-▲-MST3 siRNA基础，-□-NTC siRNA+OA,-■-MST3 siRNA+OA。
(G)由[14C]标记的OA合成TAG。
(H)由[14C]标记的葡萄糖合成TAG。
对于(C-E)和(G-H)，结果是8-10个细胞培养皿孔的均值±SEM。对于(D-E)和(G-H)，仅在与OA温育48小时后进行评估。
CE，胆固醇酯；Cer，神经酰胺(ceramide)；LPC，溶血磷脂酰胆碱(lysophosphatidylcholine)；NEFA，非酯化脂肪酸；NTC，非靶向对照；OA，油酸；PC，磷脂酰胆碱；PE，磷脂酰乙醇胺；RFU，相对荧光单位；SM，鞘磷脂(sphingomyelin)；TAG，三酰基甘油。*p＜0.05；**p<0.01(在(C)中仅显示在每种测试条件下比较MST3 siRNA相对于NTCsiRNA的统计显著性)
图2.在调节人肝细胞脂质积累中MST3功能的图解模型。与用NTC siRNA转染的细胞相比，通过siRNA消耗MST3通过增加的β-氧化和VLDL-TAG分泌来增强脂滴分解代谢，并通过抑制的游离脂肪酸(FFA)摄取和TAG合成来抑制脂滴合成代谢。因此，MST3缺失导致较低的细胞内TAG含量和减少的脂滴大小。ER，内质网；FFA，游离脂肪酸；LD，脂滴，NTC，非靶向对照；TAG，三酰基甘油；VLDL，极低密度脂蛋白。
实施例
材料和方法
永生的人肝细胞(IHH；人类起源的肝细胞系)在补充有牛胰岛素(20U/l；Sigma-Aldrich,St Louis,MO,USA)和地塞米松(dexamethasone)(50nmol/l；Sigma-Aldrich)的完全威廉姆斯E培养基(Complete William’s E medium)(Gibco，Paisley，UK)中维持。培养基中还补充了10％(体积/体积)的胎牛血清(FBS)、L-谷氨酰胺(2mmol/l)和1％(体积/体积)的青霉素/链霉素(Gibco)。转染后，将细胞与油酸(OA)温育48小时，已知油酸可在体外有效诱导脂肪变性(steatosis)(Amrutkar等人2016)。
MST3消耗
根据制造商的说明，使用Lipofectamine RNAiMax(Invitrogen,San Diego,CA,USA)通过小干扰RNA(siRNA)技术消耗MST3。使用抗MST3抗体通过蛋白质印迹证实了有效的靶标消耗。
脂质存储的测量
为了测量脂质存储，如先前所述(Amrutkar等人，2016)，用针对中性脂质的油红O染色转染的细胞。此外，对于脂质组学分析，使用Folch方法(Folch等人，1957)提取脂质，并使用超高效液相色谱/质谱和直接注入质谱法(Stahlman等人，2013)对脂质进行定量。
脂肪酸氧化的测量
为了测量β-氧化，将转染的细胞在[9,10-3H(N)]-棕榈酸存在下进行温育，并如先前所述(Nerstedt等人2012)将[3H]-标记的水测量为游离脂肪酸氧化的产物。
三酰基甘油分泌的测定
为了测量三酰基甘油(triacylglycerol)(TAG)分泌，将转染的细胞用脉冲培养基[含有18,500Bq/ml[3H]OA(PerkinElmer，Waltham，MA，USA)、360μmol/l OA(Sigma-Aldrich)，和1％体积/体积不含脂肪酸的BSA的完全威廉姆斯E]培养8小时，然后用chase培养基(补充有30％体积/体积不含脂肪酸的BSA的完全威廉姆斯E)培养长达8小时。收集培养基用于脂质提取，然后通过硅胶板上的薄层色谱分离脂质。用碘蒸气检测放射性标记的TAG，并用闪烁计数器进行定量。
油酸摄取的测量
根据制造商的说明，使用Quencher-Based Technology(QBT)Fatty Acid UptakeAssay Kit(Molecular Devices,San Jose,CA,USA)测量油酸(OA)摄取。
将油酸和葡萄糖掺入三酰基甘油中
如先前所述(Amrutkar等人，2016)，对[14C]OA和[14C]葡萄糖掺入TAG进行了测量。
来自小鼠肝脏的脂滴中的液相色谱质谱(LC-MS)分析
为C57BL6/J遗传背景的雄性小鼠喂食颗粒状高脂肪饮食(45％卡路里来自脂肪；D12451；Research Diets,New Brunswick,NJ,USA)，持续16-18周。使用Zhang等人的方法(Zhang等人，2011)将小鼠处死并从新鲜切除的肝脏中分离出脂滴(LD)。如先前所述(Chursa等人2017)进行了LC-MS分析，其具有以下修改。使用ProteoExtract Kit(Millipore,Burlington,MA,USA)沉淀来自LD提取物的蛋白质，并且在蛋白质浓度测定之前将其溶解于缓冲液[50mmol/l三乙基碳酸氢铵(triethylammonium bicarbonate)(TEAB)、4％SDS]中。在LC-MS分析之前，使用Pierce High pH Reversed-Phase PeptideFractionation Kit将总蛋白TMT 10重套(10-plex sets)分离为8个级分(fraction)。
结果
MST3与小鼠肝脏中的脂滴相关
通过LC-MS技术对从高脂肪饮食喂养的小鼠的肝脏分离出的脂滴(LD)进行整体定量磷酸化蛋白质组学，以鉴定肝LD蛋白质组的有效成分。先前已经显示，如通过LD驻留(resident)蛋白的相对富集和对应于其他细胞内区室的标记物的缺失所评估，通过这种分离方案制备的LD级分基本上没有污染(Zhang等人2011)。值得注意的是，Zhang没有将MST3鉴定为LD级分的一部分。
出乎意料的是，本研究表明MST3以及磷酸MST3(Thr172)存在于LD级分中，这提供了该激酶与小鼠肝脏LD相关的第一个证据。
人肝细胞中MST3的消耗通过增加的β-氧化和TAG分泌与减少的游离脂肪酸摄取和 脂质合成组合来抑制脂质积累
由于MST3在肝LD上的亚细胞定位，推断MST3调节肝脂质代谢。为了研究MST3对肝脏中脂质分解代谢和合成代谢的可能影响，用MST3特异性siRNA或NTC siRNA转染人IHH。在用MST3 siRNA转染的细胞中，如通过蛋白质印迹评估，MST3的蛋白质水平显著降低(图1A)。
在进行代谢评估之前，将转染的细胞与OA温育48小时，已知其可在体外有效诱导脂肪变性。值得注意的是，在有和没有OA温育的情况下都研究了细胞脂质存储和FFA摄取。
如通过油红O染色对中性脂质的评估，敲低MST3显著降低肝细胞内脂质的积累(图1B)。脂质组学分析还证实，与NTC siRNA相比，用MST3 siRNA转染的细胞中几种脂质种类的水平显著降低，这在基础条件下和OA攻击后均很明显(图1C)。siRNA介导的MST3沉默导致β-氧化明显增加(图1D)。此外，用MST3 siRNA转染的细胞中TAG到培养基中的分泌显著更高(图1E)。还发现MST3缺陷型细胞中的FFA流入(influx)明显减少(图1F)，并且在消耗了MST3的肝细胞中，培养基来源的[14C]标记的OA和[14C]标记的葡萄糖向细胞内TAG的掺入较低(图1G-H)。
讨论
在这项研究中，本发明人提供了MST3与肝脏中的细胞内脂滴相关的第一个证据。曾经被认为只是惰性能量储存库(depot)的肝脂滴越来越多地被认为是细胞器，其在调节肝脂质分配中起着关键作用(Mashek等人，2015)，为线粒体β-氧化和极低密度脂蛋白(VLDL)-三酰基甘油(TAG)的分泌提供了基质(substrate)。由于MST3定位在肝脂滴上，我们假设MST3调节肝脂质储存。实际上，证明在人类培养的肝细胞中MST3的siRNA敲低显著降低了细胞内脂质积累。有几种可能的机制，其可能是其中MST3缺失的肝细胞中脂质存储被抑制的基础：(i)脂质吸收和合成的速率降低，(ii)如富含TAG的VLDL的脂质分泌增加，(iii)脂肪酸氧化的提高的水平--或这些机制的任何组合。实际上，证明MST3调节肝细胞中脂质分解代谢相对于脂质合成代谢的代谢平衡，因为证明MST3的消耗刺激β-氧化和TAG分泌并抑制FFA流入和TAG合成(图2)。
总之，本公开提供了脂滴相关蛋白MST3在调节肝脂质储存中的细胞特异性作用的一致证据。重要的是，大量最新证据表明，肝脏中的异位(ectopic)脂质积累(也称为非酒精性脂肪性肝病(NAFLD))加剧了肝和全身性胰岛素抵抗，并积极促进了2型糖尿病和代谢综合征的发病病程(Anstee等人2013)。
此外，肝脂质存储是发生侵袭性肝病非酒精性脂肪性肝炎(NASH)的主要危险因素(Anstee等人，2013)。
因此，本公开强调了MST3调控剂作为用于预防和治疗NAFLD/NASH和相关复杂代谢疾病的潜在候选药物。
参考文献
