A new strategy to inhibit the excision reaction catalyzed by HIV-1 reverse transcriptase: compounds that compete with the template-primer
Résumé
Inhibitors of the excision reaction catalyzed by HIV-1 reverse transcriptase (RT) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme toward chain-terminating nucleotides. Only a limited number of compounds have demonstrated to inhibit this reaction to date, including nonnucleoside RT inhibitors (NNRTIs) and certain pyrophosphate analogs. We have previously found that 2-O-galloylpunicalin (2GP), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the reverse transcriptase and the ribonuclease H activities of HIV-1 RT without affecting cell proliferation or viability. Herein we show that 2GP also inhibited the ATP- and PP i}-dependent phosphorolysis catalyzed by wild-type and AZT-resistant enzymes at submicromolar concentrations. Kinetic and direct binding analysis showed that 2GP was a noncompetitive inhibitor against the nucleotide substrate, while it competed with the binding of RT to the template/primer (K d} = 85 nM). As expected from their mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalyzed phosphorolysis was responsible for the synergy found. While other RT inhibitors that compete with the template/primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogs.
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