Dataset Information

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening.

ABSTRACT: Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. Many currently known MC activators are inadequate for in vivo applications, however, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by using high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistically evaluated using quality control rules and the B-score calculation, and compounds with B-scores of >3.0 were chosen as mast cell activators (hits). These hits were re-evaluated with secondary and tertiary HTS assays, followed by further statistical analysis. From these hits, we selected 15 compounds that caused degranulation in murine and human MCs, with potential for flexible chemical modification for further study. Among these 15 compounds, ST101036, ST029248, and ST026567 exhibited higher degranulation potency than other hit compounds in both human and mouse MCs. In addition, the 15 compounds identified promote de novo synthesis of cytokines and induce the release of eicosanoids from human and mouse MCs. HTS enabled us to identify small-molecule MC activators with unique properties that may be useful as vaccine adjuvants.

SUBMITTER: Choi HW

PROVIDER: S-EPMC8278568 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening.

Choi Hae Woong HW Chan Cliburn C Shterev Ivo D ID Lynch Heather E HE Robinette Taylor J TJ Johnson-Weaver Brandi T BT Shi Jianling J Sempowski Gregory D GD Kim So Young SY Dickson John K JK Gooden David M DM Abraham Soman N SN Staats Herman F HF

SLAS discovery : advancing life sciences R & D 20190327 6

Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. Many currently known MC activators are inadequate for in vivo applications, however, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by using high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistical ...[more]

PMID: 30917061

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Project description:Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

Dataset Information

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening.

Publications

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening.

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