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Comparison of 3 Cytotoxicity Screening Assays and Their Application to the Selection of Novel Antibacterial Hits

Lek Pharmaceuticals d.d., Ljubljana, Slovenia, luka.peternel{at}sandoz.com

Miha Kotnik

Lek Pharmaceuticals d.d., Ljubljana, Slovenia

Andrej Preelj

Lek Pharmaceuticals d.d., Ljubljana, Slovenia

Uro Urleb

Lek Pharmaceuticals d.d., Ljubljana, Slovenia

Cytotoxicity screening of new chemical entities in antibacterial drug discovery discerns between cytotoxic and antimicrobial activity, thus providing predictive evidence for selective toxicity. The objective of this study was to evaluate 3 cytotoxicity assays in identifying novel antibacterial hits with desired safety margins. The endpoints in assays comprised adenylate kinase (AK) release rate as an indicator of membrane rupture (Toxilight^TM), intracellular adenosine triphosphate (CellTiter-Glo^TM), and reduction of resazurin (CellTiter-Blue^TM) both as indicators of cell metabolic activity. In the CellTiter-Glo^TM and the CellTiter-Blue^TM assays, 7 of 8 selected compounds showed cytotoxicity, whereas in the Toxilight^TM assay, 3 of 8 compounds significantly reduced cell viability in the ChoK1 and the JurkatE6.1 cell line. The CellTiter-Glo^TM assay proved to be the most sensitive among the evaluated assays, and excellent Z' values were obtained in the 96-well plate (Z' > 0.83). The CellTiter-Glo^TM assay was clearly superior to the CellTiter-Blue^TM and the Toxilight^TM assay for the initial cytotoxicity screening. Moreover, the application of the CellTiter-Glo^TM assay to determine mammalian cell toxicity versus the antibacterial effect ratio contributed to early identification of antibacterial hits with desired safety margins. The chemical structures of these novel antibacterial hits are disclosed herein. (Journal of Biomolecular Screening 2009:142-150)

Key Words: cytotoxicity screening assay • ATP • antibacterial hits • safety margins

This version was published on February 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 2, 142-150 (2009)
DOI: 10.1177/1087057108329452


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