title: Development of a fluorescence-based HIV-1 integrase DNA binding assay for identification of novel HIV-1 integrase inhibitors.
abstract: Human immunodeficiency virus integrase (HIV-1 IN) inhibitors that are currently approved or are in advanced clinical trials specifically target the strand transfer step of integration. However, considerable cross-resistance exists among some members of this class of IN inhibitors. Intriguingly, though, HIV-1 IN possesses multiple sites, distinct from those involved in the strand transfer step, that could be targeted to develop new HIV-1 IN inhibitors. We have developed a fluorescent HIV-1 IN DNA binding assay that can identify small molecules termed IN binding inhibitors (INBIs) that inhibit IN binding to viral DNA. This assay has been optimized with respect to concentrations of each protein, long terminal repeat (LTR) DNA substrate, salt, and time, and has been used successfully to measure the HIV-1 IN DNA binding activity of a well-characterized INBI termed FZ41. In addition, we have used the assay to screen a small library of natural products, resulting in the identification of nigranoic acid as a new INBI. The proposed fluorescence assay is easy and inexpensive, and provides a high-throughput detection method for determination of HIV-1 IN DNA binding activity, monitoring of enzyme kinetics, and high-throughput screening for the identification of new INBIs.
authors.affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Ying-Shan
authors.initials: YS
authors.lastname: Han
authors.forename: Wei-Lie
authors.initials: WL
authors.lastname: Xiao
authors.forename: Peter K
authors.initials: PK
authors.lastname: Quashie
authors.forename: Thibault
authors.initials: T
authors.lastname: Mesplède
authors.forename: Hongtao
authors.initials: H
authors.lastname: Xu
authors.forename: Eric
authors.initials: E
authors.lastname: Deprez
authors.forename: Olivier
authors.initials: O
authors.lastname: Delelis
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Pu
authors.forename: Han-Dong
authors.initials: HD
authors.lastname: Sun
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wainberg
mesh_terms: D001667:Binding, Competitive; D004279:DNA, Viral / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029633:Schisandraceae / Q000378:metabolism; D012680:Sensitivity and Specificity; D012717:Sesquiterpenes / Q000737:chemistry; D014315:Triterpenes / Q000302:isolation & purification
chemical_list: D004279:DNA, Viral; D005456:Fluorescent Dyes; D019428:HIV Integrase Inhibitors; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D014315:Triterpenes; C100860:nigranoic acid; D019427:HIV Integrase