Browsing by Author "Palkowitz, Alan D."

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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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    Identification of PLCG2 activators for the treatment of Alzheimer’s disease
    (Wiley, 2025-01-09) Clayton, Brent; Massey, Steven M.; Beck, Daniel E.; Putt, Karson S.; Utsuki, Tada; Visvanathan, Ramya; Mesecar, Andrew D.; Lendy, Emma K.; Kaiser, Bridget L.; Chu, Shaoyou; Mason, Emily R.; Lamb, Bruce T.; Palkowitz, Alan D.; Richardson, Timothy I.; Pharmacology and Toxicology, School of Medicine
    Background: The goal of the TREAT‐AD Center is to enable drug discovery by developing assays and providing tool compounds for novel and emerging targets. The role of microglia in neuroinflammation has been implicated in the pathogenesis of Alzheimer’s disease (AD). Genome‐wide association studies, whole genome sequencing, and gene‐expression network analyses comparing normal to AD brain have identified risk and protective variants in genes essential to microglial function. among them. The P522R variant of phospholipase C gamma2 (PLCγ2) is associated with reduced risk for AD and has been characterized as a functional hypermorph. Carriers of P522R with mild cognitive impairment exhibited a slower cognitive decline rate. Conversely the M28L variant increases risk. Therefore, activation of the protein PLCγ2 with small molecules has been proposed as a therapeutic strategy to reduce the rate of disease progression and cognitive decline in AD patients. Method: We performed a high‐throughput screen using affinity selection mass spectrometry (ASMS) to identify novel small molecules that bind to the full‐length protein PLCγ2. A Cellular Thermal Shift Assay (CETSA) was developed to confirm target engagement in cells. A liposomal‐based, fluorogenic reporter biochemical assay was implemented to evaluate activity of the enzyme. A high‐content imaging assay measuring phagocytosis, cell number, and nuclear intensity was carried out using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Structure activity relationship (SAR) studies were performed to synthesize analogs and optimize for binding and cellular pharmacology. Optimized compounds have been studied in vivo to assess pharmacokinetic properties and drug likeness. Result: Novel PLCγ2 activators have been discovered and preliminary optimization has been completed. These compounds have shown positive results for target engagement, biochemical activity, and cellular pharmacology. In silico predictions indicated the molecule structures are suitable CNS drug discovery program starting points. Conclusion: Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds capable of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead‐like molecules for clinical studies will be made.
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    IUSM‐Purdue TREAT‐AD Center Capabilities and Strategy to Enable and Advance Novel Therapeutic Targets for the Treatment of Alzheimer’s Disease
    (Wiley, 2025-01-09) Mowery, Stephanie A.; Huang, Kun; Mesecar, Andrew D.; Dage, Jeffrey L.; Clayton, Brent; Lamb, Bruce T.; Palkowitz, Alan D.; Richardson, Timothy I.; Neurology, School of Medicine
    Background: The TaRget Enablement to Accelerate Therapy Development of Alzheimer’s Disease (TREAT‐AD) Centers are dedicated to identifying and validating targets from the NIH Accelerating Medicines Partnership for Alzheimer’s Disease (AMP‐AD). The centers develop Target Enabling Packages (TEPs) to explore new therapeutic target hypotheses, moving beyond the traditional focus on amyloid or tau pathologies. In accordance with open science principles, data, methods, and tools are freely shared with the research community via an open‐access platform, the AD Knowledge Portal. The Indiana University School of Medicine and Purdue University TREAT‐AD (IUSM Purdue TREAT‐AD) Center comprises four technical cores: Bioinformatics and Computational Biology (BCB), Structural Biology and Biophysics Core (SBB), Assay Development and High Throughput Screening (ADHTS), and Medicinal Chemistry and Chemical Biology (MCCB). These cores collaborate to develop research tools that are used to validate biological targets and assess their druggability with an initial focus on understanding the role of neuroinflammation in AD. Method: The BCB Core supports target selection and validation with data and analysis. The SBB Core provides proteins for assay development, biophysical assays, and structural studies to aid in mode of action and Structure Activity Relationship (SAR) studies. The ADHTS Core develops in vitro and in vivo assays for SAR studies and translational PD biomarker strategies to assist in determining early phase clinical dosing regimens. The MCCB Core selects therapeutic modalities (small molecules, antibodies, siRNA) and discovers pharmacological tools, employing strategies for SAR studies to balance pharmacological and drug‐like properties. Result: Target Enabling Packages (TEPs) are now available via the AD Knowledge Portal for microglia targets that were prioritized for early drug discovery studies. TEPs include bioinformatics analysis, biological reagents and protocols, protein production methods, and recommended chemical probes with detailed information (Figure 1). Novel small molecule hits and leads were identified for SHIP1, PLCG2, SHP1 and LYN/HCK. Conclusion: A pipeline of prioritized microglia targets were selected and enabled for early drug discovery. The IUSM Purdue TREAT‐AD Center is now working with AMP‐AD researchers to explore biological hypothesis in addition to the role of neuroinflammation in 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.