Browsing by Author "Mason, Emily R."
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Item AD Informer Set: Chemical tools to facilitate Alzheimer's disease drug discovery(Wiley, 2022-04-20) Potjewyd, Frances M.; Annor-Gyamfi, Joel K.; Aubé, Jeffrey; Chu, Shaoyou; Conlon, Ivie L.; Frankowski, Kevin J.; Guduru, Shiva K.R.; Hardy, Brian P.; Hopkins, Megan D.; Kinoshita, Chizuru; Kireev, Dmitri B.; Mason, Emily R.; Moerk, Charles T.; Nwogbo, Felix; Pearce, Kenneth H.; Richardson, Timothy I.; Rogers, David A.; Soni, Disha M.; Stashko, Michael; Wang, Xiaodong; Wells, Carrow; Willson, Timothy M.; Frye, Stephen V.; Young, Jessica E.; Axtman, Alison D.; Medicine, School of MedicineIntroduction: The portfolio of novel targets to treat Alzheimer's disease (AD) has been enriched by the Accelerating Medicines Partnership Program for Alzheimer's Disease (AMP AD) program. Methods: Publicly available resources, such as literature and databases, enabled a data-driven effort to identify existing small molecule modulators for many protein products expressed by the genes nominated by AMP AD and suitable positive control compounds to be included in the set. Compounds contained within the set were manually selected and annotated with associated published, predicted, and/or experimental data. Results: We built an annotated set of 171 small molecule modulators targeting 98 unique proteins that have been nominated by AMP AD consortium members as novel targets for the treatment of AD. The majority of compounds included in the set are inhibitors. These small molecules vary in their quality and should be considered chemical tools that can be used in efforts to validate therapeutic hypotheses, but which will require further optimization. A physical copy of the AD Informer Set can be requested on the Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) website. Discussion: Small molecules that enable target validation are important tools for the translation of novel hypotheses into viable therapeutic strategies for AD.Item Differential Inhibition of Human Nav1.2 Resurgent and Persistent Sodium Currents by Cannabidiol and GS967(MDPI, 2020-04) Mason, Emily R.; Cummins, Theodore R.; Pharmacology and Toxicology, School of MedicineMany epilepsy patients are refractory to conventional antiepileptic drugs. Resurgent and persistent currents can be enhanced by epilepsy mutations in the Nav1.2 channel, but conventional antiepileptic drugs inhibit normal transient currents through these channels, along with aberrant resurgent and persistent currents that are enhanced by Nav1.2 epilepsy mutations. Pharmacotherapies that specifically target aberrant resurgent and/or persistent currents would likely have fewer unwanted side effects and be effective in many patients with refractory epilepsy. This study investigated the effects of cannbidiol (CBD) and GS967 (each at 1 μM) on transient, resurgent, and persistent currents in human embryonic kidney (HEK) cells stably expressing wild-type hNav1.2 channels. We found that CBD preferentially inhibits resurgent currents over transient currents in this paradigm; and that GS967 preferentially inhibits persistent currents over transient currents. Therefore, CBD and GS967 may represent a new class of more targeted and effective antiepileptic drugs.Item Epilepsy Mutations in Different Regions of the Nav1.2 Channel Cause Distinct Biophysical Effects(2020-06) Mason, Emily R.; Cummins, Theodore; Sullivan, William J., Jr.; Brustovetsky, Nickolay; Sheets, Patrick; Hashino, EriWhile most cases of epilepsy respond well to common antiepileptic drugs, many genetically-driven epilepsies are refractory to conventional antiepileptic drugs. Over 250 mutations in the Nav1.2 gene (SCN2A) have been implicated in otherwise idiopathic cases of epilepsy, many of which are refractory to traditional antiepileptic drugs. Few of these mutations have been studied in vitro to determine their biophysical effects on the channels, which could reveal why the effects of some are refractory to traditional antiepileptic drugs. The goal of this dissertation was to characterize multiple epilepsy mutations in the SCN2A gene, which I hypothesized would have distinct biophysical effects on the channel’s function. I used patch-clamp electrophysiology to determine the biophysical effects of three SCN2A epilepsy mutations (R1882Q, R853Q, and L835F). Wild-type (WT) or mutant human SCN2A cDNAs were expressed in human embryonic kidney (HEK) cells and subjected to a panel of electrophysiological assays. I predicted that the net effect of each of these mutations was enhancement of channel function; my results regarding the L835F and R1882Q mutations supported this hypothesis. Both mutations enhance persistent current, and R1882Q also impairs fast inactivation. However, examination of the same parameters for the R853Q mutation suggested a decrease of channel function. I hypothesized that the R853Q mutation creates a gating pore in the channel structure through which sodium leaks into the cell when the channel is in its resting conformation. This hypothesis was supported by electrophysiological data from Xenopus oocytes, which showed a significant voltage-dependent leak current at negative potentials when they expressed the R853Q mutant channels. This was absent in oocytes expressing WT channels. Overall, these results suggest that individual mutations in the SCN2A gene generate epilepsy via distinct biophysical effects that may require novel and/or tailored pharmacotherapies for effective management.Item Identification of Chemical Tool Compounds to Investigate the Role of Lyn Kinase in TREM2‐Mediated Microglia Activation and Phagocytosis(Wiley, 2025-01-09) Weerawarna, Pathum M.; Robo, Michael T.; Chu, Shaoyou; Mason, Emily R.; Davis, Chris; Angus, Steven P.; Richardson, Timothy I.; Medicine, School of MedicineBackground: Lyn kinase, a member of the Src family of tyrosine kinases, predominantly phosphorylates ITIM and ITAM motifs linked to immune receptors and adaptor proteins, and is emerging as a target for Alzheimer’s disease (AD). The role of Lyn in TREM2‐mediated microglial activation and phagocytosis, a critical pathway for clearing Aβ plaques, remains unclear and potent, selective, and brain penetrant Lyn inhibitors are unavailable. In this study, we report the characterization of Lyn kinase inhibitors from the literature as well as the establishment of an advanced virtual screening platform at the IUSM‐Purdue‐TREAT‐AD center to identify new type II Lyn inhibitors suitable as molecular probes. Method: We first performed a thorough literature survey and found 14 reported Lyn kinase inhibitors. We then validated their Lyn inhibitor activities and Lyn selectivities using the HotSpot kinase assay. We tested these compounds for microglia activation in a high‐content imaging assay using HMC3 (human) and BV2 (mouse) microglia‐like cell lines. We also performed kinome profiling in these cells to evaluate cellular target engagement and selectivity. Finally, we screened a million‐compounds using a computational pipeline that combined molecular docking, shape‐based screening, and MD simulations to identify novel and potent type II Lyn kinase inhibitors. Result: Our findings revealed that Type I inhibitors, particularly Saracatinib and Bosutinib, potently inhibit Lyn within the picomolar (pM) range. On the other hand, Type II inhibitors, such as Masitinib and Imatinib, displayed pronounced >20‐fold selectivity for Lyn over Hck with low nM Lyn inhibitor activities. Saracatinib and Bosutinib significantly induced phagocytosis in HMC3 cells, whereas Type II inhibitors demonstrated moderate activity in both HMC3 and BV2 cells. Our virtual screening platform identified a new type II Lyn inhibitor with picomolar activity and good Lyn/Hck selectivity. Conclusion: We have successfully evaluated previously reported inhibitors and introduced a novel type II Lyn kinase inhibitor with picomolar (pM) activities suitable for use as chemical probes to investigate the role of Lyn in TREM2‐mediated microglial activation.