Browsing by Author "Hamdani, Adam K."

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    SHIP1 phosphatase as a Late‐Onset Alzheimer’s Disease therapeutic target
    (Wiley, 2025-01-09) Singhal, Kratika; Hamdani, Adam K.; Jesudason, Cynthia D.; Beck, Daniel E.; Clayton, Brent; Richardson, Timothy I.; Mesecar, Andrew D.; Medicine, School of Medicine
    Background: Alzheimer’s disease (AD) is a highly complex neurological disorder, with Late‐Onset AD (LOAD) being its most common form. INPP5D has been identified as a risk gene for AD and is involved in the TREM2 signaling pathway, which is crucial for microglial activity. INPP5D encodes SHIP1, a protein phosphatase that disrupts TREM2 signaling by converting PIP3 into PIP2, thereby inhibiting the PI3K‐mediated activation of Akt‐dependent signaling, which is essential for the clearance of amyloid oligomers, fibrils, and plaques. SHIP1 is a large, multidomain protein, and many aspects of its structure and function are poorly understood. Method: We have expressed, purified, and characterized the kinetic and biophysical properties of various domain constructs of SHIP1 to better understand the roles of individual domains. Ongoing work involves screening of inhibitors using a range of biochemical and biophysical assays with different constructs of SHIP1. Result: The response of different SHIP1 domain constructs with different substrates surprisingly revealed no significant differences in kinetic parameters between different domain constructs with the same substrate suggesting that the various domains surrounding the catalytic domain do not influence catalysis in solution. However, use of a designed chemical probe with a covalent warhead that targets SHIP1 allosterically between the catalytic and C2 domains shows significant inhibition of SHIP1 (in the absence of its SH2 domain) identifying a potential druggable site. X‐ray crystallography was used to confirm the binding pose within this site. Binding affinity with additional compounds has been determined for different domain constructs using enzyme kinetics and biophysical methods including Microscale Thermophoresis (MST) and Differential Scanning Fluorescence (DSF). Conclusion: SHIP1 is highly active in vitro (solution) without much regulation of its catalytic activity by surrounding domains. A potential druggable site has been identified between the SHIP1 catalytic and C2 domains that can be targeted allosterically by small molecule compounds. These discoveries will aid in identifying new molecules that can inhibit SHIP1 as a potential therapeutic target for AD.
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    SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease
    (Wiley, 2023) Jesudason, Cynthia D.; Mason, Emily R.; Chu, Shaoyou; Oblak, Adrian L.; Javens-Wolfe, June; Moussaif, Mustapha; Durst, Greg; Hipskind, Philip; Beck, Daniel E.; Dong, Jiajun; Amarasinghe, Ovini; Zhang, Zhong-Yin; Hamdani, Adam K.; Singhal, Kratika; Mesecar, Andrew D.; Souza, Sarah; Jacobson, Marlene; Di Salvo, Jerry; Soni, Disha M.; Kandasamy, Murugesh; Masters, Andrea R.; Quinney, Sara K.; Doolen, Suzanne; Huhe, Hasi; Sukoff Rizzo, Stacey J.; Lamb, Bruce T.; Palkowitz, Alan D.; Richardson, Timothy I.; Medicine, School of Medicine
    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. Discussion: 3-((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-containing 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.