Background: L-708,906, a diketo acid derivative, has been described as a selective inhibitor of HIV-1 integration. We confirmed the inhibitory activity of L-708,906 against the replication of HIV-1 in cell culture (50% effective concentration (EC50): 1.5 µg/ml) has been previously reported on HIV-1 resistance selection demonstrating that loss of susceptibility required the acquisition of integrase active site mutations, T66I and S153Y or S153Y and N155S. We now have selected a new drug-resistant HIV-1 strain.

Methods: L-708,906-resistant HIV-1 strains were obtained after sequential passaging of HIV-1(IIIB) in the presence of increasing concentrations of the compound. DNA sequence analysis of the integrase-encoding region was performed. The integrase (in) gene of several resistant strains was recombined in a wild-type NL4.3 virus, deleted for in. The reduced susceptibility of the selected and the recombined virus strains to the antiviral effect of L-708,906 and reference anti-HIV compounds was analysed.

Results: Treatment of HIV-1(IIIB) with L-708,906 (up to 10 µg/mL after 30 passages) resulted in the emergence of T66I mutant virus. On further passaging (up to 70 passages) the virus retained the T66I mutation but acquired L74M and S230R mutations in the in genome in addition to the T66I mutation. This multiple-mutant virus proved highly resistant to L-708,906, but retained full sensitive to HIV entry (bicyclam AMD3100), nucleoside (zidovudine, abacavir) and non-nucleoside (nevirapine) reverse transcriptase and protease (ritonavir) inhibitors. The resistance profile of the multiple-mutant recombined virus was the same as the respective resistant strain selected in vitro. The mutations were introduced in recombinant integrase to confirm their respective contribution to the observed drug-resistance.

Conclusions: Novel mutations in HIV-1 integrase that confer resistance to diketo acids were identified. The resistance phenotype could completely be rescued by the recombination of the in gene.