Amrutkar M,Kern M,Nunez-Duran E,Stahlman M,Cansby E,Chursa U,Stenfeldt E,Boren J,Bluher M,and Mahlapuu M.2016.Protein kinase STK25controlslipid partitioning in hepatocytes and correlates with liver fat content inhumans.Diabetologia 59,341-353.
Anstee QM,Targher G,and Day CP.2013.Progression of NAFLD to diabetesmellitus,cardiovascular disease or cirrhosis.Nat.Rev.Gastroenterol.Hepatol.10,330-344.
Chursa U,Nunez-Duran E,Cansby E,Amrutkar M,Sutt S,Stahlman M,Olsson BM,Boren J,Johansson ME,Backhed F,Johansson BR,Sihlbom C,and Mahlapuu M.(2017)Overexpression of protein kinase STK25 in mice exacerbates ectopic lipidaccumulation,mitochondrial dysfunction and insulin resistance in skeletalmuscle.Diabetologia 60,553-567.
Creasy CL,Ambrose DM,and Chernoff J.1996.The Ste20-like proteinkinase,Mst1,dimerizes and contains an inhibitory domain.J.Biol.Chem.271:21049–21053.
Davoli T,Xu AW,Mengwasser KE,Sack LM,Yoon JC,Park PJ,and ElledgeSJ.2013.Cumulative haploinsufficiency and triplosensitivity drive aneuploidypatterns and shape the cancer genome.Cell.155:948–962.
Folch J,Lees M,and Sloane Stanley GH.1957.A simple method for theisolation and purification of total lipides from animaltissues.J.Biol.Chem.226,497-509.
Glatter T,Wepf A,Aebersold R,and Gstaiger M.2009.An integratedworkflow for charting the human interaction proteome:insights into the PP2Asystem.Mol.Syst.Biol.5:237.
Huang CY,Wu YM,Hsu CY,Lee WS,Lai MD,Lu TJ,Huang CL,Leu TH,Shih HM,Fang HI,et al.2002.Caspase activation of mammalian sterile 20-like kinase 3(Mst3).Nuclear translocation and induction of apoptosis.J.Biol.Chem.277:34367–34374.
Kean MJ,Ceccarelli DF,Goudreault M,Sanches M,Tate S,Larsen B.GibsonLC,Derry WB,Scott IC,Pelletier L,et al.2011.Structure-function analysis ofcore STRIPAK Proteins:a signaling complex implicated in Golgipolarization.J.Biol.Chem.286:25065–25075.
Lee KK,Murakawa M,Nishida E,Tsubuki S,Kawashima S,Sakamaki K,andYonehara S.1998.Proteolytic activation of MST/Krs,STE20-related proteinkinase,by caspase during apoptosis.Oncogene.16:3029–3037.
Lee KK,Ohyama T,Yajima N,Tsubuki S,and Yonehara S.2001.MST,aphysiological caspase substrate,highly sensitizes apoptosis both upstream anddownstream of caspase activation.J.Biol.Chem.276:19276–19285.
Lee WS,Hsu CY,Wang PL,Huang CY,Chang CH,and YuanCJ.2004.Identification and characterization of the nuclear import and exportsignals of the mammalian Ste20-like protein kinase 3.FEBS Lett.572:41–45.
Lu TJ,Lai WY,Huang CY,Hsieh WJ,Yu JS,Hsieh YJ,Chang WT,Leu TH,ChangWC,Chuang WJ,et al.2006.Inhibition of cell migration by autophosphorylatedmammalian sterile 20-like kinase 3(MST3)involves paxillin and protein-tyrosine phosphatase-PEST.J.Biol.Chem.281:38405–38417.
Madsen CD,Hooper S,Tozluoglu M,Bruckbauer A,Fletcher G,Erler JT,BatesPA,Thompson B,and Sahai E.2015.STRIPAK components determine mode of cancercell migration and metastasis.Nat.Cell Biol.17:68–80.
Mashek D G,Khan SA,Sathyanarayan A,Ploeger JM,and FranklinMP.2015.Hepatic lipid droplet biology:Getting to the root of fattyliver.Hepatology 62,964-967.
Nerstedt A,Cansby E,Andersson CX,et al.2012.Serine/threonine proteinkinase 25(STK25):a novel negative regulator of lipid and glucose metabolismin rodent and human skeletal muscle.Diabetologia 55:1797-1807.
Olesen SH,Zhu JY,and Martin MP.2016.Discovery of Diverse Small-Molecule Inhibitors ofMammalian Sterile20-like Kinase 3(MST3).Chem.Med.Chem.11,1137-1144.
Stahlman M,Fagerberg B,Adiels M,Ekroos K,Chapman JM,Kontush A,andBoren J.2013.Dyslipidemia,but not hyperglycemia and insulin resistance,isassociated with marked alterations in the HDL lipidome in type 2 diabeticsubjects in the DIWA cohort:impact on small HDL particles.BiochimBiophys Acta1831,1609-161.
Stockton RA,Shenkar R,Awad IA,and Ginsberg MH.2010.Cerebral cavernousmalformations proteins inhibit Rho kinase to stabilize vascularintegrity.J.Exp.Med.207:881–896.
