A new strategy to inhibit the excision reaction

CRUCHAGA C; ANSO E; FONT M; MARTINO V; ROUZAUT A; MARTINEZ IRUJO J

BIOCHEMICAL JOURNAL

Año: 2007 vol. 405 p. 161 - 167

0264-6021

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain­terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophos­phate analogues. We have found previously that 2GP (2-0-gal­loylpunicalin), an antiviral compound extracted from the leaves of Terminalia trifiora, was able to inhibit both the RT and the RN ase H activities of HIV- f RT without affecting cell prolifera­tion or viabilíty. In the present study, we show that 20P also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-Iype and AZT (3'-azido-3'-deoxylhymidine)-resislant en­zymes at sub-micromolar concentrations. Kinetic and direct­binding analysis showed that 20P was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (Kd = 85 nM). As expected from its 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-catalysed phosphorolysis was responsib1e for the synergy found. Although other RT inhibitors that compete with the template-primer liave 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 analogues.