Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, U.K.
ACS Chem Neurosci. 2023 May 3;14(9):1672-1685. doi: 10.1021/acschemneuro.3c00135. Epub 2023 Apr 21.
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 compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). 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. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog bound to human CDKL5. A structurally similar analog () proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β, making it a suitable negative control. 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 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.
尽管周期素依赖性激酶样 5（CDKL5）在大脑中调节着多个重要过程，包括在发育过程中，但它仍然是一种特征描述不足的人类蛋白激酶。因此，其底物、功能和调节机制尚未完全描述。我们意识到，一种针对 CDKL5 的有效且选择性的小分子探针的可用性可以使其在正常发育以及因突变而异常的疾病中的作用得到阐明。我们制备了 AT-7519 的类似物，该化合物已进入 II 期临床试验，是几种细胞周期蛋白依赖性激酶（CDKs）和细胞周期蛋白依赖性激酶样激酶（CDKLs）的已知抑制剂。我们发现类似物 是一种针对 CDKL5/GSK3（糖原合酶激酶 3）的高活性和细胞活性的化学探针。对其激酶组广泛选择性的评估证实，类似物 对 CDKL5 和 GSK3α/β具有优异的选择性，仅保留了对 GSK3α/β的亲和力。接下来，我们证明了其对下游 CDKL5 和 GSK3α/β信号的抑制作用，并解析了与人类 CDKL5 结合的类似物 的共晶结构。结构相似的类似物 () 证明缺乏对 CDKL5 的亲和力，并且保持对 GSK3α/β的强大且选择性抑制作用，使其成为合适的阴性对照。最后，我们使用我们的化学探针对（和）证明抑制 CDKL5 和/或 GSK3α/β可促进暴露于内质网应激的人运动神经元的存活。我们已经证明了我们的化学探针对诱导的神经保护表型，并举例说明了我们的化合物在神经元内外表征 CDKL5/GSK3 作用的用途。
