The Nucleoside Analog BMS-986001 Shows Greater In Vitro Activity against HIV-2 than against HIV-1 Robert A. Smith,a Dana N. Raugi,a Vincent H. Wu,a Sally S. Leong,a Kate M. Parker,a Mariah K. Oakes,a Papa Salif Sow,b* Selly Ba,b Moussa Seydi,b Geoffrey S. Gottlieb,a,c for the University of Washington-Dakar HIV-2 Study Group Center for Emerging and Reemerging Infectious Diseases and Department of Medicine, Division of Allergy and Infectious Diseases,a and Department of Global Health,c University of Washington, Seattle, Washington, USA; Service des Maladies Infectieuses, CHNU de Fann, Dakar, Senegalb

Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2=,3=-didehydro-3=-deoxy-4=-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4=-ethynyl stavudine, or 4=-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ⴞ 18 nM versus 610 ⴞ 200 nM, respectively; mean ⴞ standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture.

D

uring the past 3 decades, improvements in the potency and breadth of antiretroviral therapy (ART) have had a profound impact on the medical management of human immunodeficiency virus type 1 (HIV-1) infection. Beginning with zidovudine (AZT; 1987), a total of 27 antiretroviral (ARV) drugs spanning six distinct classes have been approved by the United States Food and Drug Administration (FDA) for use in HIV-1-infected individuals (1). In resource-rich settings, patients who initiate ART early in infection and exhibit durable suppression of HIV-1 viremia now have an average life expectancy of 70 to 80 years, essentially the same as their HIV-negative counterparts (2, 3). In comparison, treatment of human immunodeficiency virus type 2 (HIV-2) infection has advanced at a much slower pace. HIV-2 infection and HIV-1/2 dual infection are largely confined to West Africa, together with a few locales with socioeconomic ties to the region, and account for ⬃3 to 5% of the global burden of HIV (4). Currently, ART for HIV-2 relies entirely on compounds that were developed, optimized, and licensed for use for the treatment of HIV-1 (specifically, HIV-1 group M, subtype B) (5). The limited activity of many of these drugs against HIV-2 is a major obstacle to treatment; HIV-2 is intrinsically resistant to nonnucleoside reverse transcriptase (RT) inhibitors, the fusion inhibitor enfuvirtide (T-20), and the majority of the protease inhibitors (PIs) used for HIV-1 ART (6–11) but is sensitive to both integrase strand transfer inhibitors (INSTIs) and nucleoside reverse transcriptase inhibitors (NRTIs), with the 50% effective concentrations (EC50s) of these drugs for HIV-2 being comparable to those seen for HIV-1 (12–17). In West Africa, the most widely used regimen for first-line HIV-2 treatment is ritonavir-boosted lopinavir (LPV/r) plus two NRTIs (typically, AZT and lamivudine [3TC]). This choice is primarily based on cost and the need to maintain stocks of generic LPV/r for second-line HIV-1 ART. Treatment of HIV-2 infection is further hampered by the

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emergence of drug resistance. Mutations that diminish the activity of NRTIs, PIs, and INSTIs have been reported in HIV-2 isolates from ARV-treated patients (9, 18–27), and up to 30% of HIV-2infected patients living in West Africa show evidence of multiclass (NRTI and PI) resistance (19, 22). Once resistance emerges, HIV2-infected individuals are left with few (if any) options for effective treatment. Although the Senegalese national HIV treatment program (ISAARV) recently added raltegravir and darunavir to its list of available ARVs, the vast majority of HIV-2-infected patients in West Africa have no access to second-line agents and by default remain on PI-based regimens long after the onset of virologic failure and drug resistance. These difficulties underscore the pressing need to identify new ARVs for treatment of patients infected with HIV-2. BMS-986001 (2=,3=-didehydro-3=-deoxy-4=-ethynylthymidine; also known as festinavir, censavudine, 4=-ethynyl stavudine, 4=ethynyl-d4T, and OBP-601) is a novel NRTI that is structurally

Received 5 June 2015 Returned for modification 13 July 2015 Accepted 11 September 2015 Accepted manuscript posted online 21 September 2015 Citation Smith RA, Raugi DN, Wu VH, Leong SS, Parker KM, Oakes MK, Sow PS, Ba S, Seydi M, Gottlieb GS, for the University of Washington-Dakar HIV-2 Study Group. 2015. The nucleoside analog BMS-986001 shows greater in vitro activity against HIV-2 than against HIV-1. Antimicrob Agents Chemother 59:7437–7446. doi:10.1128/AAC.01326-15. Address correspondence to Robert A. Smith, [email protected]. * Present address: Papa Salif Sow, Bill and Melinda Gates Foundation, Seattle, Washington, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01326-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Thymidine

d4T (Stavudine)

BMS-986001

FIG 1 Chemical structures of deoxythymidine (thymidine), d4T (stavudine; 2=,3=-didehydro-3=-deoxythymidine), and BMS-986001 (2=,3=-didehydro-3=deoxy-4=-ethynyl-thymidine).