Thompson BJ and Sahai E.2015.MST kinases in development anddisease.J.Cell Biol.210(6),871-882.
Tozluoglu M,Mao Y,Bates PA,and Sahai E.2015.Cost-benefit analysis ofthe mechanisms that enable migrating cells to sustain motility upon changesin matrix environments.J.R.Soc.Interface.12.10.1098/rsif.2014.1355.
Ultanir SK,Yadav S,Hertz NT,Oses-Prieto JA,Claxton S,Burlingame AL,Shokat KM,Jan LY,and Jan YN.2014.MST3 kinase phosphorylates TAO1/2 to enableMyosin Va function in promoting spine synapse development.Neuron.84:968–982.
Zheng X,Xu C,Di Lorenzo A,Kleaveland B,Zou Z,Seiler C,Chen M,Cheng L,Xiao J,He J,et al.2010.CCM3 signaling through sterile 20-like kinases playsan essential role during zebrafish cardiovascular development and cerebralcavernous malformations.J.Clin.Invest.120:2795–2804.
Zhang H,Wang Y,Li J,Yu J,Pu J,Li L,Zhang H,Zhang S,Peng G,Yang F,andLiu P.2011.Proteome of skeletal muscle lipid droplet reveals association withmitochondria and apolipoprotein a-I.J.Proteome.Res 10,4757-4768.
序列表
<110> 亚历克塞拉公司(ALEXERA AB)
<120> 用于鉴定与STK24相互作用的治疗剂的方法
<130> MST3
<150> SE 1730249-8
<151> 2017-09-13
<160> 2
<170> PatentIn版本3.5
<210> 1
<211> 4579
<212> DNA
<213> 智人（Homo sapiens）
<220>
<221> CDS
<222> (274)..(1605)
<400> 1
agacacgaca ttgcagcact ggtgaccgga tccagagcgg agccaggctg cagtggtggg 60
gaggaccggg caggtccaga tgactttgaa gggactccag aggaaggaga gagtggagga 120
ggagggcttt gggttaggga gagtgctttc gtttgtttta aatgggagaa actggagcat 180
gttgccaagg gcagagagcc agcagagagg ggtgaatgga agaaggagcg agaagggggt 240
tactgacgaa gccttatcct ggaggagaga agg atg gac tcc aga gcc cag ctt 294
Met Asp Ser Arg Ala Gln Leu
1 5
tgg gga ctg gcc ttg aat aaa agg agg gcc act cta cct cat cct gga 342
Trp Gly Leu Ala Leu Asn Lys Arg Arg Ala Thr Leu Pro His Pro Gly
10 15 20
ggg agc acg aac cta aag gca gac cca gaa gag ctt ttt aca aaa cta 390
Gly Ser Thr Asn Leu Lys Ala Asp Pro Glu Glu Leu Phe Thr Lys Leu
25 30 35
gag aaa att ggg aag ggc tcc ttt gga gag gtg ttc aaa ggc att gac 438
Glu Lys Ile Gly Lys Gly Ser Phe Gly Glu Val Phe Lys Gly Ile Asp
40 45 50 55
aat cgg act cag aaa gtg gtt gcc ata aag atc att gat ctg gaa gaa 486
Asn Arg Thr Gln Lys Val Val Ala Ile Lys Ile Ile Asp Leu Glu Glu
60 65 70
gct gaa gat gag ata gag gac att caa caa gaa atc aca gtg ctg agt 534
Ala Glu Asp Glu Ile Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Ser
75 80 85
cag tgt gac agt cca tat gta acc aaa tat tat gga tcc tat ctg aag 582
Gln Cys Asp Ser Pro Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Lys
90 95 100
gat aca aaa tta tgg ata ata atg gaa tat ctt ggt gga ggc tcc gca 630
Asp Thr Lys Leu Trp Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Ala
105 110 115
cta gat cta tta gaa cct ggc cca tta gat gaa acc cag atc gct act 678
Leu Asp Leu Leu Glu Pro Gly Pro Leu Asp Glu Thr Gln Ile Ala Thr
120 125 130 135
ata tta aga gaa ata ctg aaa gga ctc gat tat ctc cat tcg gag aag 726
Ile Leu Arg Glu Ile Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Lys
140 145 150
aaa atc cac aga gac att aaa gcg gcc aac gtc ctg ctg tct gag cat 774
Lys Ile His Arg Asp Ile Lys Ala Ala Asn Val Leu Leu Ser Glu His
155 160 165
ggc gag gtg aag ctg gcg gac ttt ggc gtg gct ggc cag ctg aca gac 822
Gly Glu Val Lys Leu Ala Asp Phe Gly Val Ala Gly Gln Leu Thr Asp
170 175 180
acc cag atc aaa agg aac acc ttc gtg ggc acc cca ttc tgg atg gca 870
Thr Gln Ile Lys Arg Asn Thr Phe Val Gly Thr Pro Phe Trp Met Ala
185 190 195
ccc gag gtc atc aaa cag tcg gcc tat gac tcg aag gca gac atc tgg 918
Pro Glu Val Ile Lys Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Trp
200 205 210 215
tcc ctg ggc ata aca gct att gaa ctt gca aga ggg gaa cca cct cat 966
Ser Leu Gly Ile Thr Ala Ile Glu Leu Ala Arg Gly Glu Pro Pro His
220 225 230
tcc gag ctg cac ccc atg aaa gtt tta ttc ctc att cca aag aac aac 1014
