Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions.
The kinetic parameters governing the inhibition by Nevirapine of the RNA-dependent DNA synthesis catalyzed by HIV-1 reverse transcriptase have been determined by steady-state kinetic analysis with the wild-type enzyme and with mutant reverse transcriptases containing the single amino acid substitutions L100I, K103N, V106A, V179D, Y181I and Y188L. While the mutant V179D was inhibited by Nevirapine as the wild-type enzyme, all the other mutations displayed a 17 to 90-fold reduced sensitivity to the drug in the order: Y181I<(i.e. less sensitive) Y188L < V106A < L100I < K103N < wild-type. Determination of the rate constants for Nevirapine binding (kon) and dissociation (koff) for the mutant and wild-type enzymes showed that mutations L100I and V106A increased the koff values by 12 and 8.5-fold, respectively, without significantly affecting the kon, whereas mutation K103N decreased the kon 5-fold without increasing the koff. Mutations Y181I and Y188L, on the other hand, conferred resistance to Nevirapine affecting both koff and kon values. In addition, mutations L100I and Y181I reduced the catalytic potential of HIV-1 RT. Thus, Nevirapine resistance could arise from a combination of loss of stabilizing interactions and emergence of steric and thermodynamic barriers for drug binding, depending on the particular amino acid substitution involved.
Maga
G
G
Institute of Biochemical and Evolutionary Genetics I.G.B.E.-C.N.R., Pavia, I-27100, Italy.
Amacker
M
M
Ruel
N
N
Hübscher
U
U
Spadari
S
S
D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000502:physiology; D019829:Nevirapine / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics
D019380:Anti-HIV Agents; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; D054303:HIV Reverse Transcriptase