It is well accepted that HIV can overcome drug therapy through the accumulation of resistance mutations. Clinical data indicate that resistant viruses against the first-generation INI drugs (RAL and EVG) have indeed arisen already (18
). We have engineered a new INI, S/GSK1349572, that is highly active against both wild-type HIV and many INI-resistant mutants and that has the potential for a higher genetic barrier to resistance. The EC50
of S/GSK1349572 against HIV-1 in PBMCs and various other cells (data not shown) is in the low-nanomolar to subnanomolar range. In addition, the median EC50
against nine clinical isolates of HIV-2 was 0.8 nM (10
). Cellular toxicity was in the micromolar range for a variety of cell types, indicating that the observed antiviral effect of S/GSK1349572 was not due to cytotoxicity.
Based on the data presented, the mechanism of inhibition is strongly suggested to be at the strand transfer step of HIV integrase, thus preventing the insertion of viral DNA into host DNA. In addition, integration reaction products obtained using blunt-end DNA as a substrate were resolved with a DNA sequencing gel, and S/GSK1349572 showed minor inhibition of 3′-end processing versus strand transfer inhibition (data not shown), indicating that S/GSK1349572 is a strand transfer inhibitor. S/GSK1349572 inhibited both the HIV integration reaction strand transfer step in vitro and HIV replication in cells with similar potencies. The inhibitor had no effect on total viral DNA synthesis in infected cells but blocked the integration of viral DNA into host DNA with the same potency as its antiviral effect. In addition, this INI increased the appearance of viral 2-LTR circles, a predicted by-product of integrase inhibition, with the same potency. The compound had potency against mutant viruses resistant to NRTIs, NNRTIs, and PIs similar to its potency against the wild type and consistent with action on a different antiretroviral target. The passage of virus in an S/GSK1349572-containing medium selected for viruses with mutations in the vicinity of the integrase active site, though the FCs in the EC50s against the mutants were very low. Finally, S/GSK1349572 had decreased potency against a small subset of mutant viruses resistant to known INIs.
passage with S/GSK1349572 followed a pathway significantly different from that with RAL, ELV, or lamivudine. While viruses with the ability to replicate at micromolar levels of RAL, ELV, or lamivudine were readily selected, the concentration of S/GSK1349572 that allowed for virus replication could not be raised above 32 nM. In addition, only four substitutions or combinations of substitutions (T124A, S153Y, T124A/S153Y, and L101Y/T124A/S153Y) were observed on passage with S/GSK1349572 up to day 112, and these amino acid substitutions did not cause a high FC in susceptibility to S/GSK1349572 in the phenotype assay. Although amino acid position 153 is conserved, amino acid positions 101 and 124 in HIV-1 integrase are polymorphic (8
). It was also demonstrated that the selection of resistant mutations from a wild-type population was greatly delayed in S/GSK1349572-containing medium in contrast with that for first-generation INIs. Taken together, these results demonstrate that S/GSK1349572 possesses a resistance profile distinct from that of the first-generation INIs and suggest that S/GSK1349572 may possess a higher genetic barrier to the development of resistance.
The substitutions observed in the passage study with S/GSK1349572 had no effect or minimal effects on susceptibility to S/GSK1349572 or other INIs when they were introduced into a laboratory strain by site-directed mutagenesis. Moreover, the substitutions described here have not been observed so far in passage studies with other INIs, except for T124A, which is a polymorphism in the integrase region; viruses with T124A have been isolated with S-1360, S/GSK364735, RAL, L-870,810, and EVG (24
). Notably, the passage with HIV-1 IIIB utilized a virus population derived from a chronically infected cell line, and at the initiation of passage, the T124A variant was present as approximately 40% of the quasispecies population. The T124A substitution alone did not contribute to virus resistance and, like L101I, is a naturally occurring polymorphism (26
Importantly, S/GSK1349572 showed potency against all integrase-resistant single mutants in our panel at a level similar to that observed against wild-type virus or with an FC as high as 3.6-fold. This result may partially explain the observation during passage in the presence of S/GSK1349572 that no virus with high resistance to S/GSK1349572 was observed with 32 nM or higher concentrations of S/GSK1349572 in the culture medium. The hypothesis would be as follows: more than one substitution is required for very high resistance, and if single mutations conferring significant resistance did not first arise, then very high resistance would not occur during passage. In contrast, during passage with RAL and EVG (as well as with the control, lamivudine), single amino acid substitutions could cause a significant fold change; then additional secondary mutations could provide increased resistance or compensate for the decreased fitness of the virus. Overall, these data are also supportive of the potential for a higher genetic barrier of S/GSK1349572 to resistance, although clinical data will be required to fully test this hypothesis. It is noteworthy that in a 10-day phase 2a monotherapy study, the plasma steady-state geometric mean concentration at the end of a dosing interval (Ctau) at once-daily dosing with 50 mg was 0.83 μg/ml, 13 times the in vitro PA-EC90 (S. Min et al., submitted for publication).
It is also striking that S/GSK1349572 had significant potency against most double or more mutants in our panel, with the exception of the E138K/Q148K and Q148R/N155H mutants (FCs, 19 and 10, respectively). While these double mutants were highly resistant to S/GSK1349572, the first mutation of these pathways (i.e., Q148K, Q148R, or N155H) was not selected in the passage study with S/GSK1349572, perhaps because the FCs for these single mutants remained similar to that for the wild type. The replication/infectivity of the E138K/Q148K mutant was higher than that with Q148K alone, but even so, its replication capacity and integration efficiency were poorer than those of the wild type (34
). The Q148R and N155H mutations were not observed in the same genome in clinical isolates but were found as mixtures (28
), and the replication capacity of the Q148R/N155H mutant molecular clone was poor in PBMCs (unpublished data). It is thus noteworthy that S/GSK1349572 had limited cross-resistance to RAL- and EVG-resistant mutants, clearly distinguishing S/GSK1349572 as a next-generation INI. To better understand the distinct resistance profile of S/GSK1349572 compared to those of RAL and EVG, studies of docking into HIV-1 IN/Mg2+
/DNA models are in progress.
Since INIs will be used in combination regimens, it is important to evaluate the potential for synergy and antagonism. S/GSK1349572 was tested in combination assays with representatives of all approved classes of HIV therapeutics, as well as with adefovir and ribavirin, drugs likely to be coadministered to HIV patients coinfected with HBV or HCV. We used ribavirin and stavudine (d4T) in the study as positive controls for antagonism (19
). Ribavirin has no anti-HIV activity itself, and when added to d4T, ribavirin led to a decrease in its potency, thus demonstrating antagonism. No antagonism or trend toward antagonism was observed when S/GSK1349572 was tested with any class of marketed HIV drug, adefovir, or ribavirin. On the other hand, with several of the combinations, either statistically significant synergism or a trend toward synergism was seen. Similar data have been presented for other INIs (27
) and raise the possibility of highly potent combinations in the clinic.
In conclusion, S/GSK1349572 has a resistance profile markedly distinct from those of RAL and ELV, and it demonstrates the potential for a higher genetic barrier to resistance. These preclinical findings formed part of the rationale for the selection of S/GSK1349572 as a candidate for clinical development, and they provide a strong foundation for its ongoing clinical investigation.