related to stavudine (d4T), but it bears an ethynyl substitution at the 4=-carbon of the thymidine nucleoside sugar (Fig. 1) (28). The active form of the analog (BMS-986001-5=-triphosphate) is recognized as a substrate for incorporation by HIV-1 reverse transcriptase, resulting in termination of viral DNA synthesis (29, 30). BMS-986001 inhibits HIV-1 replication in culture with EC50s in the nanomolar range and retains activity against a broad range of NRTI-resistant HIV-1 mutants (31–34). In a randomized phase IIb trial, the combination of lamivudine, efavirenz, and BMS986001 (400 mg once daily) resulted in virologic suppression in 89% of HIV-1-infected subjects after 48 weeks of treatment (35). Remarkably, despite the fact that BMS-986001 has progressed to clinical trials, its activity against HIV-2 has not been reported. In the present study, we evaluated the potential utility of BMS986001 for the treatment of HIV-2 infection using culture-based drug susceptibility assays and structural modeling. Our in vitro analysis included prototypic HIV-1 and HIV-2 strains, additional isolates from ART-naive individuals, HIV-1 and HIV-2 mutants with site-directed mutations in RT, and a novel clone derived from an HIV-2-infected patient (HIV-2ROD9-4.7a). The genome of the HIV-2ROD9-4.7a construct encodes multiple amino acid changes in RT that are associated with NRTI resistance. MATERIALS AND METHODS Inhibitors. BMS-986001 was obtained from Bristol-Myers Squibb (Wallingford, CT). d4T was purchased from Sigma-Aldrich Co. (St. Louis, MO). A master stock of BMS-986001 (41 mM) was prepared by dissolving 20.4 mg of the compound in 2 ml of molecular-grade water (Fisher Scientific, Fair Lawn, NJ). For d4T, a master stock (40 mM) was prepared by dissolving 28 mg powder in 3.1 ml of molecular-grade water. Working dilutions of BMS-986001 and d4T were prepared from the master stocks at concentrations 10-fold greater than those used in the single-cycle assay with MAGIC-5A cells, which are CD4⫹ CXCR4⫹ CCR5⫹ HeLa cells containing an HIV-inducible reporter gene (HIV long terminal repeat [LTR]–␤-galactosidase). All aqueous preparations of the inhibitors were stored at ⫺80°C. Plasmids, cells, and virus. pROD9, a full-length molecular clone of HIV-2 (group A), was kindly provided by Michael Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA. Full-length HIV-1 clone pNL4-3 (group M, subtype B) was obtained from Bruce Chesebro, National Institutes of Health (NIH), Rocky Mountain Laboratories, Hamilton, MT. Bacterial culture steps (i.e., single-colony isolation, starter cultures, and large-scale expansions for plasmid purification) were performed at 30°C to minimize the outgrowth of plasmids with deletions in the HIV insert (36). Transfection-grade (endonuclease-free) preparations of pROD9 and pNL4-3 were isolated using a HiSpeed plasmid maxikit

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with the buffers and reagents from an EndoFree plasmid maxikit (Qiagen, Valencia, CA). 293tsA1609neo clone 17 (293T/17) cells were purchased from the American Type Culture Collection (Manassas, VA). MAGIC-5A cells were obtained from Michael Emerman. 293T/17 and MAGIC-5A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Manassas, VA) supplemented with 4 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Gibco, Life Technologies Corp., Grand Island, NY) and with 10% fetal bovine serum (heat inactivated at 70°C for 30 min; HyClone, Logan, UT). CEMss cells are continuously replicating, non-human T-cell leukemia virus-transformed lymphoblasts that were originally isolated from a pediatric patient with acute T cell leukemia (37); CEMss is a cloned cell line derived from this population (38). CEMss cells were obtained from the NIH AIDS Reagent Program (ARP; National Institute of Allergy and Infectious Diseases, Division of AIDS, Pathogenesis and Basic Research Branch, Germantown, MD) and were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco) containing 4 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 10% fetal bovine serum. All virus cultures and uninfected cell lines were maintained in a humidified incubator at 37°C in a 5% CO2 atmosphere. Stocks of HIV-1NL4-3 and HIV-2ROD9 were generated by transient transfection of chloroquine-treated 293T/17 cells with pNL4-3 and pROD9 as previously described (9, 39). Supernatants from transfected 293T/17 cell cultures were clarified by centrifugation at 218 ⫻ g for 10 min at room temperature and frozen in 1-ml aliquots at ⫺80°C. Samples from the 293T/17-derived stocks were used directly for infections of MAGIC-5A and CEMss cells without additional expansion. HIV-2EHO was kindly provided by Jan McClure, University of Washington (UW), Seattle, WA. The remaining HIV-1 and HIV-2 strains in our panel were obtained from the NIH ARP. Site-directed mutants and patient-derived HIV-2 clone pROD94.7a. Mutants with site-directed mutations in HIV-2 RT were generated by use of the full-length pROD9 molecular clone as previously described (25). All plasmids were sequenced to ensure the absence of unintended mutations in the HIV-2-encoding region. Clone pROD9-4.7a was generated using RT sequences amplified from an HIV-2-infected patient. This individual began treatment with AZT-3TC-indinavir in June 2003 and was subsequently switched to AZT-3TC-LPV/r in November 2008. The patient was continuing LPV-based treatment with a plasma HIV-2 load of 3,400 RNA copies/ml and a CD4 count of 336 at the time of plasma specimen collection (January 2010). Collection of data and patient samples for this study was performed with the approval of the UW Institutional Review Board and the Senegal Ethics Committee. HIV-2 pol gene sequences were amplified from the plasma sample by RT-PCR using the primers and reaction conditions described elsewhere (22, 40). We then subjected the pol products to a third round of PCR using primers 5=RTf (5=-TAAAAATAATGCTTAAGCCAGGGAAAGATGG-3=) and cRTr (5=CTCTGCTTCTGCTAGCTCTGTCCACTGTAC-3=); these primers introduced unique AflII and NheI restriction sites (underlined) near the 5=and 3= termini of the amplicon. To clone these products into pROD9, we first engineered AflII and NheI restriction sites at nucleotide positions 2968 and 3826 of the HIV-2 sequence, respectively. These sites were introduced via point mutations A2971T, A3826G, and T3827C, which are synonymous mutations at codons 12, 297, and 298 of RT, respectively. The resultant clone was digested with AflII and NheI, and the region between the restriction sites was replaced with a 22-bp synthetic linker to yield clone pROD9⌬5=RT (the linker sequence is available upon request). Finally, the patient sample PCR products were digested and ligated into the AflII and NheI sites of pROD9⌬5=RT; a single clone from this ligation (pROD9-4.7a) was isolated for sequencing and preparation of transfection-grade DNA. pROD9-4.7a carries the NRTI resistance-associated changes K65R, Q151M, and M184V, as well as two other changes associated with NRTI-based ART (N69S and V111I) (41, 42). In addition, relative to the amino acid sequence of the parental pROD9 clone, pROD-

