Fig 1.
SYK topology and activation mechanism with Fc receptors.
(A) Domain representation of SYK. (N)SH2 domain (residues 8–118), interdomain A (IA), (C)SH2 domain (residues 163–264), interdomain B (IB), and the catalytic kinase domain (364–620). (B) The activation mechanisms of autoinhibited SYK via both autophosphorylation and engaging p-ITAM on Fc receptors. Tyrosines phosphorylated via these mechanisms are highlighted.
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 < 0.05). For all plots no point plotted indicates that the gene was not scored or measured in that dimension. The AD Biological Domains shows the fraction of all terms annotated to a biological domain to which the gene is annotated, and the size of the point indicates the number of terms. The SEA-AD Cell Type Expression panel shows the mean expression values for the gene per cell subclass, stratified by cognitive status of the donor, where expression is measured as the natural log [number of unique molecular identifiers (UMIs) for each gene in a given cell divided by the total number of UMIs in the same cell divided by 10,000] plus 1]. The size of each point represents the fraction of cells from each cell type expressing the gene. (C) mRNA expression levels of SYK and FCER1G in hiPSC-derived neurons and microglia.
Fig 3.
Compound screening cascade and the development and use of a biochemical TR-FRET assay to evaluate inhibition of the PPI between SYK-tSH2 and the FCERG1G p-ITAM peptide.
(A) Compound screening cascade. (B) and (C) TR-FRET assay with 6xHis-SYK-tSH2 and a p-ITAM-FITC tagged peptide. Unlabeled FCER1G p-ITAM can displace p-ITAM-FITC, while FCER1G ITAM does not. (D) Primary uHTS of 138,214 compounds with 294 primary positive identified.
Fig 4.
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.
Table 1.
Biochemical and biophysical data for compounds 37, 13, 42, 43, and 44.
The estimated IC50 for GST-PD is calculated via the western blot densitometry. a Solubility calculated with a nephelometer (S5 Fig). b Kinetic solubility calculated by Analiza, Inc. An unmodified blot used to generate GST-PD IC50 values is included in S7 Fig.
Fig 5.
Compounds 13 and 37 react covalently with SYK-tSH2.
(A) Mass spectrometry analysis of SYK incubated with compounds 37 and 13 at 100 μM for 1 h at room temperature. (B) Hypothesized mechanism of inhibition of 37 and 13 with SYK-tSH2.
Fig 6.
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.
Fig 7.
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.