A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

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 analog2as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog2demonstrates 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 analog2bound 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 (2and4) 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.

Illumination of understudied ciliary kinases

“…CDKL5, also known as serine/threonine kinase 9 (STK9), is the only member of the CDKL family with strong connections to ciliogenesis in humans. CDKL5 is ubiquitously expressed in human tissues, but is observed at higher levels in the hippocampus, cerebellar, striatum, and cortex regions of the CNS in humans and mice, consistent with its roles in dendritic spine growth, brain development, and excitatory synapse composition (Rusconi et al, 2008;Fagerberg et al, 2014;Canning et al, 2018;Ong et al, 2023). A study using RNAi targeting CDKL5, which caused reduced neurite growth and dendritic arborization in rat cortical neurons, confirmed that this kinase regulates neuronal morphogenesis (Chen et al, 2010).…”

“…CDKL kinases are a family of five relatively underexplored serine/threonine human kinases: CDKL1, CDKL2, CDKL3, CDKL4, and CDKL5. This family has the highest sequence similarity to cyclin-dependent kinases (CDKs) (Canning et al, 2018;Ong et al, 2023). CDKLs contain a cyclin binding domain, although no cyclin-dependent functions have been ascribed.…”

“…The C-termini of CDKL2 and CDKL3 were found to have an atypical αJ helix necessary for their catalytic functions, which is absent in CDKL1 and CDKL5 (Canning et al, 2018). Notably, for CDKL5, the C-terminus is involved in trafficking CDKL5 to its subcellular compartments during various developmental stages (Rusconi et al, 2008;Canning et al, 2018;Ong et al, 2023).…”

Identifying dysregulated regions in amyotrophic lateral sclerosis through chromatin accessibility outliers

“…Similarly, the CDKL5 gene exhibits overexpression with an over-accessible promoter region (protein levels are unavailable). Suppressing CDKL5 expression using a small molecule probe enhances the survival of human motor neurons under endoplasmic reticulum stress conditions 50 . Another outlier gene we identified, HIF1A, contributes to motor neuron degeneration through hypoxic stress, and prolonged survival observed in ALS mice suggests up-regulation of HIF1A as a potential therapeutic target 51 .…”

“…We manually curated a list of ALS genes from literature 1,11,[42][43][44][45][46][47][49][50][51][73][74][75][76][77][78][79][80][81][82] and ALSOD 41 . The curated list is available at 10.5281/zenodo.8331545.…”