title: Simple and rapid determination of the enzyme kinetics of HIV-1 reverse transcriptase and anti-HIV-1 agents by a fluorescence based method.
abstract: Highly sensitive fluorescence dye, PicoGreen, can detect a small amount of polynucleotide duplex. The DNA/RNA duplex is a reaction product of HIV-1 reverse transcriptase (HIV-RT), which demonstrated that the HIV-1 RT kinetics and anti-HIV agent efficiency could be determined rapidly by a fluorometric method using PicoGreen dye. A small amount of dye is enough to test a minimal quantity of substrate to rapidly determine the enzyme kinetic properties K(m) and V(max). This method was applied to screen HIV-RT inhibitors. The enzymatic inhibition of 11 dipyridodiazepinone derivative inhibitors was compared. The IC(50) of all compounds was determined and it was found that two showed high inhibition efficiency. In addition, this method is applied for screening of many natural agents belonging to diverse sources. The proposed fluorometric method using PicoGreen dye is an easy, rapid, and sensitive detection method to determine HIV-RT activity, monitor enzyme kinetics and perform high-throughput screening of anti-HIV-1 RT agents.
authors.affiliation: Interdisciplinary Graduated Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkean, Bangkok 10900, Thailand.
authors.forename: Kun
authors.initials: K
authors.lastname: Silprasit
authors.forename: Ratsupa
authors.initials: R
authors.lastname: Thammaporn
authors.forename: Supanna
authors.initials: S
authors.lastname: Tecchasakul
authors.forename: Supa
authors.initials: S
authors.lastname: Hannongbua
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D009930:Organic Chemicals; C099476:PicoGreen; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase