Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965.
Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965.
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研究ULK1/AMPK抑制剂SBI-0206965的特异性和作用机理。
DOI：
10.1042/bcj20210284
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发表时间：
2021-08-13
期刊：
影响因子：
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通讯作者：
Sakamoto K
中科院分区：
文献类型：
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作者：
Ahwazi D;Neopane K;Markby GR;Kopietz F;Ovens AJ;Dall M;Hassing AS;Gräsle P;Alshuweishi Y;Treebak JT;Salt IP;Göransson O;Zeqiraj E;Scott JW;Sakamoto K
SBI-0206965最初被认为是一种自噬起始蛋白激酶ULK1的抑制剂，最近被报道为一种比广泛使用的混杂抑制剂化合物C/多索吗啡更有效和更具选择性的AMP激活蛋白激酶抑制剂。在这里，我们研究了sbi-0206965对多种细胞类型的AMPK信号和代谢读数的影响，包括肝细胞、骨骼肌细胞和脂肪细胞。我们观察到SBI-0206965剂量依赖地减弱AMPK激活剂(991)刺激的Acc的磷酸化和对肝细胞脂肪生成的抑制。SBI-0206965(≥25 μM)可抑制C2C12肌管上的AMPK信号转导，但也抑制胰岛素信号转导、胰岛素介导/AMPK非依赖性葡萄糖摄取和AicA核苷摄取。我们在体外对一组140个人的蛋白激酶进行了sbi-0206965的扩展筛选，结果显示sbi-0206965抑制了几种激酶，包括AMPK相关激酶(NUAK1，Mark3/4)的成员，其抑制作用与AMPK或ULK1相同或更强。这一筛选结合分子模拟显示，大多数对sbi-0206965敏感的激酶都含有一个大的守门人残基，并在这个位置偏爱蛋氨酸。我们观察到，守门人蛋氨酸突变为较小的侧链氨基酸(苏氨酸)，使AMPK和ULK1对SBI-0206965抑制产生抗性。这些结果表明，尽管SBI-0206965可以用于描述AMPK或ULK1信号和细胞功能，但该化合物有效地抑制了其他几种激酶和关键的细胞功能，如葡萄糖和核苷摄取。我们的研究表明，守门人残基作为抑制剂敏感性的决定因素，以及具有抑制剂抗性的突变形式可以被用作潜在的对照，以探索SBI-0206965的特定细胞效应。
SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.