Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China.
Cell Commun Signal. 2019 Aug 30;17(1):111. doi: 10.1186/s12964-019-0425-4.
Distinctive from their normal counterparts, cancer cells exhibit unique metabolic dependencies on glutamine to fuel anabolic processes. Specifically, pancreatic ductal adenocarcinoma (PDAC) cells rely on an unconventional metabolic pathway catalyzed by aspartate transaminase 1 (GOT1) to rewire glutamine metabolism and support nicotinamide adenine dinucleotide phosphate (NADPH) production. Thus, the important role of GOT1 in energy metabolism and Reactive Oxygen Species (ROS) balance demonstrates that targeting GOT1 may serve as an important therapeutic target in PDAC.
To assay the binding affinity between Aspulvinone O (AO) and GOT1 proteins, the virtual docking, microscale thermophoresis (MST), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) methods were employed. GOT1 was silenced in several PDAC cell lines. The level of OCR and ECR were assayed by seahorse. To evaluate the in vivo anti-tumor efficacy of AO, the xenograft model was built in CB17/scid mouse.
Screening of an in-house natural compound library identified the AO as a novel inhibitor of GOT1 and repressed glutamine metabolism, which sensitizes PDAC cells to oxidative stress and suppresses cell proliferation. Virtual docking analysis suggested that AO could bind to the active site of GOT1 and form obvious hydrophobic interaction with Trp141 together with hydrogen bonds with Thr110 and Ser256. Further in vitro validation, including MST, CETSA and DARTS, further demonstrated the specific combining capacity of AO. We also show that the selective inhibition of GOT1 by AO significantly reduces proliferation of PDAC in vitro and in vivo.
Taken together, our findings identify AO as a potent bioactive inhibitor of GOT1 and a novel anti-tumour agent for PDAC therapy.
与正常细胞不同，癌细胞对谷氨酰胺表现出独特的代谢依赖性，以提供合成代谢过程所需的能量。具体而言，胰腺导管腺癌（PDAC）细胞依赖天冬氨酸转氨酶 1（GOT1）催化的非传统代谢途径，重新布线谷氨酰胺代谢，并支持烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的产生。因此，GOT1 在能量代谢和活性氧（ROS）平衡中的重要作用表明，靶向 GOT1 可能成为 PDAC 的重要治疗靶点。
为了检测 Aspulvinone O（AO）与 GOT1 蛋白的结合亲和力，采用了虚拟对接、微量热泳动（MST）、细胞热转移分析（CETSA）和药物亲和响应靶标稳定性（DARTS）方法。在几种 PDAC 细胞系中沉默 GOT1。通过 Seahorse 测定 OCR 和 ECR 的水平。为了评估 AO 的体内抗肿瘤疗效，在 CB17/scid 小鼠中建立了异种移植模型。
从内部天然化合物库中筛选出 AO 是 GOT1 的新型抑制剂，可抑制谷氨酰胺代谢，使 PDAC 细胞对氧化应激敏感，并抑制细胞增殖。虚拟对接分析表明，AO 可以结合到 GOT1 的活性位点，并与 Trp141 形成明显的疏水性相互作用，同时与 Thr110 和 Ser256 形成氢键。进一步的体外验证，包括 MST、CETSA 和 DARTS，进一步证明了 AO 的特异性结合能力。我们还表明，AO 对 GOT1 的选择性抑制可显著减少 PDAC 在体外和体内的增殖。
综上所述，我们的研究结果确定 AO 为 GOT1 的有效生物活性抑制剂，是 PDAC 治疗的一种新型抗肿瘤药物。
