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.

中文翻译：

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研究 ULK1/AMPK 抑制剂 SBI-0206965 的特异性和作用机制

SBI-0206965 最初被确定为自噬起始激酶 ULK1 的抑制剂，最近被报道为相对于广泛使用但混杂的抑制剂化合物 C/Dorsomorphin 更有效和选择性的 AMP 活化蛋白激酶 (AMPK) 抑制剂。在这里，我们研究了 SBI-0206965 对多种细胞类型（包括肝细胞、骨骼肌细胞和脂肪细胞）中 AMPK 信号传导和代谢读数的影响。我们观察到 SBI-0206965 剂量依赖性减弱 AMPK 激活剂 (991) 刺激的 ACC 磷酸化和肝细胞脂肪生成的抑制。SBI-0206965 (≥25 μM) 适度抑制 C2C12 肌管中的 AMPK 信号传导，但也抑制胰岛素信号传导、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 种人类蛋白激酶进行了 SBI-0206965 的扩展筛选，结果表明 SBI-0206965 抑制多种激酶，包括 AMPK 相关激酶（NUAK1、MARK3/4）的成员，其抑制作用与 AMPK 相同或更有效或 ULK1。该筛选与分子建模一起表明，大多数 SBI-0206965 敏感激酶包含一个大的看门人残基，在该位置偏爱甲硫氨酸。我们观察到，看门人甲硫氨酸突变为较小的侧链氨基酸（苏氨酸）使 AMPK 和 ULK1 对 SBI-0206965 抑制具有抗性。这些结果表明，尽管 SBI-0206965 可用于描述 AMPK 或 ULK1 信号传导和细胞功能，但该化合物可有效抑制其他几种激酶和关键细胞功能，如葡萄糖和核苷摄取。