title: EASY-HIT: HIV full-replication technology for broad discovery of multiple classes of HIV inhibitors.
abstract: HIV replication assays are important tools for HIV drug discovery efforts. Here, we present a full HIV replication system (EASY-HIT) for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed that measures HIV infection by two parameters relating to the early and the late phases of HIV replication, respectively. Validation of the assay with a panel of nine reference inhibitors yielded effective inhibitory concentrations consistent with published data and allowed discrimination between inhibitors of early and late phases of HIV replication. Finer resolution of the effects of reference drugs on different steps of HIV replication was achieved in secondary time-of-addition assays. The EASY-HIT assay yielded high Z' scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC(1280) library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors.
authors.affiliation: Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Kremb
authors.forename: Markus
authors.initials: M
authors.lastname: Helfer
authors.forename: Werner
authors.initials: W
authors.lastname: Heller
authors.forename: Dieter
authors.initials: D
authors.lastname: Hoffmann
authors.forename: Horst
authors.initials: H
authors.lastname: Wolff
authors.forename: Andrea
authors.initials: A
authors.lastname: Kleinschmidt
authors.forename: Sabine
authors.initials: S
authors.lastname: Cepok
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hemmer
authors.forename: Jörg
authors.initials: J
authors.lastname: Durner
authors.forename: Ruth
authors.initials: R
authors.lastname: Brack-Werner
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry; D006678:HIV / Q000187:drug effects*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results; D014779:Virus Replication / Q000187:drug effects
chemical_list: D019380:Anti-HIV Agents