Discovery of a Potent and Selective CDKL5/GSK3 Chemical Probe That 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 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 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. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) 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 (2 and 4) 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.

Identifying dysregulated regions in amyotrophic lateral sclerosis through chromatin accessibility outliers

“…Suppressing CDKL5 expression using a small molecule probe enhances the survival of human motor neurons under endoplasmic reticulum stress conditions. 54 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. 55 Finally, VPS4B is pathologically increased in familial and sporadic ALS neuronal nuclei.…”

“…We manually curated a list of ALS genes from the literature 1 , 11 , 46 , 47 , 48 , 49 , 50 , 51 , 53 , 54 , 55 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 and ALSOD. 45 The curated list is available at 10.5281/zenodo.8331545.…”

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology

“… B1 was simultaneously assayed with other CDKL5 antagonists ( Ong et al, 2023 ). ( A ) Representative graph from a CDKL5 kinase assay demonstrating that purified WT human CDKL5 retains kinase activity, while the kinase dead (KD, CDKL5 K42R) human protein is functionally inactive (n = 3).…”

“…***p<0.0001 and nonsignificant comparisons not shown. Control, DMSO, positive (AST-487), and negative (Lapatinib) controls are as in Ong et al, 2023 .…”

Identifying dysregulated regions in amyotrophic lateral sclerosis through chromatin accessibility outliers

“…Similarly, the CDKL5 gene exhibits over-expression 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 .…”