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1st International Workshop on HIV Drug Resistance & Treatment Strategies25-28 June 1997, St. Petersburg, Florida, USA |
SIGNIFICANCE OF THE 225 PRO→HIS MUTATION IN HIV-1 REVERSE TRANSCRIPTASE
Antiviral Therapy 1997;2 (Suppl 5):14 (abstract no. 21)
J Balzarini1, H Pelemans1, R Esnouf1, A Dunkler2, MA Parniak3, A-M Vandamme1, A Karlsson4, E De Clercq1 and J-P Kleim2
1Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; 2Hoechst AG, Central Pharma Research, D-65926 Frankfurt, Germany; 3Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec H3T 1E2, Canada; and 4Biochemical Laboratory, Karolinska Institute, S-171 77 Stockholm, Sweden
When HIV-1 (strain IIIB)-infected CEM cell cultures were exposed to low quinoxaline S-2720 concentrations (0.02 µg/ml) the 106 Val→Ala reverse transcriptase (RT) mutant HIV-1 strain emerged. Further exposure of this mutant virus strain to higher S-2720 concentrations (0.1 µg/ml or 0.25 µg/ml) resulted in the selection of mutant virus strains that consistently contained a 225 Pro→His mutation in addition to the 106 Val→Ala mutation. The 101 Lys→Ile and/or 181 Tyr→Cys mutation was further added to the 106 Val→Ala and 225 Pro→His genetic background under drug-escalating conditions. The double and triple mutant viruses proved highly resistant to several non-nucleoside RT inhibitors (NNRTIs) including MKC-442. Site-directed mutagenesis studies revealed that the contributions of the single 106 Val→Ala and 225 Pro→His mutations in the RT to the degree of resistance of RT to NNRTIs were additive as compared with the 106 Val→Ala plus 225 Pro→His double RT mutant. Interestingly, BHAP U90152 had an eightfold higher inhibitory effect on the 225 Pro→His RT mutant enzyme than wild-type RT, whereas other NNRTIs showed diminished inhibition of the 225 Pro→His RT mutant. These data are consistent with the structure of the RT-BHAP U90152 complex, in which the inhibitor forces the main chain of Pro 225 to swing around by approximately 180°, leaving Pro 225 exposed on the surface of the enzyme at the edge of the polymerase active site binding cleft. A change from Pro to the more hydrophilic (His) residue is likely to stabilize the RT-BHAP complex, which would then explain the increased activity of BHAP U90152. The enzyme kinetics of BHAP U90152, quinoxaline HBY 097 and MKC-442 against 225 Pro→His mutant RT and wild-type enzyme were similar with respect to the natural substrate (dGTP) and the template/primer [poly(C)·oligo(dG)].
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1997-06-25
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