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4.7a contained the following amino acid differences at RT sites not associated with NRTI resistance: K30R, K64R, P126Q, V135T, V167I, K176P, I180L, F214L, H228Q, I251V, L270I, and K277R. Stocks of HIV2ROD9-4.7a, as well as each of the site-directed RT mutants, were generated via transfection of 293T/17 cells with the corresponding full-length plasmids as described above. Titration of virus stocks. The titers of all HIV-1 and HIV-2 preparations on MAGIC-5A cells were determined using our previously described protocol (9), with slight modifications. Briefly, subconfluent MAGIC-5A cell monolayers (in 75-cm2 tissue culture flasks) were rinsed with Dulbecco’s phosphate-buffered saline without calcium or magnesium (D-PBS; HyClone) and treated with 0.05% trypsin–EDTA (Gibco). After a 3- to 5-min incubation at 37°C, the monolayers were disrupted by firmly tapping the side of the flask. The cells were then collected, centrifuged at room temperature for 5 min at 218 ⫻ g, resuspended in fresh DMEM, and transferred to 48-well tissue culture plates; each well received 1.5 ⫻ 104 cells in 200 ␮l of medium. The outer wells of the plates received 400 ␮l of phosphate-buffered saline (PBS) (rather than cells) to avoid edge effects on cell growth and viability (43). The assay plates were incubated for 18 to 20 h at 37°C in 5% CO2 prior to infection. To titrate the virus stocks, serial 4-fold dilutions of each strain were prepared in complete DMEM supplemented with 20 ␮g/ml DEAE-dextran (Sigma-Aldrich Co., St. Louis, MO). The medium was then aspirated from the 48-well assay plates, and the diluted virus preparations (100 ␮l/well) were added directly to the MAGIC-5A cell monolayers. After a 2-h incubation (37°C, 5% CO2), 300 ␮l of fresh complete DMEM was added to each well, and the plates were returned to the incubator for 40 to 44 h. The infected monolayers were fixed and stained with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside as previously described (25), and the resulting foci were counted by light microscopy. Titers (presented as the number of focus-forming units [FFU] per milliliter) were back-calculated on the basis of the numbers of foci observed in microcultures containing 100 to 300 foci per well. Single-cycle drug susceptibility assays. For single-cycle measurements of ARV drug sensitivity, MAGIC-5A cells were seeded in 48-well plates as described above for the titration of virus stocks. Following overnight incubation, the medium was removed from the monolayers and replaced with 220 ␮l of fresh complete DMEM containing 20 ␮g/ml of DEAE-dextran. The assay plates were then dosed with various concentrations of BMS-986001, d4T, or solvent for the no-drug controls (30 ␮l/ well) and returned to the incubator for 1 h to convert the nucleoside analogs to their active 5=-triphosphates. During this incubation, HIV stocks were adjusted to 20,000 to 80,000 FFU/ml in complete DMEM, and at the 1-h time point, the assay wells were inoculated with 50 ␮l (1,000 to 4,000 FFU) of virus. Incubation was then continued for 40 to 44 h. Each plate included two virus-free, solvent-only control wells to determine the background ␤-galactosidase expression in uninfected cells. To quantify HIV infection, the MAGIC-5A cell monolayers were washed twice with PBS (200 ␮l/well) and disrupted by adding cell lysis buffer (0.1% [vol/vol] Igepal CA-630 from Sigma-Aldrich in D-PBS; 100 ␮l/well). Chlorophenol red-␤-D-galactopyranoside (CPRG; 6 mM in cell lysis buffer; BioShop Canada, Burlington, ON, Canada) was then added to the cell lysates (100 ␮l/well), and incubation was allowed to proceed for 30 min to 2 h at room temperature for substrate conversion. Following the incubation period, the CPRG-lysate mixtures were transferred to 96-well flat-bottom microtiter plates (150 ␮l/well), and the absorbance at 570 nm was quantified using a Victor3 plate reader (PerkinElmer Inc., Akron, OH). Backgroundsubtracted absorbance readings were plotted in Prism software (version 6.0b; GraphPad Software Inc., La Jolla, CA), and EC50s were calculated from the data using the sigmoid dose-response regression function of Prism. Mean ⫾ standard deviation (SD) EC50s were calculated from at least three independent dose-response assays performed on different days. Multicycle drug susceptibility assays. Drug sensitivity measurements involving spreading infections of CEMss cells were performed using a modified version of our protocol for the MT-2 T cell line (16). Briefly,

