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 PPi-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 (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 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.
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Research Article|
June 13 2007
A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template–primer
Carlos Cruchaga;
Carlos Cruchaga
*Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain
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Elena Anso;
Elena Anso
*Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain
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María Font;
María Font
†Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Navarra, 31008 Pamplona, Spain
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Virginia S. Martino;
Virginia S. Martino
‡Cátedra de Farmacognosia, IQIMEFA (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, República Argentina
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Ana Rouzaut;
Ana Rouzaut
*Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain
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Juan J. Martinez-Irujo
Juan J. Martinez-Irujo
1
*Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain
1To whom correspondence should be addressed (email jjmirujo@unav.es).
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Publisher: Portland Press Ltd
Received:
December 08 2006
Revision Received:
February 19 2007
Accepted:
March 13 2007
Accepted Manuscript online:
March 13 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2007 Biochemical Society
2007
Biochem J (2007) 405 (1): 165–171.
Article history
Received:
December 08 2006
Revision Received:
February 19 2007
Accepted:
March 13 2007
Accepted Manuscript online:
March 13 2007
Citation
Carlos Cruchaga, Elena Anso, María Font, Virginia S. Martino, Ana Rouzaut, Juan J. Martinez-Irujo; A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template–primer. Biochem J 1 July 2007; 405 (1): 165–171. doi: https://doi.org/10.1042/BJ20061831
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