title: High throughput drug screening for human immunodeficiency virus type 1 reactivating compounds.
abstract: The ability of human immunodeficiency virus type 1 (HIV-1) to persist in a latent stage in memory T cells in the presence of antiretroviral therapy poses a major obstacle to the development of an HIV-1 therapy with curative intent. As latently infected cells are phenotypically not distinguishable from uninfected cells, therapeutic reactivation of the latent infection, followed by the death of the host cell induced by viral cytopathicity, is considered the only means to eliminate this viral reservoir. To identify compounds with the potential to reactivate latent HIV-1, we have developed a series of latently HIV-1-infected reporter cell lines that allow for high throughput drug screening (HTS) in a 384-well plate-based format. The latent reporter cell lines use enhanced green fluorescence protein (eGFP) as a direct and quantitative marker of HIV-1 expression. To aid identification of specific compounds, the cells are engineered to constitutively express a second, red fluorescent protein that has no spectral overlap with eGFP, which allows for the simultaneous quantification of cell viability (inversely correlated to compound toxicity). Thus, these reporters enable prioritization of compounds most likely to have a favorable therapeutic window. The high dynamic signal range and the excellent reproducibility of the primary screening assay result in a Z' -factor of 0.89, which characterizes the HTS system as very robust. The assay has been implemented for automated drug screening, and we here discuss the advantages and limitations of the HTS system based on the data obtained for 1,600 compounds during a limited proof-of-concept drug screen.
authors.affiliation: Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jones
authors.forename: John
authors.initials: J
authors.lastname: Rodgers
authors.forename: Marintha
authors.initials: M
authors.lastname: Heil
authors.forename: Jori
authors.initials: J
authors.lastname: May
authors.forename: Lucile
authors.initials: L
authors.lastname: White
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Maddry
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Fletcher
authors.forename: George M
authors.initials: GM
authors.lastname: Shaw
authors.forename: John L
authors.initials: JL
authors.lastname: Hartman
authors.forename: Olaf
authors.initials: O
authors.lastname: Kutsch
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D005470:Fluorometry; D017930:Genes, Reporter / Q000235:genetics; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D007202:Indicators and Reagents; D010957:Plasmids / Q000235:genetics; D014161:Transduction, Genetic; D014779:Virus Replication / Q000187:drug effects
chemical_list: D019380:Anti-HIV Agents; D005819:Genetic Markers; D016655:HIV Core Protein p24; D007202:Indicators and Reagents; D049452:Green Fluorescent Proteins