丙酮酸脱氢酶激酶共价抑制剂JX06抗肿瘤活性和机制研究（英文）

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	丙酮酸脱氢酶激酶共价抑制剂JX06抗肿瘤活性和机制研究（英文）
	孙文怡 ; 谢作权1 ; 刘逸夫 ; 赵丹 ; 武志翔 ; 张大东 ; 吕灏 ; 唐帅 1; 金楠 ; 蒋华良2
	2015-04-24
关键词	Glucose metabolism PDHK1 Covalent inhibitor Cysteine Responsive cancer subsets
页码	2
英文摘要	Pyruvate dehydrogenase kinase 1(PDHK1) is a metabolic enzyme critically involved in switching glucose metabolism from mitochondrial oxidation to aerobic glycolysis in cancer cells.Increasing evidences suggest that PDHK1 appears to be the Achilles heel in the reprogrammed glucose metabolism in cancer cells,thus targeting PDHK1 holds huge antitumor promise.In an effort to discover new PDHK1 inhibitors,we carried out an enzymatic screen using a small-molecule library.JX06 emerged prominently due to the remarkable activity against PDHK1.To examine whether JX06 specifically targeted PDHK1,JX06 activity was profiled in a board panel of 323 kinases,which covered most known kinases implicated in cancer malignancy.Among the tested kinases,only PDHKs were remarkably inhibited by JX06 at the concentration of 10 μM.At cellular level,JX06 decreased PDHK1 downstream in a time-and dose-dependent manner,resulting in a metabolic switch from aerobic glycolysis to oxidative phosphorylation.To further explore the molecular mechanisms,we employed the Structure-activity relationship analysis and identified the disulfide bond and the symmetrical thioamides as the key moieties of JX06 that might critically interact with PDHK1.By using multiple biochemical and cytological methods,we revealed that JX06 formed a disulfide bond with the thiol group of a conserved cysteine residue(C240) in PDHK1.Also,the mass spectrometry analysis provided the direct evidence that C240 was modified by JX06.The unique working pattern of JX06 conferred its long-lasting inhibition towards PDHK1.Molecular docking identified a sphere with a radius of 10 A around the sulphur atom of C240 as the binding pocket,in which JX06 suited quite well and formed hydrophobic interactions with the surrounding residues.Remarkably,the sulphur atom of JX06 was located within 3.8 A distance of the sulphur atom of C240,offering a possibility to form a disulfide bond between them.Also,we introduced point mutations to the key residues in the binding pocket,aiming at interfering JX06's docking.Indeed,all these point mutations affected the potency of JX06 and dual mutations had a combinative effect.These data demonstrated that the inhibitory effect of JX06 on PDHK1 required the specific recognition of the unique binding pocket.Our above data demonstrated the essential role of C240 in modulating the enzymatic activity of PDHK1.However,it remained unclear how this residue was involved.Notably,we found out that N283/R286 in PDHK1 differed dramatically in the sidechain orientation between the open and closed conformations.In addition,we noticed that R286 was located adjacent to both JX06 binding pocket and ATP binding pocket.More importantly,JX06 greatly lost its inhibitory activity towards R286 mutants,demonstrating the critical role of R286 in mediating the activity of JX06.In an effort to examine the cellular impacts of 5X06,we unexpectedly discovered that cancer cells exhibited distinct outcomes in ROS generation,the reduction of mitochondrial membrane potential and apoptosis caused by JX06.These results suggested the existence of a responsive cancer subset to PDHK1 inhibition,which was barely explored yet.The apparent association between PDHK1 protein level and the JX06 sensitivity was not observed.By measuring the two major glucose metabolic pathways in 20 cell lines,we discovered that basal glucose metabolic capacity of cancer cells might play a key role in determining the outcomes of PDHK1 inhibition.Cancer cells featuring high glycolysis tended to be sensitive to JX06 and the silencing of PDHK1.To further prove the therapeutic potential of JX06 in the responsive cancer subset,we tested its antitumor efficacy in subcutaneous xenograft mice models.A 21-day continuous treatment of 80 mg/kg JX06 considerably reduced tumor volume and endpoint tumor weights without body weight loss.In agreement with the marked antitumor efficacy of JX06,the intratumoral PDHK1 signaling was significantly inhibited as well as the decrease of plasma lactate level in JX06 treated mice.In brief,we identified the first covalent inhibitor of PDHK1 and proved its antitumor promise.Using this probe,we discovered a novel mechanism in regulating the enzymatic activity of PDHK1 which critically involved a conserved cysteine.We also identified the close correlation between the glucose metabolic states of cancer cells and their sensitivity to PDHK1 inhibition,which might be translated to prompt the responsive patients for PDHK1 inhibitors.
会议录	2015医学前沿论坛暨第十四届全国肿瘤药理与化疗学术会议摘要汇编
文献子类	Article
语种	英语
内容类型	会议论文
源URL	[http://119.78.100.183/handle/2S10ELR8/267086]
专题	药理学第一研究室药物发现与设计中心化学蛋白质组学研究中心
作者单位	1.中国科学院上海药物研究所,药理学第一研究室,上海 201203, 中国.; 2.中国科学院上海药物研究所,药物发现与设计中心,上海 201203, 中国.; 3.中国科学院上海药物研究所,化学蛋白质组学研究中心,上海 201203, 中国.
推荐引用方式 GB/T 7714	孙文怡,谢作权,刘逸夫,等. 丙酮酸脱氢酶激酶共价抑制剂JX06抗肿瘤活性和机制研究（英文）[C]. 见:.