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CEMss cells (4.8 ⫻ 106 cells per ml in complete IMDM with 20 ␮g/ml DEAE-dextran; 125 ␮l/well) were seeded into 48-well plates, treated with appropriate concentrations of d4T or BMS-986001 (25 ␮l/well), incubated for 1.5 h to activate the drugs, and inoculated with 100 ␮l/well (3,500 FFU) of HIV-1NL4-3 or HIV-2ROD9. As with the MAGIC-5A cell assays, the outer wells of the plates received 400 ␮l of PBS to avoid edge effects on cell growth (43). On the second day postinfection, an additional 360 ␮l of complete IMDM and 40 ␮l of drug were added to each well, and the incubation was continued for two more days. Supernatants were then collected from the CEMss cell cultures and transferred to MAGIC-5A cell monolayers for quantification of infectious virus. For these infections, MAGIC-5A cells were seeded as described above for the titer determination assay, incubated overnight, refed with complete DMEM containing 20 ␮g/ml DEAE-dextran (200 ␮l/well), inoculated with samples from the CEMss cell cultures (100 ␮l/well), and returned to the incubator. After 40 to 44 h, the MAGIC-5A cell monolayers were lysed and incubated with CPRG substrate to quantify HIV infection (see the previous section for details), and EC50s were calculated as described above for the single-cycle assay. Cytotoxicity assays. The cytotoxicities of stavudine and BMS-986001 were determined using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI). For these experiments, MAGIC-5A and CEMss cells were seeded and dosed with inhibitors as described above for the drug susceptibility assays. After the appropriate incubation period (40 to 44 h for MAGIC-5A cells, 4 days for CEMss cells), the cells were treated with CellTiter-Glo substrate that had been reconstituted in CellTiter-Glo buffer per the manufacturer’s instructions. For MAGIC-5A cells, 300 ␮l/well of reconstituted substrate was added directly to the culture medium. For CEMss cell assays, the upper 500 ␮l of medium was removed, and 150 ␮l of substrate solution was added to each well. The assay plates were then mixed by rocking and incubated for 10 min at room temperature to completely disrupt the cells. Samples of the resultant lysates plus the substrateonly controls were transferred to opaque-walled 96-well plates (200 ␮l per well), and luminescence was quantified using the Victor3 plate reader. Readings from the substrate-only control wells were subtracted from the readings for the wells containing cell lysates, and the resultant data were plotted in Prism to determine 50% cytotoxic concentrations (CC50s). Structure-based homology modeling of HIV-2 RT. To model the inhibition of HIV-1 and HIV-2 by BMS-986001, the files for the structures with PDB accession numbers 1RTD (HIV-1BH10 RT-polymerase complex with DNA template-primer and deoxythmidine-5=-triphosphate) (44) and 1MU2 (unliganded HIV-2ROD RT) (45) were downloaded from the RCSB Protein Data Bank (PDB; http://rcsb.org; accessed 30 March 2015) and imported into the USCF Chimera program (version 1.10; University of California, San Francisco, CA) (46). BMS-986001 (PubChem accession number 3008897; https://pubchem.ncbi.nlm.nih.gov) was then superimposed onto the incoming dTTP in the structure with PDB accession number 1RTD, and the 4=-oxygen of the analog was ligated to the ␣-phosphate of the natural substrate using the Build Structure feature of Chimera. The resulting model was submitted to the YASARA energy minimization server (www.yasara.org/minimizationserver.htm) (47) for energy minimization. To construct an analogous model of HIV-2 RT, the amino acid sequences of the structures with PDB accession numbers 1RTD and 1MU2 were aligned using the Clustal Omega algorithm (48) in the Align Chain Sequences utility of Chimera. This alignment was used to generate a homology model of HIV-2 RT via the Chimera interface to the MODELLER program (version 9.14) (49), with the nonminimized model of the structure with PDB accession number 1RTD-BMS-986001 being chosen as the reference structure. Best-fit solutions (i.e., those with the lowest root mean square deviation [RMSD] relative to that of the structure with PDB accession number 1RTD) were selected individually for the p68 and p58 subunits, and the resultant model of the HIV-2 RT/ BMS-986001 complex was submitted to the YASARA energy minimization server. Amino acid substitutions K65R, Q151M, and M184V were introduced into the YASARA-minimized models of the HIV-1 and HIV-2

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TABLE 1 Susceptibilities of HIV-1NL4-3 and HIV-2ROD9 to d4T and BMS-986001 EC50 (nM)a Inhibitor

Cell/assay type

HIV-1NL4-3

HIV-2ROD9

Fold differenceb

d4T BMS-986001 BMS-986001

MAGIC-5Ac/single cycle MAGIC-5A/single cycle CEMssd/multicycle

1,300 ⫾ 330 (4) 890 ⫾ 370 (10) 4.2 ⫾ 3.7 (4)

1,300 ⫾ 470 (3) 74 ⫾ 34 (11) 0.14 ⫾ 0.11 (3)

1 12 30

EC50, 50% effective concentration. Values are means ⫾ standard deviations. Values in parentheses indicate the number of independent determinations performed for each strain. EC50 for HIV-1NL4-3 divided by EC50 for HIV-2ROD9. c CD4⫹ CXCR4⫹ CCR5⫹ HeLa cells containing an HIV-inducible reporter gene (HIV LTR–␤-galactosidase). The CC50s in this cell type were ⬎100 ␮M for both d4T and BMS986001. d Immortalized human T lymphocytes with 4-day spreading of infections. The CC50 for BMS-986001 in this cell type was ⬎2,000 ␮M. a b

RTs using the Rotamers tool of Chimera. Appropriate rotamers were selected for the mutated residues on the basis of three criteria: (i) the closest conformational similarity to the corresponding residue in the structure with PDB accession number 1RTD, (ii) an absence of clashes with surrounding amino acid residues, and (iii) the highest probability score in the Dunbrack backbone-dependent rotamer library (50). In cases where multiple rotamers were possible, the alternative conformations are discussed below.

RESULTS

Anti-HIV-2 activity of BMS-986001 and d4T. We initially used our established MAGIC-5A indicator cell assay (15) to evaluate the activity of BMS-986001 against two prototypic HIV isolates derived from full-length molecular clones: HIV-1NL4-3 (group M, subtype B) and HIV-2ROD9 (group A). This approach quantifies drug sensitivity in a single round of HIV infection and avoids factors that could potentially influence the resultant EC50s, including strain-to-strain differences in syncytium formation, cytopathic effects, replication rates, and host cell tropism. Previous comparisons of HIV-1 and HIV-2 (with respect to both intrinsic NRTI sensitivity and the levels of NRTI resistance observed for RT mutants with site-directed mutations) showed that the results of the MAGIC-5A cell assay are consistent with those obtained with other cell types, including peripheral blood mononuclear cells (PBMCs) (11, 13, 15, 25, 51–53). We also included the related nucleoside analog d4T in head-to-head runs of the single-cycle assay with BMS-986001. Importantly, d4T and BMS-986001 differ only by the presence of a 4=-ethynyl group in the latter compound (Fig. 1), enabling a direct assessment of the effect of the 4= substitution on antiviral activity. HIV-1NL4-3 and HIV-2ROD9 were equally susceptible to d4T in the MAGIC-5A cell single-cycle assay, with the strains having nearly identical dose-response profiles with the inhibitor (see Fig. S1A in the supplemental material). Mean EC50s ⫾ standard deviations for d4T were 1,300 ⫾ 330 nM for HIV-1NL4-3 (n ⫽ 4 determinations) and 1,300 ⫾ 470 nM for HIV-2ROD9 (n ⫽ 3) (Table 1). These results are consistent with those in previous reports of the activity of d4T against HIV-1 and HIV-2 (11, 15) and are also concordant with data showing that HIV-1 and HIV-2 are comparably sensitive to all FDA-approved NRTIs in culture (11, 13, 15, 51, 52). In contrast, BMS-986001 showed greater activity against HIV-2ROD9 than against HIV-1NL4-3 (see Fig. S1B in the supplemental material); in multiple, independent determinations, the mean EC50s for BMS-986001 were 74 ⫾ 34 nM for HIV-2ROD9 (n ⫽ 11) and 890 ⫾ 370 nM for HIV-1NL4-3 (n ⫽ 10) in the single-cycle assay (Table 1; see also Fig. S1C in the supplemental material). HIV-2ROD9 also showed greater sensitivity to BMS986001 in 4-day infections of an immortalized T cell line (CEMss),

