title: A high-throughput continuous assay for screening and characterization of inhibitors of HIV reverse-transcriptase DNA polymerase activity.
abstract: The authors have devised a continuous fluorescence-based assay to measure HIV reverse transcriptase (RT) polymerase activity for both high-throughput screening (HTS) and mechanistic characterization of inhibitors. The designed substrate is composed of a recessed DNA primer annealed to a DNA template that is labeled at the 5'-terminus with a donor fluorophore (AlexaFluor 488). RT-catalyzed incorporation of an acceptor-labeled deoxyuridine (dUTP-AlexaFluor 555) at the 3'-terminus of the fully extended DNA primer juxtaposes donor and acceptor fluorophores, resulting in robust fluorescence resonance energy transfer that can be monitored kinetically in real time. The assay is sensitive, permitting the use of low enzyme concentrations (<0.5 nM), and can be miniaturized for use in 384-well HTS mode. The authors further show that this assay is capable of evaluating inhibitor mechanism of action by confirming the binding mechanism of a set of nonnucleoside RT inhibitors. Given the versatility and the lack of requirement for costly platforms or radioactivity, this assay may serve to accelerate and streamline the discovery and characterization process for future antiviral agents.
authors.affiliation: Department of In Vitro Sciences, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cauchon
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
authors.forename: Anick
authors.initials: A
authors.lastname: Auger
authors.forename: Roman A
authors.initials: RA
authors.lastname: Melnyk
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase