Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition
Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition
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DOI：
10.1016/j.celrep.2023.113184
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发表时间：
2023-09-29
期刊：
影响因子：
8.8
通讯作者：
Tomlinson，Darren C.
中科院分区：
文献类型：
--
作者：
Martin，Heather L.;Turner，Amy L.;Tomlinson，Darren C.
激酶是重要的治疗靶点，其抑制剂根据其作用机制分类，其范围从阻断ATP结合到共价抑制。在这里，通过使用在P+1口袋中结合的Affimer试剂捕获处于中间活化状态的p21活化激酶5（PAK 5）来突出抑制机制。PAK 5是从尿路上皮癌细胞中的非假设驱动的高内容成像RNAi筛选中鉴定的。PAK 5的沉默导致细胞数量减少、G1/S停滞和细胞增大，表明其在尿路上皮癌细胞系存活和增殖中是重要的。分离Affimer试剂以鉴定抑制机制。Affimer PAK 5-Af 17重现了用siRNA观察到的表型。共结晶显示PAK 5-Af 17结合在PAK 5的P+1口袋中，将激酶锁定在部分活化状态。这种抑制机制表明另一类激酶抑制剂是可能的。
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.