The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

中文翻译：

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AMPK 激活剂 SC4 的结构功能分析和强效泛 AMPK 激活剂的鉴定。

AMP 活化蛋白激酶 (AMPK) αβγ 异源三聚体是一种主要的细胞能量传感器和能量稳态的中央调节器。用小分子药物激活骨骼肌 AMPK 可改善葡萄糖摄取，并为治疗 2 型糖尿病和胰岛素抵抗的新策略提供机会，最近的遗传和药理学研究表明，α2β2γ1 异构体组合是异源三聚体复合物的主要责任。为了开发 α2β2 特异性激活剂，我们在这里对 SC4 的 2-羟基联苯基团进行结构/功能分析，SC4 是一种具有 α2 选择性倾向的激活剂，也能够有效地激活 β2 复合物。用极性取代的基于环己烯的探针取代 LHS 2-羟基苯基产生两种 AMPK 激动剂 MSG010 和 MSG011，当针对一组 AMPK 复合物进行筛选时，它没有显示出 α2 选择性。通过放射性标记激酶测定，MSG010 和 MSG011 以比泛 AMPK 激活剂 MK-8722 高一个数量级的效力激活 α2β2γ1 AMPK。与 AMPK α2β1γ1 复合的 MSG011 的晶体结构揭示了与 SC4 相似的结合模式，以及 SC4 2-羟基和 α2-Lys31 之间相互作用对指导 α2-选择性的潜在重要性。MSG011 在小鼠原代肝细胞和常用细胞系中诱导强大的 AMPK 信号传导，并且在大多数情况下，这发生在激酶激活环残基 α-Thr172（AMP 诱导的 AMPK 活性的经典标志物）磷酸化没有变化的情况下。这些发现将指导未来设计 α2β2 选择性 AMPK 激活剂，