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. (Journal of Biomolecular Screening 2009:142-150)

Keywords

cytotoxicity screening assay ATP antibacterial hits safety margins

References

Greenwood D. : Antimicrobial Chemotherapy. 4th online ed. Oxford, UK: Oxford University Press, 2000.

Green DW : The bacterial cell wall as a source of antibacterial targets. Expert Opin Ther Targets 2002;6:1-19.

Leeds JA , Dean CR : Peptide deformylase as an antibacterial target: a critical assessment . Curr Opin Pharmacol 2006;6:445-452.

Comartin DJ , Brown ED : Non-ribosomal factors in ribosome subunit assembly are emerging targets for new antibacterial drugs. Current Opin Pharmacol 2006;6:453-458.

Fernandes P. : Antibacterial discovery and development: the failure of success? Nat Biotechnol 2006;24:1497-1503.

Kramer JA , Sagartz JE , Morris DL : The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates. Nat Rev Drug Discov 2007;6:636-649.

Kola I. , Landis J. : Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 2004;3:711-715.

Bugelski PJ , Atif A. , Molton S. , Toeg I. , Lord PG , Morgan DG : A strategy for primary high throughput cytotoxicity screening in pharmaceutical toxicology. Pharm Res 2000;17:1265-1275.

Slater K. : Cytotoxicity tests for high-throughput drug discovery. Curr Opin Biotechnol 2001;12:70-74.

10.

Sepp A. , Binns RM , Lechler RI : Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity. J Immunol Methods 1996;196: 175-180.

11.

Olsson T. , Gulliksson H. , Palmeborn M. , Bergström K. , Thore A. : Leakage of adenylate kinase from stored blood cells. J Appl Biochem 1983;5:437-445.

12.

Wang XM , Terasaki PI , Rankin GW , Chia D. , Zhong HP , Hardy S. : A new microcellular cytotoxicity test based on calcein AM release . Hum Immunol 1993;37:264-270.

13.

Hamid R. , Rotshteyn Y. , Rabadi L. , Parikh R. , Bullock P. : Comparison of alamar blue and MTT assays for high through-put screening . Toxicol In Vitro 2004;18:703-710.

14.

O'Brien J. , Wilson I. , Orton T. , Pognan F. : Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267:5421-5426.

15.

Tanke HJ , van der Linden PW , Langerak J. : Alternative fluorochromes to ethidium bromide for automated read out of cytotoxicity tests. J Immunol Methods 1982 ;52:91-96.

16.

Crouch SP , Kozlowski R. , Slater KJ , Fletcher J. : The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity . J Immunol Methods 1993;160:81-88.

17.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

18.

Clinical and Laboratory Standards Institute (CLSI) : Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Approved Standard -7th ed., vol. 26. Wayne, PA: CLSI, 2006.

19.

Petinari L. , Kohn LK , de Carvalho JE , Genari SC : Cytotoxicity of tamoxifen in normal and tumoral cell lines and its ability to induce cellular transformation in vitro. Cell Biol Int 2004;28:531-539.

20.

Williams DP , Pirmohamed M. , Naisbitt DJ , Maggs JL , Park BK : Neutrophil cytotoxicity of the chemically reactive metabolite(s) of clozapine: possible role in agranulocytosis. J Pharmacol Exp Ther 1997;283:1375-1382.

21.

Hewitt NJ , Lloyd S. , Hayden M. , Butler R. , Sakai Y. , Springer R. , et al: Correlation between troglitazone cytotoxicity and drug metabolic enzyme activities in cryopreserved human hepatocytes. Chem Biol Interact 2002; 142:73-82.

22.

Munyon WH , Salo R. , Briones DF : Cytotoxic effects of neuroleptic drugs. Psychopharmacology 1987;91:182-188.

23.

Otto M. , Hansen SH , Dalgaard L. , Dubois J. , Badolo L. : Development of an in vitro assay for the investigation of metabolism-induced drug hepatotoxicity.Cell Biol Toxicol 2008 ;24:87-99.

24.

Smith GJ , Grisham JW : Cytotoxicity of monofunctional alkylating agents: methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells. Mutat Res 1983;111:405-417.

25.

Tostmann A. , Boeree MJ , Peters WH , Roelofs HM , Aarnoutse RE , van der Ven AJ , et al: Isoniazid and its toxic metabolite hydrazine induce in vitro pyrazinamide toxicity. Int J Antimicrob Agents 2008;31:577-580.

26.

Hinson JA , Reid AB , McCullough SS , James LP : Acetaminophen-induced hepatotoxicity: role of metabolic activation, reactive oxygen/nitrogen species, and mitochondrial permeability transition . Drug Metab Rev 2004;36:805-822.

27.

Xia M. , Huang R. , Witt KL , Southall N. , Fostel J. , Cho MH , et al: Compound cytotoxicity profiling using quantitative high-throughput screening. Environ Health Perspect 2008;116:284-291.

28.

Schoonen WG , de Roos JA , Westerink WM , Débiton E. : Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells: II. Mechanistic assays on NAD(P)H, ATP and DNA contents. Toxicol In Vitro 2005;19:491-503.

29.

Inglese J. , Auld DS , Jadhav A. , Johnson RL , Simeonov A. , Yasgar A. , et al: Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries . Proc Natl Acad Sci USA 2006;103:11473-11478.

30.

Niles AL , Moravec RA , Riss TL : Multiplex caspase activity and cytotoxicity assays. Methods Mol Biol 2008;414:151-162.