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with the mean EC50 for HIV-2ROD9 being 30-fold lower than that for HIV-1NL4-3 (Table 1). In both assays, the antiretroviral activity of BMS-986001 was not attributable to deleterious effects on host cell replication or viability; CC50 values for BMS-986001 (on the basis of measurements of ATP levels) were ⬎100 ␮M in MAGIC-5A cells and ⬎2,000 ␮M in cells of the CEMss cell line. To determine the effect of varying the multiplicity of infection (MOI) on BMS-986001 sensitivity, we performed a series of single-cycle dose-response assays in which MAGIC-5A indicator cells were infected with serial 2-fold dilutions of HIV-1NL4-3 and HIV2ROD9; for both strains, the MOI ranged from 0.0042 to 0.134. HIV-2ROD9 consistently showed greater sensitivity to BMS986001, with EC50s ranging from 5.7- to 11.9-fold higher than those seen for HIV-1NL4-3 (see Table S1 in the supplemental material). For both HIV-1NL4-3 and HIV-2ROD9, there was no significant trend toward an increase or decrease in the EC50 as a function of the MOI (P ⫽ 0.115 and 0.152, respectively; analysis of variance [ANOVA] with posttest for linear trend). Altogether, the aggregate EC50s (means ⫾ SDs) in these experiments were 83 ⫾ 30 nM for HIV-2ROD9 (n ⫽ 15) and 700 ⫾ 290 nM for HIV-1NL4-3 (n ⫽ 20). Inhibition of other HIV-2 isolates from ART-naive individuals. In addition to HIV-2ROD9, we tested the activity of BMS986001 against five other group A HIV-2 isolates, as well as two group B strains (HIV-2EHO and HIV-2CDC310072), using the singlecycle assay. Three additional HIV-1 isolates representing group M subtypes A and B and group O were included in the analysis. The resultant EC50s ranged from 450 to 890 nM for HIV-1, whereas they ranged from 30 to 81 nM for HIV-2 (Table 2). Within each HIV type, the difference between the least sensitive and the most sensitive strain was 1.9-fold for HIV-1 and 2.7-fold for HIV-2. Overall, the mean EC50 for HIV-2 was 9.5-fold lower than the mean value observed for HIV-1 (P ⫽ 0.003, two-tailed MannWhitney test) (Table 2). Activity against NRTI-resistant HIV-2 mutants. To examine the resistance profile of BMS-986001, we tested the activity of the inhibitor against a collection of site-directed HIV-2 RT mutants that were generated in the full-length pROD9 molecular clone. This panel represents the most common RT variants that are observed in HIV-2-infected patients receiving NRTI-containing regimens (19, 22, 26, 54). We also generated and tested a recombinant clone in which the 5= half of the RT-encoding region of HIV2ROD9 was replaced with the corresponding sequence from an HIV-2-infected patient; this individual had an extensive history of NRTI- and PI-based ART. The resultant construct (pROD9-4.7a) carried K65R, N69S, V111I, Q151M, M184V, and other polymorphisms at RT sites that are not associated with NRTI resistance

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TABLE 2 Activity of BMS-986001 against HIV-1 and HIV-2 isolates from treatment-naive individuals

TABLE 3 Susceptibilities of HIV-1 and HIV-2 RT mutants to BMS-986001

HIV type

Strain

Group/subtype

EC50 (nM)a

nb

HIV-1

NL4-3 LAI 92UG029 MVP5180-91

M/B M/B M/A O

890 ⫾ 370 610 ⫾ 340 450 ⫾ 110 470 ⫾ 94

10 3 3 3

ROD9 7924A MVP15132 60415K CBL20 CBL23 CDC310072 EHO

A A A A A A B B

74 ⫾ 34 69 ⫾ 8.5 64 ⫾ 19 78 ⫾ 9.2 81 ⫾ 10 75 ⫾ 5.4 30 ⫾ 12 42 ⫾ 9.3

11 3 3 3 3 3 3 3

HIV-2

a

EC50, 50% effective concentration. Values were measured in single-cycle doseresponse assays using MAGIC-5A indicator cells. Values are means ⫾ standard deviations from multiple assays performed on different days. The mean ⫾ standard deviation EC50s for HIV-1 and HIV-2 were 610 ⫾ 200 and 64 ⫾ 18 nM, respectively. b n, number of independent dose-response assays performed for each strain.

(see Materials and Methods for the patient’s treatment history and additional sequence information). Lastly, we included the M184V mutant of HIV-1NL4-3 in our analysis as a known BMS-906001resistant strain (32). Substitutions typically associated with thymidine analog resistance in HIV-1 (i.e., T215Y/F, M41L, and others) were not included in our experiments, as these are rarely observed in HIV-2 (19, 22, 26, 54) and do not confer measurable NRTI resistance in HIV-2ROD9 in culture (25) (data not shown); this difference is attributable to the absence of primer-unblocking activity in HIV-2 RT (55, 56). As previously reported (32), the M184V substitution in HIV1NL4-3 conferred low-level resistance to BMS-986001 in the singlecycle assay (a 3.3-fold change in the mean EC50 relative to that for the wild-type HIV-1NL4-3 clone; Table 3). In contrast, the phenotypes of the HIV-2ROD9 variants ranged from hypersusceptible (i.e., the variant was more sensitive to the drug than the wild type) to highly resistant (Table 3). The K65R replacement conferred 3.2-fold hypersusceptibility to BMS-986001 in HIV-2, whereas Q151M had no effect on BMS-986001 sensitivity. The HIV-2 M184V and K65R ⫹ M184V mutants were 15- and 16-fold resistant to the inhibitor, respectively; higher levels of resistance were observed for the Q151M ⫹ M184V and K65R ⫹ Q151M ⫹ M184V mutants (53- and 111-fold, respectively). In addition, virus from the patient-derived construct (HIV-2ROD9-4.7a) was 105fold resistant to BMS-986001 (Table 3). Structural modeling of the HIV-2 RT/BMS-986001 complex. To identify structural features that correlate with increased BMS986001 sensitivity in HIV-2, we constructed a model of BMS986001-5=-triphosphate bound to the polymerase active site of HIV-2ROD9 RT (Fig. 2A). Briefly, we superimposed BMS-986001 onto the sugar and base of dTTP in a previously published structure of the HIV-1BH10 RT/polymerase ternary complex (PDB accession number 1RTD) (44). We then used homology alignment and energy minimization to build a corresponding model of HIV2ROD9 RT in complex with the inhibitor. Both models showed BMS-986001-5=-triphosphate to be the incoming nucleotide in the RT-DNA template/primer-deoxynucleoside triphosphate (dNTP) complex and represent the stage of the polymerization