Ser Glu Leu His Pro Met Lys Val Leu Phe Leu Ile Pro Lys Asn Asn
235 240 245
cca ccg acg ttg gaa gga aac tac agt aaa ccc ctc aag gag ttt gtg 1062
Pro Pro Thr Leu Glu Gly Asn Tyr Ser Lys Pro Leu Lys Glu Phe Val
250 255 260
gag gcc tgt ttg aat aag gag ccg agc ttt aga ccc act gct aag gag 1110
Glu Ala Cys Leu Asn Lys Glu Pro Ser Phe Arg Pro Thr Ala Lys Glu
265 270 275
tta ttg aag cac aag ttt ata cta cgc aat gca aag aaa act tcc tac 1158
Leu Leu Lys His Lys Phe Ile Leu Arg Asn Ala Lys Lys Thr Ser Tyr
280 285 290 295
ttg acc gag ctc atc gac agg tac aag aga tgg aag gcc gag cag agc 1206
Leu Thr Glu Leu Ile Asp Arg Tyr Lys Arg Trp Lys Ala Glu Gln Ser
300 305 310
cat gac gac tcg agc tcc gag gat tcc gac gcg gaa aca gat ggc caa 1254
His Asp Asp Ser Ser Ser Glu Asp Ser Asp Ala Glu Thr Asp Gly Gln
315 320 325
gcc tcg ggg ggc agt gat tct ggg gac tgg atc ttc aca atc cga gaa 1302
Ala Ser Gly Gly Ser Asp Ser Gly Asp Trp Ile Phe Thr Ile Arg Glu
330 335 340
aaa gat ccc aag aat ctc gag aat gga gct ctt cag cca tcg gac ttg 1350
Lys Asp Pro Lys Asn Leu Glu Asn Gly Ala Leu Gln Pro Ser Asp Leu
345 350 355
gac aga aat aag atg aaa gac atc cca aag agg cct ttc tct cag tgt 1398
Asp Arg Asn Lys Met Lys Asp Ile Pro Lys Arg Pro Phe Ser Gln Cys
360 365 370 375
tta tct aca att att tct cct ctg ttt gca gag ttg aag gag aag agc 1446
Leu Ser Thr Ile Ile Ser Pro Leu Phe Ala Glu Leu Lys Glu Lys Ser
380 385 390
cag gcg tgc gga ggg aac ttg ggg tcc att gaa gag ctg cga ggg gcc 1494
Gln Ala Cys Gly Gly Asn Leu Gly Ser Ile Glu Glu Leu Arg Gly Ala
395 400 405
atc tac cta gcg gag gag gcg tgc cct ggc atc tcc gac acc atg gtg 1542
Ile Tyr Leu Ala Glu Glu Ala Cys Pro Gly Ile Ser Asp Thr Met Val
410 415 420
gcc cag ctc gtg cag cgg ctc cag aga tac tct cta agt ggt gga gga 1590
Ala Gln Leu Val Gln Arg Leu Gln Arg Tyr Ser Leu Ser Gly Gly Gly
425 430 435
act tca tcc cac tga aattcctttg gcatttgggg ttttgttttt ccttttttcc 1645
Thr Ser Ser His
440
ttcttcatcc tcctcctttt ttaaaagtca acgagagcct tcgctgactc caccgaagag 1705
gtgcgccact gggagccacc ccagcgccag gcgcccgtcc agggacacac acagtcttca 1765
ctgtgctgca gccagatgaa gtctctcaga tgggtgggga gggtcagctc cttccagcga 1825
tcattttatt ttattttatt acttttgttt ttaattttaa ccatagtgca catattccag 1885
gaaagtgtct ttaaaaacaa aaacaaaccc tgaaatgtat atttgggatt atgataaggc 1945
aactaaagac atgaaacctc aggtatcctg ctttaagttg ataactccct ctgggagctg 2005
gagaatcgct ctggtggatg ggtgtacaga tttgtatata atgtcatttt tacggaaacc 2065
ctttcggcgt gcataaggaa tcactgtgta caaactggcc aagtgcttct gtagataacg 2125
tcagtggagt aaatattcga caggccataa cttgagtcta ttgccttgcc tttattacat 2185
gtacattttg aattctgtga ccagtgattt gggttttatt ttgtatttgc agggtttgtc 2245
attaataatt aatgcccctc tcttacagaa cactcctatt tgtacctcaa caaatgcaaa 2305
ttttccccgt ttgccctacg ccccttttgg tacacctaga ggttgatttc ctttttcatc 2365
gatggtacta tttcttagtg ttttaaattg gaacatatct tgcctcatga agctttaaat 2425
tataattttc agtttctccc catgaagcgc tctcgtctga catttgtttg gaatcgtgcc 2485
actgctggtc tgcgccagat gtaccgtcct ttccaatacg attttctgtt gcaccttgta 2545
gtggattctg catatcatct ttcccaccta aaaatgtctg aatgcttaca caaataaatt 2605
ttataacacg cttattttgc atactccttg aaatgtgact cttcagagga cagggcacct 2665
gctgtgtatg tgtggccgtg cgtgtgtact cgtggctgtg tgtgtgtgat gagacacttt 2725
ggaagactcc agggagaagt ccccaggcct ggagctgccg agtgcccagg tcagcgccct 2785
ggactgcttg cgcacttgct caccgagatg atgcagttgg aggttgctga tctgtgcgat 2845
tgctgtagcg gttgccgggg accttaagag ttattttgct tctctggaag gggcctatgc 2905
ttgctaggca ggcagccagt gtgtctgttt ttcttggttt gctgtgggac cttgcttggc 2965
gagggggaaa atctctgggt ttctggagtg ggagggttcg tgcagcagct gttgactggt 3025
acatgaagca ttcttttatg tttgttgaag ctgatgattg acatctcccg tgggtgtgcc 3085
agttcttgtg gagttaagac aggatttttg gaagcaagga agttagtggg tgagcttggg 3145
gatgtagctc agctatctgc tggtctagtg gcctctaagc tatagggagg ggacagagcc 3205
ctgagctaca gatgcttgag tgggttattg tgtcggtttg ctagtgcagt ctggttttta 3265
