Primer extension analysis of the inhibition of purified HIV-1 reverse transcriptase by the TIBO analog, R82150.

Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.

Primer extension analysis of the inhibition of purified HIV-1 reverse transcriptase by the TIBO analog, R82150.

Abstr Annu Meet Am Soc Microbiol. 1991 May 5-9;91:335 (abstract no. T-10). Unique Identifier : AIDSLINE ASM91/0230292
Noll GJ; Frank KB; Connell EV; Sim IS; Roche Res. Ctr., Nutley, NJ.

Abstract: Recent studies have indicated that members of the TIBO (tetrahydro-imidazo [4,5,1-jk] [1,4]-benzodiazepin-2 (1H)-thione) family of compounds inhibit HIV-1 RT. We have used primer extension analysis to study the mechanism of action of one derivative. An in vitro transcript was made from a DNA construct containing the HIV-1 primer binding site, hybridized to a 5'-labeled primer and extended by HIV-1 RT in the presence of R82150 or ddCTP. Products were separated on a sequencing gel. ddCTP resulted in dose dependent chain termination at cytidines. However, R82150 gave products not terminated at any one nucleotide, but at various sites corresponding to random pause sites of HIV-1 RT. Using a cordycepin-terminated primer as competitor with our 5'-labeled primer, products banded in a PAGE pattern unlike the R82150 lanes, suggesting that the TIBO analog does not inhibit chain initiation. In parallel kinetic studies R82150 was non-competitive against all substrates examined. Human DNA polymerases alpha, beta and gamma were not inhibited. Our results suggest that R82150 does not act as a substrate analog of HIV-1 RT, but may bind as an allosteric inhibitor at a unique site of this replicase.

Keywords: Antiviral Agents/*PHARMACOLOGY Benzodiazepines/*PHARMACOLOGY DNA Polymerases/ANTAGONISTS & INHIB DNA, Viral/GENETICS Electrophoresis, Polyacrylamide Gel HIV-1/*ENZYMOLOGY Imidazoles/*PHARMACOLOGY Nucleic Acid Hybridization RNA-Directed DNA Polymerase/*ANTAGONISTS & INHIB/METABOLISM Substrate Specificity Zalcitabine/PHARMACOLOGY ABSTRACT
920830
M9281060

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