Fig. 3. Generation and validation of sCSF1R_inh, a novel small molecule CSF1R inhibitor.

a The chemical structure of the CSF1R inhibitor, ((S)-4-(3-((2-(6-methoxypyridin-3-yl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridin-6-yl)-2-methylbut-3-yn-2-amine (sCSF1R_inh). b CSF1R crystal structure in combination with sCSF1R_inh shows how the inhibitor binds in a DFG-out conformation and makes key interactions with the hinge region. c TREEspot™ interaction map is an artistic representation of the human kinome phylogenetic tree. Kinases found to bind sCSF1R_inh are highlighted with red circles where larger circles indicate higher affinity binding. d KINOMEscan™ screening platform is an active site-directed competition binding assay to quantify the interaction between sCSF1R_inh and 450 human kinases. The percent control score demonstrates that sCSF1R_inh is highly selective for CSF1R. e CSF1 (100 ng/mL) stimulation of BV2 microglia induces CSF1R and ERK1/2 phosphorylation after 5 min and sCSF1R_inh (500 nM) blocks these phosphorylation events (f) After 10 min, CSF1 stimulation of BV2 microglia leads to endocytosis of the receptor and sCSF1R_inh (500 nM) maintains CSF1R surface expression. Upon endocytosis, CSF1R is trafficked to the lysosome for degradation and sCSF1R_inh (500 nM) blocks CSF1R degradation. Data points represent quantification from a single Western Blot. Graphical columns represent the mean and standard deviation of biological triplicates. Statistical significance was determined by a one-way ANOVA and p values are indicated by ****p < 0.0001.