title: Rapid screening of HIV reverse transcriptase and integrase inhibitors.
abstract: Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains. The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors. This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.
authors.affiliation: HIV Drug Resistance Program, National Cancer Institute.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Smith
authors.affiliation: HIV Drug Resistance Program, National Cancer Institute; hughesst@mail.nih.gov.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Hughes
mesh_terms: D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057809:HEK293 Cells; D019427:HIV Integrase / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology; D006801:Humans; D020128:Inhibitory Concentration 50; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology* / Q000633:toxicity
chemical_list: D019428:HIV Integrase Inhibitors; D018894:Reverse Transcriptase Inhibitors; D019427:HIV Integrase; D054303:HIV Reverse Transcriptase
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