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 Inhibition of Lyn kinase: A novel approach to treatment of Alzheimer’s disease(Wiley, 2025-01-09) Benitah, Avi L.; Richardson, Timothy I.; Weerawarna, Pathum M.; Robo, Michael T.; Mason, Emily R.; Pharmacology and Toxicology, School of MedicineBackground: The TREAT‐AD centers aim to improve Alzheimer’s Disease (AD) research by offering free, high‐quality tools and technologies. Lyn is a tyrosine kinase that belongs to the Src family kinases. The expression of Lyn and its activity have been implicated in AD. This class of proteins is involved in TREM2 mediated microglial activation and phagocytosis, a process which is beneficial for clearing neurotoxins such as Aβ oligomers in the brain. Lyn inhibition may activate microglia. Given the relationship between accumulation of Aβ and its exacerbation of neurodegenerative diseases such as AD, selective inhibition of Lyn has been proposed as a novel therapeutic approach to treating early‐onset AD. However, potent, selective, and brain penetrant Lyn inhibitors are unavailable to test this hypothesis. Method: We screened a variety of known kinase inhibitors to determine their activity towards inhibition of Lyn using the biochemical HotSpot kinase assay. With this data in hand, we identified imatinib as a starting point for the design of novel Lyn inhibitors. Structure‐based design and computational docking models were used to propose more active and selective Lyn inhibitors, which were synthesized. The activities were determined, and multiple parameter optimization (MPO) informed iterative Structure Activity Relationship (SAR) studies. The best compounds were evaluated in assays of microglia activation, and their drug metabolism and pharmacokinetic (DMPK) properties were determined. Result: A series of novel type II inhibitors are now available for testing. The results demonstrate a unique tail group provides the novel scaffold with potent activity and selectivity towards inhibition of Lyn, exceeding that of imatinib. Conclusion: Computational models, SAR, and MPO provided potent and selective Lyn inhibitors with good DMPK properties. Further studies are under way to determine the impact of these compounds on TREM2 mediated activation of microglia both in vitro and in vivo.Item Microglial Phagocytosis/Cell Health High-Content Assay(Wiley, 2023) Mason, Emily R.; Soni, Disha M.; Chu, Shaoyou; Medicine, School of MedicineWe report a microglial phagocytosis/cell health high-content assay that has been used to test small molecule chemical probes and support our drug discovery projects targeting microglia for Alzheimer's disease therapy. The assay measures phagocytosis and cell health (cell count and nuclear intensity) simultaneously in 384-well plates processed with an automatic liquid handler. The mix-and-read live cell imaging assay is highly reproducible with capacity to meet drug discovery research needs. Assay procedures take 4 days including plating cells, treating cells, adding pHrodo-myelin/membrane debris to cells for phagocytosis, staining cell nuclei before performing high-content imaging, and analysis. Three selected parameters are measured from cells: 1) mean total fluorescence intensity per cell of pHrodo-myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (measuring compound effects on proliferation and cell death); and 3) average nuclear intensity (measuring compound induced apoptosis). The assay has been used on HMC3 cells (an immortalized human microglial cell line), BV2 cells (an immortalized mouse microglial cell line), and primary microglia isolated from mouse brains. Simultaneous measurements of phagocytosis and cell health allow for the distinction of compound effects on regulation of phagocytosis from cellular stress/toxicity related changes, a distinguishing feature of the assay. The combination of cell counts and nuclear intensity as indicators of cell health is also an effective way to measure cell stress and compound cytotoxicity, which may have broad applications as simultaneous profiling measurements for other phenotypic assays.Item Optimization of SHIP1 Inhibitors for the treatment of Alzheimer’s disease(Wiley, 2025-01-09) Jesudason, Cynthia D.; Lin, Peter Bor-Chian; Soni, Disha; Perkins, Bridget M.; Lee-Gosselin, Audrey; Ingraham, Cynthia M.; Hamilton, Will; Mason, Emily R.; El Jordi, Omar; Souza, Sarah; Jacobson, Marlene; Di Salvo, Jerry; Clayton, Brent; Chu, Shaoyou; Dage, Jeffrey L.; Oblak, Adrian L.; Richardson, Timothy I.; Neurology, School of MedicineBackground: SHIP1 is a phosphatidyl inositol phosphatase encoded by INPP5D, which has been identified as a risk gene for Alzheimer’s disease (AD). SHIP1 is expressed in microglia, the resident macrophage in brain. It is a complex, multidomain protein that acts as a negative regulator downstream from TREM2. SHIP1 possesses a phosphatase (Ptase) domain flanked by a pleckstrin‐homology (PH) domain that binds