A Chemical Probe for Dark Kinase STK17B Derives Its Potency and High Selectivity through a Unique P‑Loop Conformation

STK17B is a member of the death-associated
protein kinase family
and has been genetically linked to the development of diverse diseases.
However, the role of STK17B in normal and disease pathology is poorly
defined. Here, we present the discovery of thieno­[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe
for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity
over other kinases including the closely related STK17A. X-ray crystallography
of 11s and related thieno­[3,2-d]­pyrimidines bound to
STK17B revealed a unique P-loop conformation characterized by a salt
bridge between R41 and the carboxylic acid of the inhibitor. Molecular
dynamic simulations of STK17B revealed the flexibility of the P-loop
and a wide range of R41 conformations available to the apo-protein.
The isomeric thieno­[2,3-d]­pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold
lower activity of 19g on STK17B was attributed to the
reduced basicity of its pyrimidine N1.

STK17B为死亡相关蛋白激酶家族（death-associated protein kinase family）成员，且已通过遗传学分析证实与多种疾病的发生发展密切相关。然而，STK17B在正常生理及疾病病理进程中的具体功能仍未得到明确界定。本研究报道了噻吩并[3,2-d]嘧啶类化合物SGC-STK17B-1（11s）的发现过程，该化合物是针对这一尚未被充分研究的‘暗激酶’的高品质化学探针。11s属于ATP竞争性抑制剂（ATP-competitive inhibitor），相较于包括密切同源的STK17A在内的其他多种激酶，展现出优异的选择性。通过对结合了11s及相关噻吩并[3,2-d]嘧啶类化合物的STK17B进行X射线晶体学（X-ray crystallography）分析，研究人员揭示了一种独特的P环（P-loop）构象，其标志性特征为精氨酸残基R41与抑制剂的羧酸基团之间形成盐桥（salt bridge）。对STK17B开展的分子动力学模拟（molecular dynamic simulations）结果显示，P环具有一定的结构灵活性，且脱辅基蛋白（apo-protein）的R41残基可呈现多种不同的构象。本研究同时鉴定出异构体噻吩并[2,3-d]嘧啶类化合物SGC-STK17B-1N（19g）作为阴性对照化合物（negative control compound）。19g对STK17B的活性较11s降低了100倍以上，该活性差异可归因于其嘧啶环N1位点的碱性减弱。