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Background:  We recently reported that saquinavir (SQV) and lopinavir (LPV) can be synergistic on protease inhibitor (PI)-resistant viruses exhibiting high LPV/SQV IC₅₀ ratios. We hypothesize that this phenomenon is due to an enhancement of the intracellular activity of SQV by LPV, an effect that can only be measured if the target virus retains sufficient SQV susceptibility together with high resistance to the enhancing drug.  To further evaluate this concept, we measured the synergistic properties of atazanavir (ATV) and SQV.

Methods:  Synergistic interactions between ATV and SQV were tested on a panel of PI-resistant recombinant viruses, using a modified Phenoscript assay. By site-directed mutagenesis, 3 viruses were obtained and 10 were reconstructed with Gag-protease sequences from clinical samples. Inhibition by the combination of SQV and ATV was assessed at 4 fixed molar ATV:SQV ratios; 1:1, 2:1, 4:1, and 8:1. Interactions were calculated using the multiple drug effect equation of Chou and Talalay, yielding a combination index at inhibition rates of 50%, 75%, and 90%.

Results:  Synergy was considered significant when the whole range of the 95% confidence interval of the combination index from 3 independent experiments was < 1. No significant synergy was measured with wild type pNL4-3. Among recombinant viruses derived from clinical samples, ATV/SQV synergy were observed with only 2 viruses, which were those with the lowest SQV resistance (fold-change in IC₅₀ < 3.8). In the other viruses, which displayed parallel increase in IC₅₀ to both drugs, no synergy was measured. Among the 3 site-directed mutants, 1 virus exhibited marked ATV/SQV synergy. This virus, which carried mutations I50L and A71V, had a fold-change in IC₅₀ of 5.35 for ATV and 0.66 for SQV. The combination index for the IC₉₀ at an ATV:SQV ratio of 8:1 was 0.50 (95% confidence interval = 0.24 to 0.76). For each inhibition rate at which combination index was measured on this virus, synergy increased with increasing the ATV:SQV ratio, highly suggestive of a mechanism that implies enhancement of SQV activity by ATV.

Conclusions:  Similar to our previous findings with LPV, synergistic interaction of ATV and SQV can only be measured in viruses with near wild type SQV susceptibility and high ATV resistance. These results indicate that the antiviral activity of SQV can be enhanced by several other PI, following a mechanism that could relate to the intracellular properties of these compounds.

Keywords: saquinavir; atazanavir; synergy