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Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening.

ABSTRACT: Assessing the potential health risks of environmental chemical compounds is an expensive undertaking that has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating the mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.

SUBMITTER: Huang R

PROVIDER: S-EPMC2668196 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening.

Huang Ruili R Southall Noel N Cho Ming-Hsuang MH Xia Menghang M Inglese James J Austin Christopher P CP

Chemical research in toxicology 20080219 3

Assessing the potential health risks of environmental chemical compounds is an expensive undertaking that has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate ...[more]

PMID: 18281954

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Project description:A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d'Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM-46.0?M) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of genetic diversity are now feasible through the use of human lymphoblast cell lines.

Project description:CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

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Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening.

Publications

Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening.

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