SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease

Author:

Jesudason Cynthia D.1,Mason Emily R.2,Chu Shaoyou2,Oblak Adrian L.23,Javens‐Wolfe June4,Moussaif Mustapha4,Durst Greg1,Hipskind Philip1,Beck Daniel E.5,Dong Jiajun6,Amarasinghe Ovini6,Zhang Zhong‐Yin56,Hamdani Adam K.7,Singhal Kratika7,Mesecar Andrew D.7,Souza Sarah8,Jacobson Marlene8,Salvo Jerry Di8,Soni Disha M.2,Kandasamy Murugesh2,Masters Andrea R.2,Quinney Sara K2,Doolen Suzanne9,Huhe Hasi9,Rizzo Stacey J. Sukoff9,Lamb Bruce T.23,Palkowitz Alan D.24,Richardson Timothy I.234

Affiliation:

1. Lgenia Fortville Indiana USA

2. Indiana University School of Medicine Indianapolis Indiana USA

3. Stark Neurosciences Research Institute Indiana University School of Medicine Indianapolis Indiana USA

4. Indiana Biosciences Research Institute Indianapolis Indiana USA

5. Institute for Drug Discovery Purdue University West Lafayette Indiana USA

6. Department of Medicinal Chemistry and Molecular Pharmacology Purdue University West Lafayette Indiana USA

7. Department of Biochemistry Purdue University West Lafayette Indiana USA

8. Evotec Princeton New Jersey USA

9. University of Pittsburgh School of Medicine Pittsburgh Pennsylvania USA

Abstract

AbstractINTRODUCTIONThe 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.METHODSWe 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.RESULTSSHIP1 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.DISCUSSION3‐((2,4‐Dichlorobenzyl)oxy)‐5‐(1‐(piperidin‐4‐yl)‐1H‐pyrazol‐4‐yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies.Highlights Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain‐contaning inositol phosphatase 1 (SHIP1) target engagement and on‐target activity in cellular assays. A phenotypic high‐content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health. SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic. The chemical probe 3‐((2,4‐dichlorobenzyl)oxy)−5‐(1‐(piperidin‐4‐yl)−1H‐pyrazol‐4‐yl) pyridine is recommended to explore SHIP1 pharmacology.

Funder

National Institutes of Health

Publisher

Wiley

Subject

Psychiatry and Mental health,Neurology (clinical)

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