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Wild type K65R Q151M M184V K65R ⫹ M184V Q151M ⫹ M184V K65R ⫹ Q151M ⫹ M184V

0.074 ⫾ 0.034 0.023 ⴞ 0.002 0.078 ⫾ 0.020 1.1 ⴞ 0.29 1.2 ⴞ 0.19 3.9 ⴞ 1.7 8.2 ⴞ 3.0

11 3 3 4 3 4 4

0.3 1.1 15 16 53 111

K65R ⫹ N69S ⫹ V111I ⫹ Q151M ⫹ M184V

7.8 ⴞ 2.0

3

105

HIV-2ROD9

HIV-2ROD9-4.7a

a Viruses produced from full-length plasmids pNL4-3, pROD9, and patient-derived clone pROD9-4.7a. b The amino acid changes listed for HIV-1NL4-3 and HIV-2ROD9 were engineered via site-directed mutagenesis. The changes listed for HIV-2ROD9-4.7a were carried by the patient-derived insert; see Materials and Methods for additional sequence information for the HIV-2ROD9-4.7a construct. c EC50, 50% effective concentrations (mean ⫾ standard deviation) measured in the MAGIC-5A cell single-cycle assay. Values shown in bold are significantly different from the value for the corresponding wild-type clone (P ⬍ 0.05, ANOVA of log10transformed EC50s with Tukey’s posttest). d n, number of independent dose-response assays performed for each strain. e EC50 for the mutant divided by EC50 for the corresponding wild-type clone (wild-type HIV-2ROD9 for the patient-derived strain).

cycle immediately prior to phosphodiester bond formation (i.e., the N-site complex [57]). The energy-minimized model of HIV-2ROD9 RT with BMS986001 (Fig. 2A and B) shows an overall architecture that is similar to the original HIV-1 RT/dTTP crystal structure (PDB accession number 1RTD) and to the energy-minimized model of the HIV-1 enzyme with BMS-986001-5=-triphosphate in the polymerase active site (␣-carbon RMSDs ⫽ 0.581 and 0.611 Å, respectively). As previously suggested for HIV-1 (30), HIV-2ROD9 RT appears to accommodate the 4=-ethynyl group of BMS-986001 without significant rearrangements of amino acids in the dNTP-binding site (Fig. 2C), although subtle differences in the positioning of V111, A114, Y115, M184, and D185 are apparent in the HIV-1BH10 and HIV-2ROD9 RT models (Fig. 2D). In addition, residue F160 of the HIV-1BH10 enzyme is closely associated with two amino acids, I118 and L214, that differ in the HIV-2ROD9 sequence (V118 and F214 of HIV-2ROD9 RT, respectively; Fig. 2D). In the HIV-2ROD9 model, the longer side chains at positions 118 and 214 coincide with a rotation and shifting of the F160 phenyl group to a position that is nearly perpendicular to the 4=-ethynyl extension of BMS986001 (Fig. 2D). However, sites 118 and 214 are variable in the HIV-1 and HIV-2 RTs, including the RTs of the isolates tested in this study (see Table S2 in the supplemental material), and the identity of the amino acids at these positions does not correlate with BMS-986001 sensitivity in culture, suggesting that other residues (or, possibly, factors outside RT) are responsible for the higher sensitivity to the drug in HIV-2. Structural correlates of BMS-986001 resistance in HIV-2. To further explore the mechanisms by which HIV-2 might acquire resistance to BMS-986001, we introduced the K65R, Q151M, and M184V changes individually into the energy-minimized model of HIV-2 RT and then asked whether different rotamers of the mutated residues might affect the binding or positioning of the BMS-

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A

B

C

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986001-5=-triphosphate in the polymerase active site. For K65R, two rotamers are in the proper orientation to maintain hydrogen bonding between the arginine guanidino group and the ␥-phosphate of the inhibitor (44) (Fig. 3, rotamers a and b). Rotamer a holds a conformation similar to that of the wild-type lysine in the HIV-1 and HIV-2 models, whereas rotamer b is a better match to the wild-type residue in the original HIV-1 structure (PDB accession number 1RTD; data not shown). This uncertainty over the position of the arginine side chain precludes further insights regarding the mechanism of K65R-mediated BMS-986001 hypersusceptibility. In contrast, for Q151M and M184V, only a single rotamer of each mutant residue could be accommodated into the HIV-2 model without significant steric clashes with neighboring amino acids (Fig. 3). In agreement with our drug sensitivity analysis, the methionine replacement at position 151 did not appear to significantly affect the interaction of BMS-986001-5=-triphosphate with HIV-2 RT. However, for M184V, the valine replacement resulted in a predicted steric clash between the mutant side chain and the 4=-ethynyl group of the substrate analog (Fig. 3); accommodation of valine at position 184 would require an ⬃1-Å displacement of the 4=-ethynyl moiety toward the ␣-carbon of Y115. These findings suggest that, in HIV-2, M184V-mediated resistance to BMS986001 results from steric constraints in the area surrounding the 4= extension of the inhibitor. DISCUSSION

