Browsing by Author "Richardson, Timothy I."

Now showing 1 - 5 of 5
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    A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence
    (Taylor & Francis, 2024-01-18) Ruíz Moleón, Vera; Fotouhi, Maryam; Alende, Charles; Ayoubi, Riham; Bedford, Logan M.; Southern, Kathleen; Richardson, Timothy I.; Laflamme, Carl; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium; Pharmacology and Toxicology, School of Medicine
    Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
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    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 Medicine
    Introduction: 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.
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    Develop the Disease Specific Bioinformatics Platforms with Integrated Bioinformatics Data
    (2022-11) Liu, Jiannan; Yan, Jingwen; Zhang, Jie; Huang, Kun; Zhang, Chi; Richardson, Timothy I.; Wu, Huanmei
    With the advance of multiple types of omics technology and corresponding analytical methods, various type of bioinformatic data have become available. Mining and integrating these data for analysis will provide valuable insights for disease mechanism investigation, drug target identification and new drug development. However, most of the omics data are large size, heterogeneous, and complex, it is challenging for biomedical researchers to mine the data for relevant evidence, especially for those with limited computational skills. In this thesis, I aimed to develop disease specific platforms integrated with multimodal bioinformatic data types to provide researchers with strong bioinformatics support. To achieve this goal, I explored advanced transcriptomic data analytical methods and proposed a novel biomarker for the prediction of overall survival of colon cancer patients, then prototyped a user-friendly patient oriented clinical decision support system to provide accurate and intuitive colorectal cancer risk factor assessment. With the experience of the transcriptomic data analytical methods and the web-based application development, I further designed and implemented Cancer Gene and Pathway Explorer which is an integrative bioinformatics webserver that can be used for cancer publication trends investigation, gene set enrichment analysis with integrated data, and optimal cancer cell line identification. Based on the framework of CGPE, I developed another bioinformatics platform focusing on Alzheimer’s disease, called Alzheimer’s Disease Explorer, which is a first-of-its-kind bioinformatics server, providing rich bioinformatic support from literature, omics and chemical data to facilitate researchers in ND drug development field. By accomplishing a series of work in my thesis, I have shown that integrated disease specific bioinformatics platforms can provide great value to the research community by allowing 1.) fast and accurate investigation of currently available literature, 2.) quick hypothesis generation and validation using transcriptomic datasets, 3.) multi-dimension drug target evaluation and 4) fast querying of published bioinformatics outcomes.
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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.
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    Use of AD Informer Set compounds to explore validity of novel targets in Alzheimer's disease pathology
    (Wiley, 2022-04-12) 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., Jr.; 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 Medicine
    Introduction: A chemogenomic set of small molecules with annotated activities and implicated roles in Alzheimer's disease (AD) called the AD Informer Set was recently developed and made available to the AD research community: https://treatad.org/data-tools/ad-informer-set/. Methods: Small subsets of AD Informer Set compounds were selected for AD-relevant profiling. Nine compounds targeting proteins expressed by six AD-implicated genes prioritized for study by Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) teams were selected for G-protein coupled receptor (GPCR), amyloid beta (Aβ) and tau, and pharmacokinetic (PK) studies. Four non-overlapping compounds were analyzed in microglial cytotoxicity and phagocytosis assays. Results: The nine compounds targeting CAPN2, EPHX2, MDK, MerTK/FLT3, or SYK proteins were profiled in 46 to 47 primary GPCR binding assays. Human induced pluripotent stem cell (iPSC)-derived neurons were treated with the same nine compounds and secretion of Aβ peptides (Aβ40 and Aβ42) as well as levels of phosphophorylated tau (p-tau, Thr231) and total tau (t-tau) peptides measured at two concentrations and two timepoints. Finally, CD1 mice were dosed intravenously to determine preliminary PK and/or brain-specific penetrance values for these compounds. As a final cell-based study, a non-overlapping subset of four compounds was selected based on single-concentration screening for analysis of both cytotoxicity and phagocytosis in murine and human microglia cells. Discussion: We have demonstrated the utility of the AD Informer Set in the validation of novel AD hypotheses using biochemical, cellular (primary and immortalized), and in vivo studies. The selectivity for their primary targets versus essential GPCRs in the brain was established for our compounds. Statistical changes in tau, p-tau, Aβ40, and/or Aβ42 and blood-brain barrier penetrance were observed, solidifying the utility of specific compounds for AD. Single-concentration phagocytosis results were validated as predictive of dose-response findings. These studies established workflows, validated assays, and illuminated next steps for protein targets and compounds.