title: Comparison of 3 cytotoxicity screening assays and their application to the selection of novel antibacterial hits.
abstract: 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), intracellular adenosine triphosphate (CellTiter-Glo), and reduction of resazurin (CellTiter-Blue) both as indicators of cell metabolic activity. In the CellTiter-Glo and the CellTiter-Blue assays, 7 of 8 selected compounds showed cytotoxicity, whereas in the Toxilight assay, 3 of 8 compounds significantly reduced cell viability in the ChoK1 and the JurkatE6.1 cell line. The CellTiter-Glo 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 assay was clearly superior to the CellTiter-Blue and the Toxilight assay for the initial cytotoxicity screening. Moreover, the application of the CellTiter-Glo 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.
authors.affiliation: Lek Pharmaceuticals d.d., Ljubljana, Slovenia. luka.peternel@sandoz.com
authors.forename: Luka
authors.initials: L
authors.lastname: Peternel
authors.forename: Miha
authors.initials: M
authors.lastname: Kotnik
authors.forename: Andrej
authors.initials: A
authors.lastname: Prezelj
authors.forename: Uros
authors.initials: U
authors.lastname: Urleb
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003603:Cytotoxins / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008826:Microbial Sensitivity Tests / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects; D008954:Models, Biological; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D003603:Cytotoxins