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Development of a Rapid In Vitro Screening Assay Using Metabolic Inhibitors to Detect Highly Selective Anticancer Agents.

ABSTRACT: Traditional long exposure (24-72 h) cell viability assays for identification of potential drug compounds can fail to identify compounds that are: (a) biologically active but not toxic and (b) inactive without the addition of a synergistic additive. Herein, we report the development of a rapid (1-2 h) compound screening technique using a commercially available cell viability kit (CellTiter-Glo) that has led to the detection of compounds that were not identified as active agents using traditional cytotoxicity screening methods. These compounds, in combination with metabolic inhibitor 2-deoxyglucose, display selectivity toward a pancreatic cancer cell line. An evaluation of 11 mammalian cell lines against 30 novel compounds and two metabolic inhibitors is reported. The inclusion of metabolic inhibitors during an initial screening process, and not simply during mechanistic investigations of a previously identified hit compound, provides a rapid and sensitive tool for identifying drug candidates potentially overlooked by other methods.

SUBMITTER: Abebe FA

PROVIDER: S-EPMC8296616 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Development of a Rapid <i>In Vitro</i> Screening Assay Using Metabolic Inhibitors to Detect Highly Selective Anticancer Agents.

Abebe Felagot A FA Hopkins Megan D MD Vodnala Suraj N SN Sheaff Robert J RJ Lamar Angus A AA

ACS omega 20210702 28

Traditional long exposure (24-72 h) cell viability assays for identification of potential drug compounds can fail to identify compounds that are: (a) biologically active but not toxic and (b) inactive without the addition of a synergistic additive. Herein, we report the development of a rapid (1-2 h) compound screening technique using a commercially available cell viability kit (CellTiter-Glo) that has led to the detection of compounds that were not identified as active agents using traditional ...[more]

PMID: 34308064

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Project description:BackgroundInterpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin.MethodsSplenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 μM (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6.ResultsPhenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis.ConclusionUsing this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.

Dataset Information

Development of a Rapid In Vitro Screening Assay Using Metabolic Inhibitors to Detect Highly Selective Anticancer Agents.

Publications

Development of a Rapid <i>In Vitro</i> Screening Assay Using Metabolic Inhibitors to Detect Highly Selective Anticancer Agents.

Similar Datasets

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