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.

SJP-L-5 inhibits HIV-1 polypurine tract primed plus-strand DNA elongation, indicating viral DNA synthesis initiation at multiple sites under drug pressure

“…These results demonstrate that SJP-L-5 blocks HIV-1 infection through RT inhibition, although it does not work early in this process. This mechanism is quite different from those previously reported since all FDA-approved NNRTIs target the RDDP activity while SJP-L-5 inhibits the DDDP activity 6 , 17 .…”

“…HIV-1 RTase DDDP activity was measured via fluorescent resonance energy transfer (FRET) assay as previously described, with minor modifications 17 . The DNA template (5′-AF488-AGT CCC CCC TTT TCT TTT AAA AAG TGG CTA AGA), and the DNA primer (PPT-based) (5′-TTA AAA GAA AAG GGG GG) were synthesized by Sangon Biotech (China).…”

Formation of a Quaternary Complex of HIV-1 Reverse Transcriptase with a Nucleotide-competing Inhibitor and Its ATP Enhancer

“…200 nM of nucleotide mix containing dATP, dCTP, dGTP, and a modified dUTP were added along with INDOPY-1 (0 -30 M). The modified dUTP contains an Alexa555 quencher, which upon incorporation quenches excitation by Alexa488 present on the DNA template (23). The signal was monitored over a period of 60 min using SPECTRAmax M5 fluorometer where reductions in Alexa488 emission signals are indicative of increased DNA polymerization.…”

Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

“…There are many applications where knowledge of the chemical structure of a dye is not necessary, e.g., when using Alexa Fluor 555 in imaging [33][34][35][36][37][38][39][40][41] and spectroscopic studies [42][43][44][45] . However, Alexa Fluor 555 and Alexa Fluor 647 are dyes that are frequently and successfully used for single-molecule FRET in combination with other fluorophores [46][47][48][49][50] , or as a donor-acceptor pair [51][52][53][54][55][56] . They have become a popular choice because of their favorable performance in many assays, and this is largely due to their high water solubility and the absence of strong (unwanted) interactions between dye and target following conjugation.…”