agctctaaaa ttgaggtatt ttattagaag tggatttggg ttgaactctt aatttgtata 3325
agggatatat tttggttggg gaaatagaac tgagttgcta attcttattg tactcattac 3385
tccatacaag aatgttatgt tgaataataa aattggagaa gatttcattt tgtgtttcca 3445
gggagtattc tgtgtgggga actgtttcct tacgtgaggc cggcggcata agtcaaagat 3505
gagttttgtc cttgcgaatc acacagattg agtctgtgtt ccccagggtg tgccgttacc 3565
tgatttttaa gtgagccagg gcggacagca gcttttctga tttacagagt tcttcagatt 3625
tacaaatgga caatgacatc acagttttta gcactgaagc cagtctcatg ctagtaacag 3685
tgggtgagcc gctcgaggga ctgggttcta atgaatactg gtatgaacgg ggagtctctg 3745
cagtcgccag acaaatcata ctcagcccct tcccccgtag agcaacaagt ggttctttta 3805
gagttgactg gcagcatttc ctgtcggggg aggtggggtt tgatggagtt agaaagctcg 3865
cctctgtgta cattctctcc tgggctgtta ctttctgtag acgcacaaaa tcagccccaa 3925
tgtttttaag ggcatcttag ccaaggaagc tggcttttgt gtcgccactt ccaggcctgc 3985
attaagagag agcccaggca ccagggctac cactggaacc tgcctcagcg tcaactgctg 4045
ctggtctgta gccaggccca gcctttgaga cgggtttact gtcaccagta gcctctcagt 4105
gccagccctg agctgctcct ggctcagctg cccagagcct gcagcctggg gaggtactca 4165
gcctctggga gacgagggcc gtggactggg tggctggtag ctcctgcgtt tttgagctgt 4225
gtcctggctg gctgctgcca atgaggtgga caccagtgtg gtttggggtg cactggccac 4285
ttcttgctgg gttctgattt tcttggaagt gcatctgcct tccttatcca atagttttat 4345
ccctgcattg ctcttgtgaa gtggctggtt tggttctgta tgtagcattt tgtacctttc 4405
ctctggcaaa acactgtcag tttataaaca ttttttatat ttccctcctt taaaaacagc 4465
ttgtgtattt ctgctataaa atgtgtcagc aaaggcagag tgacctaata gggcatgttc 4525
ttaagcacag ggactgtatc atgcaggggc caataaagct caagaaaacg agta 4579
<210> 2
<211> 443
<212> PRT
<213> 智人
<400> 2
Met Asp Ser Arg Ala Gln Leu Trp Gly Leu Ala Leu Asn Lys Arg Arg
1 5 10 15
Ala Thr Leu Pro His Pro Gly Gly Ser Thr Asn Leu Lys Ala Asp Pro
20 25 30
Glu Glu Leu Phe Thr Lys Leu Glu Lys Ile Gly Lys Gly Ser Phe Gly
35 40 45
Glu Val Phe Lys Gly Ile Asp Asn Arg Thr Gln Lys Val Val Ala Ile
50 55 60
Lys Ile Ile Asp Leu Glu Glu Ala Glu Asp Glu Ile Glu Asp Ile Gln
65 70 75 80
Gln Glu Ile Thr Val Leu Ser Gln Cys Asp Ser Pro Tyr Val Thr Lys
85 90 95
Tyr Tyr Gly Ser Tyr Leu Lys Asp Thr Lys Leu Trp Ile Ile Met Glu
100 105 110
Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Glu Pro Gly Pro Leu
115 120 125
Asp Glu Thr Gln Ile Ala Thr Ile Leu Arg Glu Ile Leu Lys Gly Leu
130 135 140
Asp Tyr Leu His Ser Glu Lys Lys Ile His Arg Asp Ile Lys Ala Ala
145 150 155 160
Asn Val Leu Leu Ser Glu His Gly Glu Val Lys Leu Ala Asp Phe Gly
165 170 175
Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Lys Arg Asn Thr Phe Val
180 185 190
Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Lys Gln Ser Ala Tyr
195 200 205
Asp Ser Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile Glu Leu
210 215 220
Ala Arg Gly Glu Pro Pro His Ser Glu Leu His Pro Met Lys Val Leu
225 230 235 240
Phe Leu Ile Pro Lys Asn Asn Pro Pro Thr Leu Glu Gly Asn Tyr Ser
245 250 255
Lys Pro Leu Lys Glu Phe Val Glu Ala Cys Leu Asn Lys Glu Pro Ser
260 265 270
Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys His Lys Phe Ile Leu Arg
275 280 285
Asn Ala Lys Lys Thr Ser Tyr Leu Thr Glu Leu Ile Asp Arg Tyr Lys
290 295 300
Arg Trp Lys Ala Glu Gln Ser His Asp Asp Ser Ser Ser Glu Asp Ser
305 310 315 320
Asp Ala Glu Thr Asp Gly Gln Ala Ser Gly Gly Ser Asp Ser Gly Asp
325 330 335
Trp Ile Phe Thr Ile Arg Glu Lys Asp Pro Lys Asn Leu Glu Asn Gly
340 345 350
Ala Leu Gln Pro Ser Asp Leu Asp Arg Asn Lys Met Lys Asp Ile Pro
355 360 365
Lys Arg Pro Phe Ser Gln Cys Leu Ser Thr Ile Ile Ser Pro Leu Phe
370 375 380
Ala Glu Leu Lys Glu Lys Ser Gln Ala Cys Gly Gly Asn Leu Gly Ser
385 390 395 400
Ile Glu Glu Leu Arg Gly Ala Ile Tyr Leu Ala Glu Glu Ala Cys Pro
405 410 415
Gly Ile Ser Asp Thr Met Val Ala Gln Leu Val Gln Arg Leu Gln Arg
420 425 430
Tyr Ser Leu Ser Gly Gly Gly Thr Ser Ser His
435 440

