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 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.

Figure 1.

Comparison of CDKL5 co-crystal structure with ASC67 (left, PDB code: 4BGQ) and docking of AT-5419 (right). Key interactions, including the critical hinge binding interactions, are highlighted.

Figure 2.

SAR summary for AT-7519 analogs.

Figure 3.

(A) CDKL5 cellular and (B) CDKL5 biochemical potency for compounds 2 and 4.

Figure 4.

Selectivity data for compounds 2 and 4. PoC: percent of control.

Figure 5.

Cellular target engagement of GSK3α/β by compounds 2 and 4.

Figure 6.

Overview of the CDKL5 co-crystal structure with compound 2 (PDB code: 8CIE). N and C terminal lobes colored in aquamarine and blue, respectively. Compound 2 binds to the ATP pocket between the two lobes and is shown as orange sticks. Hydrogen bonds are indicated as green dashed lines, including interactions between the hinge region (Glu90 and Val 92, colored purple) and the pyrazole shown in the cutout.

Figure 7.

Compound 2 potently inhibits CDKL5 kinase activity. (A) Representative graph from a kinase assay showing that purified WT human CDKL5 retains kinase activity, while the kinase dead (KD, CDKL5 K42R) human protein was functionally inactive. n = 3 replicates. (B) Representative western blot showing equal levels of WT and KD proteins in the kinase assay experiments. (C-D) Purified WT human CDKL5 was used for kinase assays as described above in the presence of indicated compounds at 10 or 100 nM concentrations. n = 3 replicates. One-way ANOVA with Dunnett’s multiple comparison test was done. *** = p <0.0001. Non-significant comparisons are not illustrated.

Figure 8.

Analysis of phospho-EB2 and phospho-β-catenin expression following treatment with compound 2 or 4 confirms inhibition of downstream signaling mediated by CDKL5 and GSK3α/β. (A) Western blots showing expression of CDKL5, EB2 and tubulin, and level of S222 EB2 phosphorylation following 1 hour treatment of DIV11–14 rat primary cortical neurons with 5 nM, 50 nM, 500 nM or 5 µM of compound 2 or 4. (B) Quantification of S222 EB2 phosphorylation following treatment of DIV11–14 rat primary cortical neurons with compound 2. Two-way ANOVA with pairwise comparisons of each condition against the control condition. n = 3 replicates. (C) Quantification of S222 EB2 phosphorylation following treatment of DIV11–14 rat primary cortical neurons with compound 4. Two-way ANOVA with pairwise comparisons of each condition against the control condition. n = 3 replicates. (D) Western blots showing expression of β-Catenin, and level of S33/37/T41 β-Catenin phosphorylation following 1 hour treatment of DIV11–14 rat primary cortical neurons with 5 nM, 50 nM, 500 nM or 5 µM of compound 2 or 4. (E) Quantification of S33/37/T41 β-catenin phosphorylation following treatment of DIV11–14 rat primary cortical neurons with compound 2. Two-way ANOVA with pairwise comparisons of each condition against the control condition. n = 3 replicates. (F) Quantification of S33/37/T41 β-catenin phosphorylation following treatment of DIV11–14 rat primary cortical neurons with compound 4. Two-way ANOVA with pairwise comparisons of each condition against the control condition. n = 3 replicates. * = p ≤ 0.05 , ** = p ≤ 0.01 , *** = p ≤ 0.001, **** = p ≤ 0.0001. Non-significant (p > 0.05) comparisons are not illustrated.

Figure 9.

Compounds 2 and 4 promote survival of motor neurons in response to ER stress. Error bars represent standard error of the mean (SEM) calculated using two-way ANOVA. MN: motor neuron.

Scheme 1.

Synthesis of chemical probe compound (2)^{a a} (i) 3,5-difluorobenzoic acid, n-propanephosphonic acid anhydride, DIPEA, THF, 0 to 25 °C, 1 h; (ii) HCl, dioxane, 0 to 25 °C, 1 h.

Scheme 2.

Synthesis of negative control compound (4)^{a a}(i) 4-aminomethyltetrahydropyran, n-propanephosphonic acid anhydride, DIPEA, THF, 0 to 25 °C, 1 h.