FIG 2 Homology model of the polymerase complex of HIV-2 RT with BMS986001-5=-triphosphate as the incoming nucleotide substrate. (A) Overall structure of heterodimeric HIV-2 RT (p68 and p58 subunits, shown as light blue and gray ribbons, respectively) with double-stranded DNA templateprimer (purple and dark blue ribbons and sticks) and BMS-986001-5=triphosphate (red sticks). Green spheres represent magnesium ions. This energy-minimized model was based on the structure of HIV-1 RT in complex with deoxythymidine-5=-triphosphate (PDB accession number 1RTD) (44), as described in Materials and Methods. (B) Closeup of the polymerase domain of HIV-2 RT showing BMS-986001-5=-triphosphate (solid Corey-Pauling-Koltun [CPK]-colored spheres) paired with the complementary nucleotide of the template strand (semitransparent CPK spheres). Catalytic aspartate residues D110 and D185 are shown as CPK-colored sticks; the aromatic side chain of F160 is depicted in red. (C) Surface-rendered views of HIV-1 RT and HIV-2 RT showing the 4= pocket of each enzyme. Surface colors indicate amino acid hydrophobicity according to the Kyte-Doolittle scale (71). (D) Superimposition of BMS-986001-5=-triphosphate and amino acids proximal to the 4=ethynyl group of the inhibitor in the HIV-1 and HIV-2 RTs. For HIV-1, the carbon atoms of BMS-986001 are shown in dark gray, while the carbons in the amino acid residues are shown as tan sticks. For the HIV-2 model, the carbon atoms for BMS-986001 are depicted in light gray, and those of the amino acids are in light blue. The remaining atoms in both models are shown in CPK coloring. Labels for RT positions 118 and 214 are colored as per the carbon atoms of the corresponding residues (i.e., tan for HIV-1 and light blue for HIV-2). Labels for the remaining residues, which are identical in HIV-1 and HIV-2, are colored as per the HIV-2 model. For clarity, in panels C and D, a single magnesium ion is shown in the polymerase active site. All illustrations were generated in UCSF Chimera (version 1.10).

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Novel HIV-2-active antiretrovirals are urgently needed in West Africa and other areas where the virus is prevalent (5, 58). In the present study, we examined the ability of the investigational nucleoside analog BMS-986001 (Fig. 1) to inhibit HIV-2 replication in cell culture. Our results show that BMS-986001 is highly active against the virus; mean EC50s were 64 ⫾ 18 nM in the single-cycle MAGIC-5A cell assay (for multiple HIV-2 isolates; Table 2) and 0.14 ⫾ 0.11 nM in spreading infections of CEMss cells (for HIV2ROD9; Table 1). Remarkably, these values were 9.5- and 30-fold lower, respectively, than the corresponding EC50s for HIV-1 (Tables 1 and 2). Thus, relative to its activity against HIV-1, BMS986001 shows more potent activity against HIV-2 in culture. In addition, BMS-986001 was 18-fold more potent than d4T against HIV-2 in the single-cycle assay, whereas these two NRTIs showed comparable levels of activity against HIV-1 (Table 1). These findings suggest that the interaction between the 4=-ethynyl moiety of BMS-986001 and the polymerase active site of HIV-2 RT differs from the corresponding enzyme-inhibitor interaction in HIV-1 RT, resulting in stronger inhibition of HIV-2 replication. Using homology modeling, we attempted to identify structural features in the HIV-1 and HIV-2 RTs that might explain the observed differences in BMS-986001 susceptibility. Both models contained a well-defined hydrophobic pocket that accommodates the 4=-ethynyl group of BMS-986001-5=-triphosphate when the inhibitor is present as the incoming nucleotide substrate (i.e., in the N site of the polymerase complex) (57). The entrance to this 4= pocket, which is formed by residues A114, Y115, M184, and the main-chain atoms of D185, is slightly larger in the HIV-2ROD9 model (a difference of ⬃0.4 Å relative to its size in HIV-1BH10) (Fig. 2C). The size and shape of the 4= pocket of HIV-2 RT could potentially enhance binding of the drug at the active site and/or enable a more favorable positioning of the analog triphosphate for

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K65R a

b 2.4

2.5

1.8

Q151M

D110

V111

50° Y115 M184V

Y115 M184V F160

F160

FIG 3 Predicted structural changes resulting from the K65R, Q151M, and M184V replacements in HIV-2 RT. BMS-986001-5=-triphosphate and amino acid side chains in the wild-type HIV-2 model are colored as described in the legend to Fig. 2D. Carbon atoms in the mutant amino acids are shown in green; the remaining atoms are shown in Corey-Pauling-Koltun coloring. Stippled spheres indicate the van der Waals radii for the 4=-ethynyl moiety of BMS-986001 and the ␥-carbon of valine at position 184 of HIV-2 RT. The numbers in red indicate distance (in angstroms).

phosphodiester bond formation. Additional biochemical and structural studies are required to address these hypotheses. Our analysis of the phenotypes conferred by the most common NRTI-associated mutations in HIV-2 RT suggests that BMS986001 might exhibit a favorable resistance profile in HIV-2infected individuals. The K65R change, which confers low-level resistance to didanosine (4-fold) and moderate to high-level resistance to 3TC and emtricitabine (FTC) in HIV-2 (38- and 85-fold) (25), resulted in 3.2-fold hypersusceptibility to BMS986001 (Table 3). Q151M had no effect on BMS-986001 sensitivity, whereas the M184V replacement in RT, which produces highlevel FTC and 3TC resistance in HIV-2 (⬎200-fold) (25), resulted in moderate resistance to the investigational NRTI (15-fold; Table 3). Similar changes in BMS-986001 susceptibility have been reported for K65R, Q151M, and M184V mutants of HIV-1 (32, 34, 59). In our energy-minimized model, the M184V replacement appears to narrow the interior of the 4= pocket of HIV-2 RT; binding of BMS-986001-5=-triphosphate would require an ⬃1-Å shift in the location of the 4=-ethynyl moiety relative to that in the corresponding wild-type enzyme (Fig. 3). These observations are consistent with the idea that proper positioning of the 4=-ethynyl group is critical for antiretroviral activity. In addition, it is important to note that M184V and K65R ⫹ M184V mutants of HIV2ROD-9 exhibited EC50s for BMS-986001 that were comparable to the EC50 for wild-type HIV-1NL4-3 (differences of 1.3-fold or less; Table 3). Higher levels of BMS-986001 resistance in HIV-2 (i.e., a ⬎50-fold increase in the EC50 relative to that for the HIV-2ROD9 wild type and a ⬎4-fold increase in the EC50 compared to that for the HIV-1NL4-3 wild type) appear to require the combination of Q151M and M184V (Table 3). These findings suggest that, in many cases, BMS-986001 would retain substantial antiviral activity against NRTI-resistant mutants in HIV-2-infected individuals. However, we cannot presently rule out the possibility that other amino acid changes in HIV-2 RT might lead to high-level BMS986001 resistance. In addition, the level of in vitro resistance that correlates with virologic failure in BMS-986001-treated patients is unknown.