Claims (15)

1.用于鉴定能够调控哺乳动物脂质代谢的试剂的方法，其包括使用哺乳动物MST3，优选人MST3。
2.根据权利要求1的方法，其包括确定MST3活性和/或MST3表达。
3.根据权利要求2的方法，其中在表达MST3的哺乳动物细胞中确定MST3活性或MST表达。
4.根据权利要求3的方法，所述方法包括以下步骤：
(i)使候选化合物调控剂(modulator)与表达编码MST3多肽的多核苷酸序列的宿主细胞接触；
(ii)确定所述候选化合物调控剂对MST3的活性和/或表达的影响，由此将所述化合物鉴定为MST3调控剂，并由此将所述化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
5.根据权利要求1至4的方法，其中MST3活性是通过测量脂质沉积(deposition)、脂质分解代谢(catabolism)，例如脂肪酸氧化和/或脂质分泌，和/或脂质合成代谢(anabolism)，例如脂质摄取和/或脂质合成，和/或胰岛素刺激的葡萄糖摄取(ISGU)来确定的。
6.根据权利要求1至4的方法，其中使用定量实时PCR(qRT-PCR)、蛋白质印迹或邻近连接(proximity ligation)测定法(PLA)确定MST3表达。
7.根据权利要求2的方法，所述方法包括以下步骤：
(i)使候选化合物调控剂与MST3多肽接触；
(ii)确定所述候选化合物调控剂对MST3活性的影响，由此将所述化合物鉴定为MST3调控剂，并由此将所述化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
8.根据权利要求7的方法，其中通过测量MST3肽底物的磷酸化水平来确定MST3活性。
9.根据权利要求1的方法，其包括确定候选化合物与MST3的结合。
10.根据权利要求9的方法，所述方法包括以下步骤：
(i)使候选化合物调控剂与MST3多肽接触；
(ii)确定所述候选化合物调控剂与MST3的结合，由此将所述化合物鉴定为MST3结合剂(binder)，并由此将所述化合物鉴定为哺乳动物脂质代谢的潜在调控剂。
11.根据权利要求7至10中任一项的方法，其中所述MST3多肽是经重组产生和纯化的MST3多肽。
12.由寡核苷酸或多核苷酸组成的化合物，用于治疗代谢疾病，所述寡核苷酸或多核苷酸在长度上由8-80个碱基组成，包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列。
13.根据权利要求12中使用的化合物，其中所述代谢疾病选自肥胖、2型糖尿病(T2D)、非酒精性脂肪肝疾病(non-alcoholic fatty liver disease)(NAFLD)、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis)(NASH)、脂肪性胰腺疾病(fatty pancreasdisease)和脂肪性肾脏疾病(fatty kidney disease)。
14.用于治疗代谢疾病的方法，其包括向需要这种治疗的受试者施用药物有效量的寡核苷酸或多核苷酸，所述寡核苷酸或多核苷酸在长度上由8-80个碱基组成，包含可与核酸序列SEQ ID NO：1或其互补序列杂交的序列。
15.根据权利要求14的方法，其中所述代谢疾病选自肥胖、2型糖尿病(T2D)、非酒精性脂肪肝疾病(NAFLD)、非酒精性脂肪性肝炎(NASH)、脂肪性胰腺疾病和脂肪性肾脏疾病。
CN201880054044.9A 2017-09-13 2018-09-12 用于鉴定与stk24相互作用的治疗剂的方法 Pending CN111194355A (zh)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE1730249-8 2017-09-13
SE1730249 2017-09-13
PCT/SE2018/050918 WO2019054922A1 (en) 2017-09-13 2018-09-12 METHODS OF IDENTIFYING THERAPEUTIC AGENTS THAT INTERACT WITH STK24

