KINETICS OF DIFFERENT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE TRANSCRIPTASES RESISTANT TO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE 1-SPECIFIC REVERSE-TRANSCRIPTASE INHIBITORS

The human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors [tetrahydroimidazo[4,5,1 -jk] [1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)thymine, nevirapine, pyridinone, bis(heteroaryl)piperazine, etc.] are potent inhibitors of HIV-1 replication in cell culture. The rapid emergence of drug-resistant escape mutants in vitro (cell culture) and in vivo (patients) is predominantly linked to the Y181C mutation. Because amino acids Y181 and Y188 appear to be located within the drug binding site of the enzyme, we studied the impact of mutations of both amino acids on the enzyme kinetics and on the susceptibility of the enzyme to different HIV-1 -specific RT inhibitors. Mutations Y181C, Y181I, and Y188L led to reduced sensitivity, albeit of varying extents, to all HIV-1-specific RT inhibitors. No resistance was observed to 2',3'-dideoxyguanosine 5'-triphosphate or phosphonoformic acid. The k(cat) of the Y181C mutant was similar to that of the wild-type RT (18 sec-1 X 10(-3)). The catalytic constant of the Y181I mutant was 6-fold higher and that of the Y188L mutant was 6-fold lower. Whereas TIBO displayed a linear mixed-type (noncompetitive) inhibition with respect to the deoxynucleotide substrate when wild-type p66/p51 was used, the pattern of inhibition became competitive or uncompetitive with Y181C or Y181I, respectively. Thus, the TIBO binding site of HIV-1 RT seems to be functionally and/or spatially related to the natural deoxynucleoside triphosphate binding site.