There are currently more than 20 antiretrovirals that have been approved by the US Food and Drug Administration for the treatment of HIV infection. They fall into four categories, targeting HIV RT, protease, integrase, the entry step, and the fusion of the viral and cell membranes. RT inhibitors are either NRTIs or NNRTIs. The NRTIs such zidovudine (AZT) and lamivudine compete with the natural substrates and get incorporated into the nascent DNA chain, blocking further polymerization because they lack a 3′OH group required for DNA synthesis. NNRTIs such as nevirapine and efavirenz inhibit the polymerase activity of RT by binding at a hydrophobic pocket nearly 10 Å away from the polymerase active site (). This pocket is created after the binding of NNRTIs. The highly active antiretroviral therapy (HAART) introduced in the mid-90s contains the combination of antivirals (generally a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) targets the replication of the resistant virus.
Extended or incomplete treatments with antiretrovirals result in the emergence of drug resistance mutations. In the case of drugs that target RT, most of the resistance mutations were found to be present in the polymerase domain of RT. These resistance mutations against NRTIs function primarily with two mechanisms: (i) they reduce the binding affinity/incorporation of NRTI (34
) or (ii) enhance the selective excision of incorporated NRTI from a chain-terminated primer terminus (9
). The resistance against NNRTIs is primarily through the mutations that reduce the binding affinity of NNRTIs (7
Recent studies showed that connection subdomain mutations can confer resistance to NRTIs. Nikolenko et al. suggested that some of these mutations increase AZT resistance by reducing template RNA degradation, thereby preserving the RNA template and providing additional time for RT to excise AZT monophosphate (27
). Hachiya et al., (13
) as well as another research group (43
) identified a clinical isolate with phenotypic resistance to nevirapine (NVP) in the absence of known NNRTI mutations. This resistance was shown to be caused by N348I, a mutation at the connection subdomain of HIV-1 RT. This mutation is not a polymorphism, as it exists in more than 10% of drug-treated, but not drug-naïve HIV patients. The connection subdomain mutation N348I has been related to the altered DNA binding affinity and processivity of the mutant enzyme compared to the WT RT (4
). Ehteshami et al. showed that N348I enhances resistance to AZT through both RNase H-dependent and -independent mechanisms (10
). Since EFdA is a TDRTI and its incorporation is assumed to affect the translocation and thereby DNA binding and processivity, we investigated the susceptibility of two highly potent antiretrovirals EFdA and ENdA.
We report that both EFdA-TP and ENdA-TP are very potent inhibitors of N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M RTs. They inhibit RT primarily at the point of incorporation and since they prevent enzyme translocation they both belong to the TDRTI class of NRTIs. The D67N/K70R/L210Q/T215F set of mutations are the classical thymidine-associated mutations (TAMs), which are known to cause resistance to AZT by enhancing excision of AZT-terminated primers (1
). The A62V/V75I/F77L/F116Y/Q151M set of mutations is known as the “Q151M” complex RT, and has been known as a multidrug-resistance mutation, since the latter mutations are known to be involved in resistant variants with reduced susceptibility to dideoxynucleotides and to AZT. Unlike D67N/K70R/L210Q/T215F RT, the Q151M complex decreases susceptibility to NRTIs by decreasing incorporation efficiency of the inhibitors rather than increasing excision and unblocking of chain-terminated primers (14
). Finally, N348I is known to cause resistance to both NRTIs and NNRTIs. Hence, collectively, these mutants represent all mechanisms by which RT becomes resistant to available antivirals. Importantly, we find that they are all susceptible to the EFdA and ENdA TDRTIs. Hence, this new class of RT inhibitors should be able to efficiently block viruses that carry clinically relevant mutations, including the new connection domain mutation N348I.