Browsing by Author "Moussaif, Mustapha"

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    Generation and validation of anti‐TREM2 agonistic antibodies to enable the advancement of drug targets in the TREM2/DAP12 signaling pathway for the treatment of Alzheimer Disease
    (Wiley, 2025-01-09) Moussaif, Mustapha; Javens-Wolfe, June; Palkowitz, Alan D.; Richardson, Timothy I.; Pharmacology and Toxicology, School of Medicine
    Background: TREM2 signaling has been implicated in Alzheimer’s Disease (AD). TREM2 regulates microglial states and functions such as phagocytosis. The most prominent TREM signaling adapter is DAP12, encoded by TYROBP. Understanding functional changes of this complex, and downstream effectors such as SHIP1, PLCG2 and the Scr family kinases Lyn and Hck, is required to evaluate a broad range of therapeutic hypotheses and drug targets for prioritization and enablement. The lack of available, well validated, and openly distributed experimental tools can limit early drug discovery efforts. Therefore, the IUSM Purdue TREAT‐AD Center has generated and validated TREM2 activating antibodies to enable the advancement of drug targets in the TREM2/DAP12 signaling pathway. Method: To establish and validate anti‐TREM2 agonist antibodies, heavy and light chain variable sequences were identified from multiple publications including patent applications. Antibodies were formatted as either human IgG1, Fc null mutant IgG1 or antibody transport vehicle (ATV) Fc null mutant IgG1. They were expressed in mammalian ExpiCHO cells and tested ex vivo for agonism based on their ability to activate AKT and Syk phosphorylation in THP1 cells and TREM2/DAP12 overexpressing cells respectively. The strongest agonistic candidate was scaled, purified, and further characterized biophysically and functionally. Result: Several agonistic antibodies were identified. AL2p31 antibody showed binding specificity to human versus murine TREM2. Biophysical characterization using biolayer interferometry showed that binding kinetic parameters (KD, Kon, and Koff) were not significantly affected in LALAPG null mutant Fc background. AL2p31 specifically induced Syk phosphorylation in comparison to an isotype control. Analysis of antibodies formatted as bispecific IgG1 targeting both TREM2 and the human transferrin receptor (hTfR), confirmed that RS9‐F6 can bind both human and murine TREM2 and revealed the ATV 35‐21‐16 variant sequence as a binder for the hTfR. Conclusion: The mission of the IUSM Purdue TREAT‐AD Center is to enable and advance the next generation of drug targets for the treatment of AD. The validation of anti‐TREM2 agonistic antibodies as research tools will enable comprehensive studies of the TREM2/DAP12 signaling and potential drug targets within the pathway including SHIP1, PLCG2 and the Scr family kinase Lyn and Hck.
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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.