Background: Diketo compounds, such as the diketo acid (DKA) L‑708,906 and S‑1360, inhibit HIV replication in cell culture through inhibition of the strand transfer step of the integration process. S‑1360 is in a phase I/II clinical trial. Recently, the pyranodipyrimidines have been identified as a novel class of authentic integrase (IN) inhibitors with V‑165 as the most potent congener. We have previously selected DKA-resistant HIV‑1(III_B) strains in the presence of L‑708,906. Mutations T66I, L74M and S230R emerged successively in IN. The triple-mutant virus showed a significant (>10-fold) decrease in susceptibility to L‑708,906.

Methods: The selected strains were analysed for their phenotypic cross-resistance to S‑1360 and V‑165 by the MT-4/MTT assay. The replication kinetics of the different mutant strains were investigated. The different mutant IN enzymes were generated by site-directed mutagenesis and their susceptibility to the inhibitory effects of L-708,906, S-1360, and V-165 was evaluated in an enzymatic oligonucleotide-based IN assay.

Results: Phenotypic cross-resistance to S‑1360 was observed for strains carrying mutations T66I, T66I/L74M, or T66I/L74M/S230R, whereas only the T66I viral mutant showed diminished sensitivity to V-165. The 3 viral mutants showed substantially reduced replication fitness in comparison to wild-type (WT) HIV‑1(III_B). The relative order of replication fitness was T66I > T66I/L74M/S230R > T66I/L74M. The in vitro IN activity of the double- and triple-mutant enzymes was reduced by 2- to 3-fold as compared to WT IN activity. L‑708,906 and S-1360 inhibited the triple-mutant enzyme 2- to 3-fold less than WT IN. The T66I mutant, and to a lesser extent the S230R mutant, was partially resistant to inhibition by V-165, whereas the double- and triple‑mutants remained fully sensitive.

Conclusions: Different resistance profiles for the 3 IN inhibitors were observed. Both cell culture and enzymatic data indicated that 3 mutations were required for reduced sensitivity to L‑708,906. The mutation T66I was sufficient to cause resistance to S‑1360 in cell culture, although reduced susceptibility in the enzymatic assay required all 3 mutations. Interestingly, the complete resistance in cell culture towards DKAs could not be reproduced at the enzymatic level. The replication fitness data point to a role for S230R in the replication ability of the triple-mutant. Although the mutation T66I reduced sensitivity to V‑165 at the enzymatic level, the L74M and S230R mutations appeared to reverse the resistance phenotype. The DKA‑resistant strain remained thus sensitive to inhibition by V-165.