Optimization and Characterization of SHIP1 Ligands for Cellular Target Engagement and Activity in Alzheimer’s Disease Models

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (<b>32</b>), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response <i>in vivo</i>. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound <b>32</b> induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound <b>32</b> enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound <b>32</b> resulted in brain exposure sufficient to alter gene expression and reduce IL-1β levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimer's disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and <i>in vivo</i> activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimer's disease.