title: A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity.
abstract: We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg(2+) (5 mm) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg(2+) significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg(2+)-assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase-derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL.
authors.affiliation: Centre d'Analyses et de Recherche UR EGFEM Faculté des Sciences Université Saint-Joseph Beirut Lebanon; LBPA, UMR8113 du CNRS Ecole Normale Supérieure de Cachan Cedex Cachan France.
authors.forename: Farah F
authors.initials: FF
authors.lastname: Ammar
authors.affiliation: Centre d'Analyses et de Recherche UR EGFEM Faculté des Sciences Université Saint-Joseph Beirut Lebanon.
authors.forename: Zeina
authors.initials: Z
authors.lastname: Hobaika
authors.affiliation: LBPA, UMR8113 du CNRS Ecole Normale Supérieure de Cachan Cedex Cachan France.
authors.forename: Safwat
authors.initials: S
authors.lastname: Abdel-Azeim
authors.affiliation: LBPA, UMR8113 du CNRS Ecole Normale Supérieure de Cachan Cedex Cachan France.
authors.forename: Loussinée
authors.initials: L
authors.lastname: Zargarian
authors.affiliation: Centre d'Analyses et de Recherche UR EGFEM Faculté des Sciences Université Saint-Joseph Beirut Lebanon.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Maroun
authors.affiliation: LBPA, UMR8113 du CNRS Ecole Normale Supérieure de Cachan Cedex Cachan France; Chemistry and Biology, Nucleo(s)tides and Immunology for Therapy UMR8601 CNRS Paris Cedex 06 France.
authors.forename: Serge
authors.initials: S
authors.lastname: Fermandjian
keywords: DNA; HIV‐1; inhibitors; integrase; interaction; intercalation