Ong Han Wee, Liang Yi, Richardson William, Lowry Emily R, Wells Carrow I, Chen Xiangrong, Silvestre Margaux, Dempster Kelvin, Silvaroli Josie A, Smith Jeffery L, Wichterle Hynek, Pabla Navjot S, Ultanir Sila K, Bullock Alex N, Drewry David H, Axtman Alison D
Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.
Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7DQ, United Kingdom.
bioRxiv. 2023 Feb 10:2023.02.09.527935. doi: 10.1101/2023.02.09.527935.
Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog demonstrates excellent selectivity and only retains GSK3α/β affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog bound to CDKL5. A structurally similar analog ( ) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β. Finally, we used our chemical probe pair ( and ) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
尽管细胞周期蛋白依赖性激酶样 5（CDKL5）在大脑中介导了多个重要过程，包括在发育过程中，但它仍然是一种特征不明的人类蛋白激酶。因此，其底物、功能和调节机制尚未得到充分描述。我们意识到，一种针对 CDKL5 的强效且选择性的小分子探针，能够揭示其在正常发育以及因突变而异常的疾病中的作用。我们制备了 AT-7519 的类似物，AT-7519 是几种细胞周期蛋白依赖性和细胞周期蛋白依赖性激酶样激酶的已知抑制剂，已进入 II 期临床试验。我们鉴定出类似物 作为 CDKL5/糖原合酶激酶 3（GSK3）的一种高效且具有细胞活性的化学探针。对其全激酶组选择性的评估证实，类似物 表现出优异的选择性，仅保留对 GSK3α/β 的亲和力。作为我们的化学探针可用于定向生物学研究的高质量工具的证据，我们证明了对下游 CDKL5 和 GSK3α/β 信号传导的抑制，并解析了类似物 与 CDKL5 结合的共晶体结构。一种结构相似的类似物（ ）被证明缺乏 CDKL5 亲和力，并保持对 GSK3α/β 的强效且选择性抑制。最后，我们使用我们的化学探针组合（ 和 ）来证明抑制 CDKL5 和/或 GSK3α/β 可促进暴露于内质网（ER）应激的人类运动神经元的存活。我们已经证明了我们的化学探针组合所引发的神经保护表型，并举例说明了我们的化合物在表征 CDKL5/GSK3 在神经元及其他方面的作用方面的实用性。
