A targeted DNA substrate mechanism for the inhibition of HIV‐1 integrase by inhibitors with antiretroviral activity

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.