Browsing by Author "Mesecar, Andrew D."
Now showing 1 - 7 of 7
Results Per Page
Sort Options
Item Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer(AACR Publications, 2016-09) Vickman, Renee E.; Crist, Scott A.; Kerian, Kevin; Eberlin, Livia; Cooks, R. Graham; Burcham, Grant N.; Buhman, Kimberly K.; Hu, Chang-Deng; Mesecar, Andrew D.; Cheng, Liang; Ratliff, Timothy L.; Pathology and Laboratory Medicine, School of MedicineCholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity.Item Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer(American Association for Cancer Research, 2019-06) Vickman, Renee E.; Yang, Jiang; Lanman, Nadia A.; Cresswell, Gregory M.; Zheng, Faye; Zhang, Chi; Doerge, R. W.; Crist, Scott A.; Mesecar, Andrew D.; Hu, Chang-Deng; Ratliff, Timothy L.; Medical and Molecular Genetics, School of MedicineCholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells is not understood. In these studies, single-cell mRNA sequencing (scRNA-seq) was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed (DE) genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases tumor necrosis factor alpha (TNF) expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4–2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further support this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including TNF, CD40LG, FADD, and NFKB1. Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer.Item Genetic Variants of Phospholipase C-γ2 Alter the Phenotype and Function of Microglia and Confer Differential Risk for Alzheimer’s Disease(Elsevier, 2023) Tsai, Andy P.; Dong, Chuanpeng; Lin, Peter Bor-Chian; Oblak, Adrian L.; Di Prisco, Gonzalo Viana; Wang, Nian; Hajicek, Nicole; Carr, Adam J.; Lendy, Emma K.; Hahn, Oliver; Atkins, Micaiah; Foltz, Aulden G.; Patel, Jheel; Xu, Guixiang; Moutinho, Miguel; Sondek, John; Zhang, Qisheng; Mesecar, Andrew D.; Liu, Yunlong; Atwood, Brady K.; Wyss-Coray, Tony; Nho, Kwangsik; Bissel, Stephanie J.; Lamb, Bruce T.; Landreth, Gary E.; Medical and Molecular Genetics, School of MedicineGenetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aβ clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.Item 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 MedicineBackground: 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.Item 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 MedicineBackground: 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.Item 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 MedicineBackground: 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.Item 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 MedicineIntroduction: 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.