Introduction

The risk of developing Alzheimer’s disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain–containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P₃ to PI(3,4)P₂, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target.

Methods

We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies.

Results

SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration.

By Cynthia D. Jesudason, Emily R. Mason, Shaoyou Chu, Adrian L. Oblak, June Javens-Wolfe, Mustapha Moussaif, Greg Durst, Philip Hipskind, Daniel E. Beck, Jiajun Dong, Ovini Amarasinghe, Zhong-Yin Zhang, Adam K. Hamdani, Kratika Singhal, Andrew D. Mesecar, Sarah Souza, Marlene Jacobson, Jerry Di Salvo, Disha M. Soni, Murugesh Kandasamy, Andrea R. Masters, Sara K Quinney, Suzanne Doolen, Hasi Huhe, Stacey J. Sukoff Rizzo, Bruce T. Lamb, Alan D. Palkowitz, Timothy I. Richardson