Background:  Nucleotide-competing reverse transcriptase inhibitors (NcRTI) are potent inhibitors of HIV replication with a unique mechanism of action. They bind the active site of RT and compete with the next incoming nucleotide. To further investigate the effect of RTI-resistance-associated mutations on the activity of NcRTI, the susceptibility of >6000 recent clinical isolates for a prototype compound (NcRTI-1, EC₅₀ for wild type HIV-1 = 30 nM) was determined.

Methods:  Virological methods included phenotyping, genotyping, constructing site-directed-mutant (SDM) strains and in vitro resistant virus selection.

Results:  More than 80% of the profiled clinical isolates remained susceptible for NcRTI-1 (fold change in EC₅₀ [FC] <4). No cross-resistance was observed between NcRTI-1 and efavirenz (EFV), nevirapine (NVP), tenofovir (TDF), zidovudine (AZT), stavudine (d4T), abacavir (ABC), zalcitabine (ddC), or didanosine (ddI). Some cross-resistance with lamivudine (3TC) and emtricitabine (FTC) could be detected (Pearson Correlation Coefficient = 0.59). Analysis of the genotype of >1700 of these viruses showed that the combination of M184V + Y115F correlated most with reduced susceptibility to NcRTI-1. This was confirmed in SDM strains:  single mutations M184V and Y115F showed a FC of 5.0 and 7.9, respectively. The combination of both resulted in a FC of 39. Analysis of the dataset also indicated that the K65R mutation is associated with hypersusceptibility to NcRTI-1 and that it reverses M184V-induced resistance. This was confirmed using a M184V + K65R SDM strain (FC = 0.63). The interplay between K65R and M184V was also explored by replicating wild type HIV-1 in the presence of both NcRTI-1 and TDF. This showed that, in contrast to 3TC or ABC, NcRTI-1 prevents the selection of K65R. Further breakdown of the dataset showed that NcRTI-1 activity is not influenced by the presence of thymidine analog mutations (TAM), the 69ins-MDR complex, the Q151M-MDR complex, or mutations associated with NNRTI-resistance.

Conclusions:  Analysis of HIV-1 clinical isolates and SDM strains showed that the combination of RT mutations M184V and Y115F is associated with a decreased susceptibility to NcRTI. In addition, amino acid change K65R was found to restore the susceptibility of M184V carrying viruses to NcRTI. These data, together with the observation that neither TAM, nor the 69ins-MDR complex, nor the Q151M-MDR complex influences susceptibility to NcRTI, warrant further exploration of this novel class of HIV inhibitors.