Publications (1)

Publication Number Publication Date
CN111194355A true CN111194355A (zh) 2020-05-22

Family

ID=63722740

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201880054044.9A Pending CN111194355A (zh) 2017-09-13 2018-09-12 用于鉴定与stk24相互作用的治疗剂的方法

Country Status (7)

Country Link
US (2) US20200182860A1 (zh)
EP (1) EP3682034A1 (zh)
JP (1) JP2020533013A (zh)
CN (1) CN111194355A (zh)
AU (1) AU2018333813A1 (zh)
CA (1) CA3073406A1 (zh)
WO (1) WO2019054922A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030225527A1 (en) * 2002-03-22 2003-12-04 Antonysamy Stephen Suresh Crystals and structures of MST3
US20060014184A1 (en) * 2004-06-14 2006-01-19 Reginald Brys Methods for identification, and compounds useful for the treatment of degenerative & inflammatory diseases
CN101273144A (zh) * 2005-07-27 2008-09-24 肿瘤疗法科学股份有限公司 诊断食道癌的方法
WO2012024640A2 (en) * 2010-08-20 2012-02-23 Cedars-Sinai Medical Center Mst1 as a prognostic biomarker and therapeutic target in human cancer
CN105695562A (zh) * 2014-11-27 2016-06-22 中国科学院上海生命科学研究院 Mst4基因诊断及细胞治疗感染性疾病的用途及其相关药物

