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

Abstract

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3b, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3b activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3b activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3b. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

Charting the Progress of Epilepsy Classification: Navigating a Shifting Landscape

“…Optimizing treatment response: Precision medicine involves continuous monitoring and adjustment of treatment plans based on individual patient responses. Biomarkers, including genetic markers and neuroimaging findings, can help assess treatment effectiveness and guide modifications [35]. For example, EEG monitoring can detect changes in seizure frequency and patterns, allowing for timely adjustments in medication dosages or the exploration of alternative treatment strategies.…”

“…Disease-modifying therapies: In some epilepsy syndromes, disease-modifying therapies can target the underlying mechanisms rather than symptom control. Precision medicine facilitates the identification of suitable candidates for such therapies [35]. For example, individuals with genetic mutations leading to mTOR pathway activation may be candidates for mTOR inhibitors like everolimus [36].…”

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells

“…This enzyme is expressed in cells fused to a protein target of interest to detect binding of the fluorescent probe and evaluate competition by unlabeled small molecules. This method was recently used to characterize small molecule engagement of the kinases CDKL5, p38α, and JAK3, and the lysosomal ion channel TRPML1 . However, a drawback is that fusion of proteins to other proteins or peptides has the potential to affect their function .…”

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2

“…Because CDKL5 is not inhibited by our compound, we expected to see a maximal 20–25% reduction of EB2 phosphorylation by the CDKL2 probe. We evaluated a CDKL5 chemical probe (SGC-CAF382–1) in parallel at a concentration (50 nM) where ∼80% reduction in EB2 phosphorylation was previously observed . Neurons were treated with increasing concentrations of compound 9 alone or 50 nM of SGC-CAF382–1 with increasing concentrations of compound 9 to explore whether these two compounds could completely inhibit EB2 phosphorylation when dosed together.…”

“…We evaluated a CDKL5 chemical probe (SGC-CAF382−1) in parallel at a concentration (50 nM) where ∼80% reduction in EB2 phosphorylation was previously observed. 25 Neurons were treated with increasing concentrations of compound 9 alone or 50 nM of SGC-CAF382−1 with increasing concentrations of compound 9 to explore whether these two compounds could completely inhibit EB2 phosphorylation when dosed together. Negative control compound 16 was evaluated in parallel to explore chemotype-induced effects on EB2 phosphorylation.…”