Martino, V.S. (Virginia S.)
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- A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer(Portland Press, 2007) Rouzaut, A. (Ana); Cruchaga, C. (Carlos); Font, M. (María); Anso, E. (Elena); Martinez-Irujo, J.J. (Juan José); Martino, V.S. (Virginia S.)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 chain-terminating 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-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=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 responsible for the synergy found. Although 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 analogues.
- Argentine plant extracts active against polymerase and ribonuclease H activities of HIV-1 reverse transcriptase(John Wiley and Sons, 1999) Ferraro, G. (Gabriela); Sanroman, M. (Marcos); Font, M. (María); Coussio, J. (Jorge); Monge, A. (Antonio); Hnatyszyn, O. (Oksana); Herrera, G. (Gilda); Martinez-Irujo, J.J. (Juan José); Martino, V.S. (Virginia S.); Santiago, E. (Esteban); Broussalis, A. (Adriana); Cuevas, M.T. (María T.); Muschietti, L. (Liliana); Lasarte, J.J. (Juan José)Lipophilic and hydrophilic extracts of four Argentine plants (Gamochaeta simplicaulis Cabr. 1, Achyrocline flaccida Wein. D. C. 2, Eupatorium buniifolium H. et A. 3, and Phyllanthus sellowianus Muell. Arg. 4) were examined in vitro for their ability to inhibit the polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (wild and Y181C mutant types). The active extracts were also examined as inhibitors of viral replication in HLT4LacZ-1IIIB cell cultures, evaluating their cytotoxicity in parallel. Infusions 2I and 4I, among the crude extracts, showed the highest activity. These extracts were refractioned into four fractions; 2I4 and 4I4 were active as inhibitors of DNA-polymerase (wild and Y181C types) and RNase H activities. These fractions were potent as inhibitors of viral replication and were not cytotoxic. Refractionation of 2I4 yielded five new fractions, two of which, 2I4-4 and 2I4-5, showed notable activity. Refractionation of 4I4 yielded for new fractions; of these, 4I4-3 and 4I4-4 were active. The marked biological activity found in the infusion of A. flaccida and P. sellowianus makes them sufficiently attractive to be considered in the combined chemotherapy of the disease.
- Inhibitory effect against polymerase and ribonuclease activities of HIV-reverse transcriptase of the aqueous leaf extract of Terminalia triflora(John Wiley and Sons, 2002) Sanroman, M. (Marcos); Font, M. (María); Coussio, J. (Jorge); Monge, A. (Antonio); Lopez-Casas, P. (Pedro); Martinez-Irujo, J.J. (Juan José); Martino, V.S. (Virginia S.); Santiago, E. (Esteban); Cuevas, M.T. (María T.); Lasarte, J.J. (Juan José)Dichloromethane, methanol and aqueous extracts from the leaves of Terminalia triflora were investigated for their inhibitory effect on polymerase and ribonuclease activities of HIV reverse transcriptase.The most potent activity was found in the aqueous extract, which inhibited both polymerase and ribonuclease activities of the enzyme with an IC50 of 1.6 micro g/mL and 1.8 micro g/mL respectively. The antiinfective activity of the extract was demonstrated in HLT4LacZ-IIIB cell culture with an IC50 of 1.0 micro g/mL. The extract was submitted to a purification process by extractive and chromatographic methods. The activity remained in the hydrophillic fraction. Tannins present in this active purified fraction, as determined by TLC and HPLC methods, could account for the anti HIV-RT activity found in the aqueous extract.