Insights into the Molecular Mechanism of Inhibition and Drug Resistance for HIV-1 RT with Carbovir Triphosphate

Abstract

Abacavir (1592U89, or Ziagen) is a powerful and selective inhibitor of HIV-1 viral replication that has been approved by the FDA for treatment of acquired immunodeficiency syndrome. Abacavir is metabolized to the active compound carbovir triphosphate (CBVTP). This compound is a guanosine analogue containing a 2',3'-unsaturation in its planar carbocyclic deoxyribose ring that acts on HIV-1 reverse transcriptase (RT(WT)) as a molecular target, resulting in chain termination of DNA synthesis. A single amino acid change from methionine 184 to valine in HIV-1 RT (RT(M184V)) has been observed clinically in response to abacavir treatment. The ability of the natural substrate, dGTP, or CBVTP to be utilized during DNA- and RNA-directed polymerization by RT(WT) and RT(M184V) was defined by pre-steady-state kinetic parameters. In the case of RT(WT), CBVTP was found to be a surprisingly poor substrate relative to dGTP. In both DNA- and RNA-directed polymerization, a decrease in the efficiency of CBVTP utilization with respect to dGTP was found with RT(M184V), suggesting that this mutation confers resistance at the level of CBVMP incorporation. The relatively low incorporation efficiency for RT(WT) was unanticipated considering earlier studies showing that the triphosphate form of a thymidine nucleoside analogue containing a planar 2',3'-unsaturated ribose ring, D4TTP, was incorporated with high efficiency relative to the natural substrate, dTTP. The difference may be related to the isosteric replacement of oxygen in the deoxyribose ring with carbon. This hypothesis was tested by synthesizing and evaluating D4GTP (the planar 2',3'-unsaturated deoxyribose guanosine analogue that is complementary to D4TTP). In contrast to CBVTP, D4GTP was found to be an excellent substrate for RT(WT) and no resistance was conferred by the M184V mutation, thus providing novel insight into structure-activity relationships for nucleoside-based inhibitors. In this work, we illustrate how an understanding of the molecular mechanism of inhibition and drug resistance led to the discovery of a novel prodrug of D4G. This compound shows promise as a potent antiviral especially with the drug resistant M184V HIV-1 RT that is so often encountered in a clinical setting.

Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

“…Such aphidicolin-resistant viruses exhibit altered sensitivities to other substrate analogues (17). Sequence analysis and marker transfer experiments located point mutations within the conserved regions of both HSV and vaccinia virus DNApols, which conferred resistance to aphidicolin (10,22,47 (4,16,35,36,38,45).The aim of this study was to select and characterize AcMNPV mutants resistant to antiviral compounds that target the viral DNApol enzyme. We hypothesized that "escape" mutants selected from the serial passage of the parental AcMNPV in the presence of increasing concentrations of these drugs would harbor mutation(s) in the AcMNPV genome, particularly within the DNA polymerase gene (dnapol).…”

“…Such aphidicolin-resistant viruses exhibit altered sensitivities to other substrate analogues (17). Sequence analysis and marker transfer experiments located point mutations within the conserved regions of both HSV and vaccinia virus DNApols, which conferred resistance to aphidicolin (10,22,47 (4,16,35,36,38,45).…”

“…Abacavir, also referred to as 1592U89 (44) or Ziagen, is a potent and selective NRTI of HIV and is currently included in the multiple regimen of highly active antiretroviral therapy (HAART) (40). Like other antiviral drugs, mutations in HIV RT following prolonged passage or treatment with abacavir can confer resistance to a magnitude of 11-fold (36). Abacavirresistant mutants and those of other NRTIs such as lamivudine have been mapped to the nucleoside triphosphate binding site of the RT enzyme (4,16,35,36,38,45).…”

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

“…Its efficiency of incorporation is six-and 9.6-fold less than dCTP during DNA-and RNA-directed incorporation, respectively (Table 1). This is in contrast to D-D4TTP and D-D4GTP which are effective at mimicking their corresponding natural nucleotides (Vaccaro et al, 1999;Ray et al, 2002b). These results suggest that there may be base specific interactions important in nucleotide analogue incorporation.…”

“…In general, a number of studies with HIV-1 RT have noted that the efficiency is higher for RNA-directed versus DNA-directed incorporation although the molecular mechanism has remained elusive (Kerr & Anderson, 1997;Vaccaro et al, 1999;Ray et al, 2002b). An unpublished crystal structure of RT bound to TTP and a DNA/RNA primer/template offers no further clues, as the distances between many important amino acids and TTP remain unchanged (SC Harrison, Harvard University, Mass., USA, personal communication).…”