肿瘤代谢关键激酶丙酮酸脱氢酶激酶（PDHK）抗肿瘤作用研究及敏感群体发现

Cancer cells feature a unique metabolic profile of high aerobic glycolysis, which is characterized by the enhanced conversion of glucose into lactate even in the presence of oxygen. Aerobic glycolysis confers a significant growth advantage of cancer cells by supplying essential ATP production, generating precursors for biosynthesis, and providing reducing equivalents for antioxidant defense. Intervention of aerobic glycolysis has emerged as an new strategy for cancer therapy. Mitochondrial pyruvate dehydrogenase kinase 1 (PDHK1) is a metabolic enzyme critically involved in switching glucose metabolism from mitochondrial oxidation to aerobic glycolysis in cancer cells. PDHK1 converges the signaling of various upstream oncogenic proteins to and appears to be the Achilles heel in the reprogrammed glucose metabolism in cancer cells. Indeed, PDHK1 is one of the most investigated anticancer targets in the aerobic glycolysis pathway, but most inhibitors are still at early stage preclinical research and are largely limited by their compromised potency, selectivity or drug-like properties. Our previous studies focused on a novel irreversible PDHK inhibitor, designated as JX06, which was previously discovered during a high-throughput screen targeting PDHK1 and exhibited improved potency against PDHK compared with reported inhibitors. This study aims to further identify new PDHK inhibitors with drug-like properties, elucidate their molecular mechanism, and assess their anticancer efficacy. In parallel, this study plans to explore the responsive cancer subset to PDHK1 inhibition and identify relevant molecular biomarkers. This study may potential facilitate the translation of PDHK1 inhibition to therapeutic benefits by gaining mechanistic insights into the enzymatic regulation of PDHK1, obtaining PDHK1 inhibitors with new moecular mechanism, and providing molecular signatures to stratify responsive cancer subsets to PDHK1 inhibition.

肿瘤细胞的能量代谢异常，即有氧糖酵解的增强和氧化磷酸化的抑制，是新近揭示的肿瘤十大特征之一。能量代谢异常为肿瘤快速增殖提供能量保证的，为核酸、蛋白等生物大分子的合成提供了重要前体。靶向肿瘤代谢是当前备受关注的新兴的抗肿瘤策略。丙酮酸脱氢酶激酶（PDHK）位于糖酵解和氧化磷酸化通路的关键枢纽，是调控肿瘤能量代谢的重要激酶，是极具潜质的肿瘤代谢抗肿瘤靶点。目前PDHK抑制剂的研究还非常有限，仅有的几个PDHK抑制剂存在活性弱、选择性差、成药性差等问题。本项目前期发现了首个活性更优的不可逆PDHK抑制剂JX06。本研究拟揭示JX06抑制PDHK酶活的作用机制，评价JX06逆转肿瘤细胞代谢特性、抑制肿瘤细胞增殖的抗肿瘤活性，并以JX06为基础通过定向设计发现成药性更优的PDHK抑制剂。同步探索指征PDHK抑制剂敏感群体的生物标志物，旨在为靶向PDHK的抗肿瘤策略提供重要的理论基础和候选化合物。

肿瘤细胞的能量代谢异常又称有氧糖酵解，是肿瘤十大特征之一。靶向肿瘤代谢异常是近年来备受关注的新兴抗肿瘤策略。丙酮酸脱氢酶激酶（PDHK）位于糖酵解和氧化磷酸化通路的关键枢纽，是调控肿瘤能量代谢的重要激酶，是极具潜质的肿瘤代谢抗肿瘤靶点。针对PDHK抑制剂研发的现状，本项目重点开展了针对PDHK的新型共价抑制剂的研发和作用机制研究，并针对肿瘤代谢异质性的挑战，开展了针对该类抑制剂的敏感群体研究。本项目执行期间，在上述三个方面均取得了显著进展：1）以前期高通量筛选发现的首个PDHK共价抑制剂JX06为探针，系统研究了共价抑制PDHK激酶活性的分子机制。发现JX06能特异识别PDHK的ATP结合域附近的一个疏水性结合口袋，通过二硫键共价结合到PDHK的一个保守的半胱氨酸残基（PDHK1的Cys240），通过范德华力诱导286位精氨酸（Arg286）构象发生变化，阻碍ATP进入PDHK的催化口袋，从而影响PDHK的酶催化活性，发挥酶活抑制作用。2）面对PDHK抑制剂敏感肿瘤不明的问题，通过对大量肿瘤细胞筛选结合代谢表型表征，发现肿瘤细胞产酸率（ECAR）与耗氧率（OCR）的比值是一个重要的参数，可以作为肿瘤细胞是否对PDHK抑制剂敏感的判定标准，并阐明其分子机制是PDHK抑制剂能激活线粒体氧化呼吸，引起ROS释放和细胞凋亡。3）针对JX06对PDHK亚型的选择性较差、毒副作用大的问题，本项目与药物化学家合作，开展了针对PDHK1亚型选择性共价抑制剂的研究。设计合成了近100个新结构衍生物，发现了具有良好亚型选择性的PDHK1共价抑制化合物3a（JX27），该化合物对PDHK2、3和4的选择性显著提高，分别为PDHK1的42倍、46倍和~100倍。分子对接显示3a与PDHK1-4的相互作用模式，从口袋的契合程度及与共价反应位点Cys残基的距离两个方面系统阐明了3a特异抑制PDHK1的共价机制，证实3a是安全性和选择性显著提升的先导化合物。本项目的研究成果对于新一代PDHK共价抑制剂的研发奠定了重要的基础，对于该类抑制剂在研发阶段敏感模型的选择和临床应用阶段获益群体的筛选提供了依据。项目执行期间，在J Med Chem、Nat Commun等杂志共发表标注本项目资助论文7篇，其中通讯作者论文6篇，申请PCT发明专利1项。