Abstract
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 2 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 2 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 2 bound to CDKL5. A structurally similar analog ( 4 ) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β. Finally, we used our chemical probe pair ( 2 and 4 ) 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.