phosphatidylinositol (3,4,5)‐trisphosphate[PI(3,4,5)P3] and a C2 domain that binds phosphatidylinositol (3,4)‐bisphosphate [PI(3,4)P2]. The Ptase domain converts PI(3,4,5)P3 to PI(3,4)P2. SHIP1 also has an SH2 domain that binds to ITIMs and ITAMs where it competes with kinases. Inhibiting SHIP1 is hypothesized to have potential therapeutic benefits, as it may improve TREM2‐mediated microglial responses to neurotoxins and promote an overall neuroprotective microglial phenotype to maintain a more resilient brain and slow the rate of cognitive decline in AD patients. Method: The IUSM Purdue TREAT‐AD Center recently evaluated SHIP1 inhibitors and proposed 3‐((2,4‐Dichlorobenzyl)oxy)‐5‐(1‐(piperidin‐4‐yl)‐1H‐pyrazol‐4‐yl)pyridine for target validation studies. Structurally related analogs were synthesized and tested for SHIP1 enzyme inhibition, AKT signaling, and microglia activation in a high‐content imaging assay using HMC3 and BV2 microglia‐like cell lines. Primary microglia were treated with an optimized SHIP1 inhibitor, and subsequent changes in fibril Aβ uptake and cell viability were assessed. The NanoString nCounter Neuroinflammation assay was used to measure transcriptomic profiles. For comparison primary microglial derived from both wild‐type and Inpp5d‐haploinsufficient mice were assessed. Result: Novel SHIP1 inhibitors have been discovered and preliminary Structure Activity Relationship (SAR) studies have been completed. These compounds have shown positive results for biochemical activity, target engagement and cellular pharmacology. Both Inpp5d deficiency and pharmacological inhibition increase amyloid uptake and cell viability in primary microglia. Elevated ERK and AKT phosphorylation, after amyloid exposure, were decreased by Inpp5d deficiency. Functional pathways associated with phagocytosis, apoptosis, cytokine production, and complement system activity were altered. Conclusion: These data demonstrate that SHIP1 inhibition promotes amyloid uptake through the complement system. SHIP1 inhibition also enhances cell survival and homeostasis in primary microglia. Further studies of SHIP1 inhibition and INPP5D knockdown in animal models may provide a potential therapeutic strategy for Alzheimer’s disease.Item Resurgent and Gating Pore Currents Induced by De Novo SCN2A Epilepsy Mutations(Society for Neuroscience, 2019-10-16) Mason, Emily R.; Wu, Fenfen; Patel, Reesha R.; Xiao, Yucheng; Cannon, Stephen C.; Cummins, Theodore R.; Pharmacology and Toxicology, School of MedicineOver 150 mutations in the SCN2A gene, which encodes the neuronal Nav1.2 protein, have been implicated in human epilepsy cases. Of these, R1882Q and R853Q are two of the most commonly reported mutations. This study utilized voltage-clamp electrophysiology to characterize the biophysical effects of the R1882Q and R853Q mutations on the hNav1.2 channel, including their effects on resurgent current and gating pore current, which are not typically investigated in the study of Nav1.2 channel mutations. HEK cells transiently transfected with DNA encoding either wild-type (WT) or mutant hNav1.2 revealed that the R1882Q mutation induced a gain-of-function phenotype, including slowed fast inactivation, depolarization of the voltage dependence of inactivation, and increased persistent current. In this model system, the R853Q mutation primarily produced loss-of-function effects, including reduced transient current amplitude and density, hyperpolarization of the voltage dependence of inactivation, and decreased persistent current. The presence of a Navβ4 peptide (KKLITFILKKTREK-OH) in the pipette solution induced resurgent currents, which were increased by the R1882Q mutation and decreased by the R853Q mutation. Further study of the R853Q mutation in Xenopus oocytes indicated a reduced surface expression and revealed a robust gating pore current at negative membrane potentials, a function absent in the WT channel. This not only shows that different epileptogenic point mutations in hNav1.2 have distinct biophysical effects on the channel, but also illustrates that individual mutations can have complex consequences that are difficult to identify using conventional analyses. Distinct mutations may, therefore, require tailored pharmacotherapies in order to eliminate seizures.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.