Week 144 resistance analysis of elvitegravir/cobicistat/emtricitabine/tenofovir DF versus atazanavir+ritonavir+emtricitabine/tenofovir DF in antiretroviral-naïve patients.

OBJECTIVE: To describe baseline and emergent HIV-1 resistance to elvitegravir/ cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF) and ritonavir-boosted atazanavir/emtricitabine/tenofovir DF (ATV+RTV+FTC/TDF) in HIV-1-infected, treatment-naïve subjects through 144 weeks. METHOD: This was a randomized, double-blind, phase 3 study. HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening. Genotypic and phenotypic analyses were performed at virologic failure confirmation and retrospectively at baseline for PR, RT, and integrase (IN) for patients with virologic failure through week 144. RESULTS: In the EVG/ COBI/FTC/TDF group through week 144, HIV-1 from 8 patients (2.3%; 8/353 treated patients) developed primary IN strand transfer inhibitor (INSTI) (n = 6) and/or nucleoside RT inhibitor (NRTI) resistance substitutions (n = 7). The emergence of resistance decreased after the first year, with 5 patients developing HIV-1 resistance through week 48, 1 from weeks 48-96, and 2 from weeks 96-144. Emergent substitutions were E92Q, N155H, or Q148R (n = 2 each) and T66I or T97A (n = 1 each) in IN and M184V/I (n = 7) and K65R (n = 1) in RT. All 8 isolates had reduced susceptibility to EVG, FTC, or TDF. Virus with EVG phenotypic resistance showed cross-resistance to raltegravir. In the ATV+RTV+FTC/TDF group, HIV-1 from 2 patients (0.6%; 2/355 treated patients; both at week 144) developed the resistance substitution M184V/I in RT. CONCLUSIONS: Resistance development to EVG/COBI/FTC/TDF was infrequent (2.3%) through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

Mechanism of resistance to GS-9148 conferred by the Q151L mutation in HIV-1 reverse transcriptase.

GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2',3'-dihydrofuran ring structure that contains a 2'-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2'-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.

The A62V and S68G mutations in HIV-1 reverse transcriptase partially restore the replication defect associated with the K65R mutation.

BACKGROUND: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. METHODS: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. RESULTS: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. CONCLUSIONS: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

Design and profiling of GS-9148, a novel nucleotide analog active against nucleoside-resistant variants of human immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate prodrug, GS-9131.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (K(i) = 0.8 muM) and exhibits low inhibitory potency against host polymerases including DNA polymerase gamma. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 microM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

Effects of the translocation status of human immunodeficiency virus type 1 reverse transcriptase on the efficiency of excision of tenofovir.

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3'-azido-3'-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.

The balance between NRTI discrimination and excision drives the susceptibility of HIV-1 RT mutants K65R, M184V and K65r+M184V.

The HIV-1 reverse transcriptase (RT) resistance mutations K65R and M184V occur individually and in combination, and can contribute to decreased treatment responses in patients. In order to understand how these mutations interact with one another to confer drug resistance, the susceptibilities and underlying resistance mechanisms of these mutants to nucleoside RT inhibitors (NRTIs) were determined. Virus carrying K65R have reduced susceptibility to most NRTIs, but retain full susceptibility to zidovudine (AZT). M184V mutants have reduced susceptibility to lamivudine (3TC), emtricitabine (FTC) and didanosine (ddl), and contribute to reduced susceptibility to abacavir; however, they remain fully susceptible to tenofovir (TFV), AZT and stavudine (d4T). In cell culture, the K65R+M184V virus showed slightly increased susceptibility to TFV, AZT and d4T compared with K65R alone, but showed further decreases in susceptibility to 3TC, FTC, ddl and abacavir. There are two major biochemical mechanisms of resistance: altered NRTI binding/incorporation and altered NRTI excision after incorporation. For most NRTIs, the primary mechanism of resistance by K65R, M184V and K65R+M184V mutant RTs is to disrupt the NRTI-binding/incorporation steps. In the case of AZT, however, decreased binding/incorporation by K65R and K65R+M184V was counteracted by decreased AZT excision resulting in wild-type susceptibility. For TFV, decreased excision by K65R and K65R+M184V may partially counteract the K65R-driven decrease in incorporation relative to wild-type resulting in only low levels of TFV resistance. The K65R-mediated effect on decreasing NRTI excision was stronger than for M184V. These studies show that both mechanisms of resistance (binding/incorporation and excision) must be considered when defining resistance mechanisms.

Molecular mechanisms of tenofovir resistance conferred by human immunodeficiency virus type 1 reverse transcriptase containing a diserine insertion after residue 69 and multiple thymidine analog-associated mutations.

Two amino acids inserted between residues 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rare mutations that may develop in viruses containing multiple thymidine analog (zidovudine [AZT], stavudine)-associated mutations and that confer high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). The two known mechanisms of resistance to NRTIs are decreased incorporation and increased excision. The mechanism used by RT insertion mutants has not been described for tenofovir (TFV), a recently approved agent in this class. A patient-derived HIV-1 strain (strain FS-SSS) that contained an insertion mutation in a background of additional resistance mutations M41L, L74V, L210W, and T215Y was obtained. A second virus (strain FS) was derived from FS-SSS. In strain FS the insertion and T69S were reverted but the other resistance mutations were retained. The FS virus showed strong resistance to AZT but low-level changes in susceptibilities to other NRTIs and TFV. The FS-SSS virus showed reduced susceptibilities to all NRTIs including TFV. Steady-state kinetics demonstrated that the relative binding or incorporation of TFV was slightly decreased for FS-SSS RT compared to those for wild-type RT. However, significant ATP-mediated excision of TFV was detected for both mutant RT enzymes and followed the order FS-SSS RT > FS RT > wild-type RT. The presence of physiological concentrations of the +1 nucleotide inhibited TFV excision by the wild-type RT and slightly inhibited excision by the FS RT, whereas the level of excision by the FS-SSS RT remained high. Computer modeling suggests that the increased mobility of the beta3-beta4 loop may contribute to the high-level and broad NRTI resistance caused by the T69 insertion mutation.

Molecular mechanisms of resistance to human immunodeficiency virus type 1 with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutations K65R and M184V result in changes in susceptibility to several nucleoside and nucleotide RT inhibitors. K65R-containing viruses showed decreases in susceptibility to tenofovir, didanosine (ddI), abacavir, and (-)-beta-D-dioxolane guanosine (DXG; the active metabolite of amdoxovir) but appeared to be fully susceptible to zidovudine and stavudine in vitro. Viruses containing the K65R and M184V mutations showed further decreases in susceptibility to ddI and abacavir but increased susceptibility to tenofovir compared to the susceptibilities of viruses with the K65R mutation. Enzymatic and viral replication analyses were undertaken to elucidate the mechanisms of altered drug susceptibilities and potential fitness defects for the K65R and K65R+M184V mutants. The relative inhibitory capacities (K(i)/K(m)) of the active metabolites of tenofovir, ddI, and DXG were increased for the RT containing the K65R mutation compared to that for the wild-type RT, but the relative inhibitory capacity of abacavir was only minimally increased. For the mutant viruses with the K65R and M184V mutations, the increase in tenofovir susceptibility compared to that of the mutants with K65R correlated with a decrease in the tenofovir inhibitory capacity that was mediated primarily by an increased K(m) of dATP. The decrease in susceptibility to ddI by mutants with the K65R and M184V mutations correlated with an increase in the inhibitory capacity mediated by an increased K(i). ATP-mediated removal of carbovir as well as small increases in the inhibitory capacity of carbovir appear to contribute to the resistance of mutants with the K65R mutation and the mutants with the K65R and M184V mutations to abacavir. Finally, both the HIV-1 K65R mutant and, more notably, the HIV-1 K65R+M184V double mutant showed reduced replication capacities and reduced RT processivities in vitro, consistent with a potential fitness defect in vivo and the low prevalence of the K65R mutation among isolates from antiretroviral agent-experienced patients.