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

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 has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. 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 postsynaptic 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

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer\u27s disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer\u27s disease (AD) traits and microglia. These proteins are more abundant in Alzheimer\u27s patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PI

Compounds 13, 37, and 44 (100 μM) bind covalently to TBXT and MSN via mass spectrometry.

Number of mass adducts of the compound are indicated in red.</p

Compound 44 covalently modifies SYK-tSH2.

(A) Chemical structures of initial HTS hit 39 and analogue 43. (B) Synthesis of 44 from analogue 42. (C) Mass spectrometry of SYK-SH2 incubated with covalent inhibitor 1 and old versus fresh stocks of 42 and 43 at 100 μM for 1 h at room temperature. Only the old DMSO stock of 42 reacts covalently with SYK-tSH2. (D) Mass spectrometry of SYK-tSH2 incubated with 42 and 44 at 100 μM for 1 h at room temperature. I Dose-response of 44 in covalently labelling SYK-tSH2, assessed by mass spectrometry. (F) Dose response curve of 44 in the TR-FRET assay (IC50 = 0.27 ± 0.02 μM). (G) Hypothesized mechanism of inhibition of 44 with SYK-tSH2. (H) Compound 44 disrupted the PPI via GST-pulldown in a dose-response fashion. An unmodified blot corresponding with panel (H) is included in S7 Fig.</p

Identification of active disulfide compounds which form in DMSO solutions of compounds 42 and 43.

(A) LCMS analysis of 42 as a fresh solution in MeCN indicates the monomer is present. LCMS Calculated for [M+H]+ C7H9N4O2S: 213.04; observed: 213.1 [M+H]+. (B) LCMS analysis of 42 10 mM stock solution in MeCN indicates the disulfide dimer is present. LCMS Calculated for [M+H]+ C14H15N8O4S2: 423.06; observed: 423.0 [M+H]+. (C) LCMS analysis of 43 as a fresh solution in MeCN indicates the monomer is present. LCMS Calculated for [M+H]+ C10H15N4O2S: 255.08; observed: 255.1 [M+H]+. (D) LCMS analysis of 43 10 mM stock solution in MeCN indicates the disulfide dimer is present. LCMS Calculated for [M+H]+ C20H27N8O4S2: 507.15; observed: 507.1 [M+H]+. (TIFF)</p

Identification of 3 compounds that inhibit the interaction of SYK and FCER1G in a GST-SYK and FCER1G-Flag pulldown assay.

(A) Optimization of the GST-SYK and FCER1G-Flag pulldown assay in HEK293 cells. (B) Compounds 13, 24, and 37 were able to inhibit the interaction between SYK-GST and FCER1G-Flag in a pulldown assay at a single concentration (100 μM). (C) Compounds 13, 24, and 37 were tested via GST-pulldown in a dose-response fashion. (D) Chemical structures of compounds 13, 24, and 37. Unmodified blots corresponding with panels (A), (B), and (C) are included in S7 Fig.</p

Second biological replicate of GST-PD data displayed in Fig 4B and dose-response GST-PD for compounds 4 and 32.

(A) Compounds 4, 13, 24, 32, and 37, 4, were able to inhibit the interaction between SYK-GST and FCER1G-Flag in a pulldown assay at a single concentration (100 μM). Second biological replicate displayed. (B) Dose-response GST-PD of 4 and 32. Unmodified gels corresponding with panels (A) and (B) are included in S7 Fig. (TIFF)</p

Gel filtration fractions and intact mass deconvolution of purified SYK proteins.

(A) SYKA-c020 His tagged, (B) SYKA-c020 His tag cleaved, and (C) SYKA-c020 His tag cleaved and biotinylated. (TIFF)</p

Fig 2 -

TREAT-AD bioinformatic summaries for SYK (A) and FCER1G (B). For each target, the composite Target Risk Score (Overall, top left) is a sum of the Genetics and Omics risk dimensions. The Genetics Summary includes the average rank of gene-level significance values from GWAS and QTL studies, variant severity analysis, and phenotype summaries for both human genes (Hsap) and orthologous genes. The Omics Summary represents the results from meta-analyses of AMP-AD transcriptomic and proteomic datasets showing the effect size (log fold change) and significance (red points are detected at an FDR (C) mRNA expression levels of SYK and FCER1G in hiPSC-derived neurons and microglia.</p