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By virtue of its 4=-ethynyl extension, BMS-986001 is chemically related to other nucleoside analogs bearing a 4= modification of the ribosyl group. Of these, 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is highly active against HIV-1 in culture, with EC50s in the low to subnanomolar range, and shows minimal cytotoxicity in PBMCs (CC50, ⬎10 ␮M) (60, 61). Unlike BMS986001, EFdA bears an extendable 5=-hydroxyl group and is therefore not an obligate chain terminator. Instead, EFdA inhibits HIV-1 RT through multiple mechanisms; incorporation of the analog slows RT translocation and reduces the efficiency of addition of the next nucleotide, but in cases where the next nucleotide is polymerized, the presence of EFdA at the penultimate site of the primer strand results in delayed chain termination by blocking further DNA synthesis (62, 63). HIV-1 RT also misincorporates EFdA at template sites other than thymidine, resulting in terminal mismatches that are poor substrates for the primer-unblocking activity of RT (62). Of note, EFdA has been reported to inhibit HIV-2EHO replication in MT-2 cells at subnanomolar concentrations (60); the activity of EFdA against a broader range of HIV-2 isolates, as well as its mechanism of inhibition of HIV-2 RT, remains to be determined. Historically, in West Africa, ART for HIV-2 infection has been characterized by high rates of immunovirologic failure and multiclass (NRTI and PI) drug resistance (22, 58, 64–66). Newer ARVs, including generic FTC and tenofovir disoproxil fumarate (TDF), ritonavir-boosted darunavir, and the INSTI raltegravir, have recently been made available to a limited number of HIV-2infected patients in some West African HIV treatment programs. While these compounds may be beneficial, concerns regarding the extensive cross-resistance seen within the NRTI and PI classes in HIV-2 remain (5, 7, 9, 19, 21, 22, 25, 54, 67, 68), and emergent resistance to raltegravir has been reported in HIV-2-infected individuals (18, 20, 23, 24, 26, 27, 69, 70). Our analysis suggests that BMS-986001 might help fill the need for newer HIV-2-active ARVs. Among the eight structurally diverse NRTIs tested in our laboratory, BMS-986001 is second only to TDF in terms of antiHIV-2 activity (Fig. 4). To our knowledge, BMS-986001 is the first

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REFERENCES

10

EC50 ( M)

1

0.100

0.010

ddI

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ABC

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3TC

FTC

AZT

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DRV

LPV

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FIG 4 Antiviral activity of BMS-986001 relative to the activities of other ARVs. All values in this figure were obtained using HIV-2ROD9 in the singlecycle MAGIC-5A cell assay and are the means from three or more independent determinations. EC50s for d4T and BMS-986001 are from the present study (Tables 1 and 2). Values for the remaining ARVs are from previously published studies by our group (9, 17, 25, 72). Abbreviations for drug classes and names are as follows: INSTI, integrase strand-transfer inhibitor; PI, protease inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; DTG, dolutegravir; RAL, raltegravir; EVG, elvitegravir; SQV, saquinavir; LPV, lopinavir; DRV, darunavir; TDF, tenofovir disoproxil fumarate; AZT, zidovudine; FTC, emtricitabine; d4T, stavudine; 3TC, lamivudine; ABC, abacavir; PMPA, tenofovir [unmodified; (R)-9-(2-phosphonylmethoxypropyl)adenine]; ddI, didanosine.

and only NRTI that, when tested against a diverse panel of HIV-1 and HIV-2 isolates, exhibits more potent inhibition of HIV-2 replication in culture. BMS-986001 also retains full or partial activity against HIV-2 variants that harbor treatment-associated single amino acid changes in RT (specifically, K65R, Q151M, and M184V). Collectively, our findings suggest that BMS-986001 and perhaps other 4=-ethynyl nucleoside analogs should be considered for further preclinical and clinical testing as potential components of ART for HIV-2-infected individuals. ACKNOWLEDGMENTS These studies were supported by grants to G.S.G. from the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH, NIAID; R01AI120765 and 2R01-AI060466), the UW Center for AIDS Research (CFAR; an NIH-funded program; P30 AI027757), and the UW Royalty Research Fund (A92723). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). We thank Bristol-Myers Squibb for the provision of BMS-986001. Additional University of Washington-Dakar HIV-2 Study Group members are as follows: Fatima Sall, Fatou Traore, Khadim Faye, Marie Pierre Sy, Bintou Diaw, Ousseynou Ndiaye, Amadou Bale Diop, and Marianne Fadam Diome (Clinique des Maladies Infectieuses Ibrahima DIOP Mar, Centre Hospitalier Universitaire de Fann, Universite’ Cheikh Anta Diop de Dakar, Dakar, Senegal); Alassane Niang, ElHadji Ibrahima Sall, Ousseynou Cisse, Ibrahima Tito Tamba, Jean Philippe Diatta, Raphael Bakhoum, Jacque Francois Sambou, and Juliette Gomis (Région Médicale de Ziguinchor, Ziguinchor, Casamance, Senegal); and Stephen Hawes, John Lin, Ming Chang, Robert Coombs, James Mullins, and Nancy Kiviat (University of Washington, Seattle, Washington). G.S.G. has received research grants and support from the U.S. NIH, UW, Gilead Sciences, Alere Technologies, and Abbott Molecular Diagnostics. No other disclosures are reported.

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