Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics.

Akt 是细胞中促生长信号的主要调节剂。Akt 被破坏激酶和 pleckstrin 同源 (PH) 结构域之间的自身抑制界面的磷酸肌醇激活，然后在 T308 和 S473 被磷酸化。Akt 过度激活是致癌的，这刺激了有效和选择性抑制剂作为治疗剂的发展。使用氢氘交换质谱法 (HDX-MS)，我们研究了结合 Akt ATP 竞争性和变构抑制剂后的构象变化。我们将抑制剂与 Akt1 的三种不同状态进行了比较。变构抑制剂引起实质性构象变化并限制膜结合。ATP 竞争性抑制剂引起广泛的变构构象变化，改变自抑制界面并导致膜结合增加，表明 PH 结构域更容易进行膜结合。这项工作提供了对 PH 和激酶结构域的自身抑制构象以及 Akt 抑制剂诱导的构象变化的独特见解，并且对 Akt 靶向治疗的设计具有重要意义。