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007062380A2 (en) * 2005-11-21 2007-05-31 Isis Pharmaceuticals, Inc. Modulation of eif4e-bp2 expression
WO2007067812A2 (en) * 2005-12-09 2007-06-14 Metaproteomics, Llc Protein kinase modulation by hops and acacia products
CA2634940A1 (en) * 2005-12-21 2007-07-05 Schering Corporation Treatment of nonalcoholic fatty liver disease using cholesterol lowering agents and h3 receptor antagonist/inverse agonist
JP2011514327A (ja) * 2008-02-13 2011-05-06 ノバルティス アーゲー Ｆａｒｐ２およびＳｔｋ２５およびその使用
JP2010006730A (ja) * 2008-06-25 2010-01-14 Keio Gijuku 抗腫瘍剤
WO2010034670A2 (en) * 2008-09-26 2010-04-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Host cell kinases as targets for antiviral therapies against hcv infection
WO2011038763A1 (en) * 2009-09-30 2011-04-07 Franco Maria Buonaguro Method for biomolecular detection of human liver diseases compositions and kits used in said method
SG10201602286RA (en) * 2010-12-23 2016-04-28 Amazentis Sa Compositions and methods for improving mitochondrial function and treating neurodegenerative diseases and cognitive disorders

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030225527A1 (en) * 2002-03-22 2003-12-04 Antonysamy Stephen Suresh Crystals and structures of MST3
US20060014184A1 (en) * 2004-06-14 2006-01-19 Reginald Brys Methods for identification, and compounds useful for the treatment of degenerative & inflammatory diseases
CN101273144A (zh) * 2005-07-27 2008-09-24 肿瘤疗法科学股份有限公司 诊断食道癌的方法
WO2012024640A2 (en) * 2010-08-20 2012-02-23 Cedars-Sinai Medical Center Mst1 as a prognostic biomarker and therapeutic target in human cancer
CN105695562A (zh) * 2014-11-27 2016-06-22 中国科学院上海生命科学研究院 Mst4基因诊断及细胞治疗感染性疾病的用途及其相关药物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AMRUTKAR M等: ""Protein kinase STK25 controls lipid partitioning in hepatocytes and correlates with liver fat content in humans"" *

Also Published As

Publication number Publication date
US20200182860A1 (en) 2020-06-11
AU2018333813A1 (en) 2020-02-06
CA3073406A1 (en) 2019-03-21
WO2019054922A1 (en) 2019-03-21
JP2020533013A (ja) 2020-11-19
EP3682034A1 (en) 2020-07-22
US20210123904A1 (en) 2021-04-29

Similar Documents

Publication Publication Date Title
O'regan et al. Mitotic regulation by NIMA-related kinases
US7863436B2 (en) Induction of apoptosis by inhibition of sirtuin SIRT1 expression
Yasuhara et al. Wnt/β-catenin and retinoic acid receptor signaling pathways interact to regulate chondrocyte function and matrix turnover
Hribal et al. Regulation of insulin-like growth factor–dependent myoblast differentiation by Foxo forkhead transcription factors
Avruch et al. Protein kinases of the Hippo pathway: regulation and substrates
Héron-Milhavet et al. Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding
Bellido et al. Transcriptional activation of the p21WAF1, CIP1, SDI1 gene by interleukin-6 type cytokines: a prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells
Sarkissian et al. Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3
Perdiguero et al. Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38α in abrogating myoblast proliferation
An et al. Enhanced mitochondrial biogenesis contributes to Wnt induced osteoblastic differentiation of C3H10T1/2 cells
Uebi et al. Phosphorylation of the CREB-specific coactivator TORC2 at Ser307 regulates its intracellular localization in COS-7 cells and in the mouse liver
Bernacchioni et al. Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation
Yoneda et al. A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II
Oyesanya et al. Transcriptional and post-transcriptional mechanisms for lysophosphatidic acid-induced cyclooxygenase-2 expression in ovarian cancer cells
Caputo et al. STE20‐type protein kinase MST4 controls NAFLD progression by regulating lipid droplet dynamics and metabolic stress in hepatocytes
Chen et al. The emerging role of KCl cotransport in tumor biology
Mangili et al. Beta-arrestin 2 is required for dopamine receptor type 2 inhibitory effects on AKT phosphorylation and cell proliferation in pituitary tumors
Zhang et al. AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression
Broderick et al. Guanosine 3′, 5′-cyclic monophosphate (cGMP)/cGMP-dependent protein kinase induce interleukin-6 transcription in osteoblasts
Alisi et al. PKR is a novel functional direct player that coordinates skeletal muscle differentiation via p38MAPK/AKT pathways
Cocco et al. Nuclear inositides: PI-PLC signaling in cell growth, differentiation and pathology
CN111194355A (zh) 用于鉴定与stk24相互作用的治疗剂的方法
Giardino et al. Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation
Castillo-Badillo et al. Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions
KR102181813B1 (ko) 단백질 카이네이즈 d1 저해제를 포함하는 유방암 치료제 조성물

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination