The Balance between NRTI Discrimination and Excision Drives the Susceptibility of HIV-1 RT Mutants K65R, M184V and K65R+M184V

Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype. C urrently, over 35 million people are living with HIV, according to the World Health Organization (http://www.who.int /hiv/en/). Genetic diversity is characteristic of HIV-1 due to the error-prone nature of its reverse transcriptase (RT) enzyme and its high viral replication rate. The HIV-1 epidemic has rapidly evolved to include 9 major known circulating subtypes (A to D, F to H, J, and K) and over 60 known circulating recombinant forms (CRFs) that show 25 to 35% overall genetic variation; this includes 10 to 15% diversity in RT (1-4). HIV-1 subtype C accounts for 50 to 55% of all HIV infections worldwide, while HIV-1 subtype B is the most prevalent subtype in developed countries and accounts for ϳ12% of global HIV infections. CRF01_AE and CRF02_AG are two globally predominant CRFs that are found in Southeast Asia and West/Central Africa, respectively.Current standard anti-HIV-1 therapy, termed highly active antiretroviral therapy (HAART), consists of three or more antiretroviral compounds from six distinct classes, including nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, entry inhibitors, and integrase inhibitors (INIs) (for reviews, see references 5 and 6). Most currently available antiretrovirals (ARVs) were developed based on the ability to block replication of subtype B viruses, but the development of resistance to all ARVs is a major obstacle in the face of long-term treatment success (7,8). The high genetic diversity of HIV-1 subtypes may lead to distinct pathways to drug resistance (9-12), necessitating the development of novel effective ARVs that possess distinct mechanisms and superior resistance profiles for all HIV-1 subtypes.Inhibitors that target RT constitute the largest class of ARVs and are ke...

Section: Discussionsupporting

confidence: 83%

Subtype-Specific Analysis of the K65R Substitution in HIV-1 That Confers Hypersusceptibility to a Novel Nucleotide-Competing Reverse Transcriptase Inhibitor

Colby-Germinario

Quashie

et al. 2015

“…These comparator agents were chosen either because they are the most commonly used NRTIs in clinical practice today or because of structural similarity to BMS-986001. The data reported here for the comparator agents are in line with their known activity profile (12,14,15,20,22,23,25,26,28,29,33,(35)(36)(37)(38)(41)(42)(43). The HIV protease inhibitor NFV was included as a control in all experiments and exhibited little variation throughout.…”

Section: Discussionmentioning

confidence: 53%

“…Susceptibility to FTC was decreased by K65R; however, the FC-EC 50 was relatively low (14.2-or 13.0-fold) compared with that observed when K65R/ M184V was analyzed (Ͼ384-fold; P Յ 0.05). The addition of M184V to K65R further decreased susceptibility to ABC (FC-EC 50 ϭ 9.9 in the clinical isolate and 23.5 in the SDM virus) compared with K65R alone, as has been noted in clinical samples (26). Addition of M184V to K65R had little effect on the susceptibility of clinical isolates to TFV and d4T; however, in recombinant viruses, addition of M184V to K65R appeared to increase susceptibility to the drugs compared with K65R alone.…”

Section: Potency Of Bmsmentioning

confidence: 86%

In VitroCross-Resistance Profile of Nucleoside Reverse Transcriptase Inhibitor (NRTI) BMS-986001 against Known NRTI Resistance Mutations

Terry

Olds

et al. 2013

“…The mutant also displayed clear chain termination in the presence of GS-9148-DP, although full-length-product formation increased noticeably compared with that for WT RT. However, it is conceivable that K65R may also diminish excision of GS-9148, as has been shown for zidovudine (AZT) and tenofovir (20,27), which would help to explain the lack of reduced susceptibility data obtained in cell culture (5).…”

Section: Resultsmentioning

confidence: 91%

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

Scarth

White

Chen

et al. 2011

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 DNA polymerase activity of HIV-1 reverse transcriptase (RT) is targeted by nucleos(t)ide analogue RT inhibitors [N(t)RTIs], which represent the backbone of frequently used drug regimens. N(t)RTIs compete with natural deoxynucleoside triphosphate (dNTP) pools for incorporation and cause chain termination. There are currently seven approved NRTIs available for the treatment of HIV-1 infection (7). The only approved NtRTI, i.e., tenofovir, is a phosphonate with an acyclic linker attached to the adenine base (7). An investigational NtRTI, GS-9148, has recently been shown to be active against HIV-1 in cell culture and to possess a promising resistance profile in addition to a low nephrotoxic potential (4, 5). GS-9148 is the intracellularly metabolized form of the orally bioavailable phosphonate GS-9131 (29) (Fig. 1). GS-9148 undergoes two phosphorylations to become GS-9148-diphosphate (GS-9148-DP), which, like tenofovir-DP, is the active metabolite that is incorporated by HIV-1 RT. In contrast to tenofovir, GS-9148 is composed of a 2Ј,3Ј-dihydrofuran that contains a 2Ј-fluoro group (2) (Fig. 1).In vitro selection experiments in cell culture revealed the emergence of two different resistance pathways. The compound selects either for Q151L, with high-level resistance to GS-9148 (16), or for a combination of K70E, D123N, and T165I, which shows low-level resistance to . Q151L is a potential intermediate in the development of...