title: FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein.
abstract: During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: Kamal K
authors.initials: KK
authors.lastname: Sharma
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Przybilla
authors.affiliation: Institute für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France Département d'Information Médicale et de Biostatistiques, Hôpitaux Universitaires de Strasbourg, 1, pl de l'Hôpital, 67400 Strasbourg, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Godet
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France yves.mely@unistra.fr.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019829:Nevirapine / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D015215:Zidovudine / Q000494:pharmacology*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054301:gag Gene Products, Human Immunodeficiency Virus; D015215:Zidovudine; D019829:Nevirapine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase