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Antimicrobial Agents and Chemotherapy, March 2009, p. 1194-1203, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.00984-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Preclinical Evaluation of GS-9160, a Novel Inhibitor of Human Immunodeficiency Virus Type 1 Integrase{triangledown} ,{dagger}

Gregg S. Jones, Fang Yu, Ameneh Zeynalzadegan, Joseph Hesselgesser, Xiaowu Chen, James Chen, Haolun Jin, Choung U. Kim, Matthew Wright, Romas Geleziunas, and Manuel Tsiang*

Gilead Sciences, Foster City, California 94404

Received 23 July 2008/ Returned for modification 24 August 2008/ Accepted 16 December 2008


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ABSTRACT
 
GS-9160 is a novel and potent inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN) that specifically targets the process of strand transfer. It is an authentic inhibitor of HIV-1 integration, since treatment of infected cells results in an elevation of two-long terminal repeat circles and a decrease of integration junctions. GS-9160 has potent and selective antiviral activity in primary human T lymphocytes producing a 50% effective concentration (EC50) of ~2 nM, with a selectivity index (50% cytotoxic concentration/EC50) of ~2,000. The antiviral potency of GS-9160 decreased by 6- to 10-fold in the presence of human serum. The antiviral activity of GS-9160 is synergistic in combination with representatives from three different classes of antiviral drugs, namely HIV-1 protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Viral resistance selections performed with GS-9160 yielded a novel pattern of mutations within the catalytic core domain of IN; E92V emerged initially, followed by L74M. While E92V as a single mutant conferred 12-fold resistance against GS-9160, L74M had no effect as a single mutant. Together, these mutations conferred 67-fold resistance to GS-9160, indicating that L74M may potentiate the resistance caused by E92V. The pharmacokinetic profile of GS-9160 in healthy human volunteers revealed that once-daily dosing was not likely to achieve antiviral efficacy; hence, the clinical development of this compound was discontinued.


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INTRODUCTION
 
After human immunodeficiency virus type 1 (HIV-1) entry and uncoating, the viral RNA is reverse transcribed by the viral reverse transcriptase into a double-stranded linear DNA. Both ends of this linear DNA are then processed at the 3' termini by the integrase (IN) enzyme. Specifically, IN removes a dinucleotide from each 3' terminus through a reaction referred to as 3' processing. The IN-DNA complex is then transported into the nucleus where IN performs concerted integration of both viral DNA ends into host chromosomal DNA by a reaction referred to as strand transfer. The integration of viral DNA into host chromosomal DNA is essential for HIV-1 replication, making the inhibition of HIV-1 IN function an attractive antiviral strategy (9, 35, 36, 42).

Historically, treatment of individuals infected with HIV-1 has relied on agents targeting two of the viral enzymes, reverse transcriptase and protease. Despite important clinical results achieved through the use of combinations of these agents, the continuous emergence of drug resistance remains a significant problem which fuels the need to discover novel drugs targeting other steps of the HIV-1 life cycle. IN is the third virally encoded enzyme essential for HIV-1 replication, and inhibitors of the IN strand transfer activity have recently been validated clinically. Raltegravir (MK-0518) was approved for clinical use in 2007 and is dosed twice daily (8, 41), while elvitegravir (GS-9137) is in late-stage clinical development and is dosed once daily with ritonavir (47).

In a 10-day monotherapy dose-ranging study performed with raltegravir dosed twice daily for treatment-naïve patients, the mean decrease in HIV RNA levels from baseline ranged from 1.7 to 2.2 log10 copies/ml. (31). In a subsequent study of raltegravir dosed twice daily in combination with 300 mg lamivudine (3TC) and 300 mg tenofovir dosed once daily, raltegravir demonstrated durable HIV-1 RNA decline (32). Clinical trials with raltegravir conducted with treatment-experienced patients showed superior efficacy compared to trials conducted with placebo plus optimized baseline therapy (17, 8, 41). A 10-day dose-ranging study conducted with elvitegravir in treatment-naïve patients demonstrated that 50 mg ritonavir dosed once daily resulted in mean reductions from baseline in HIV-1 RNA of 1.99 log10 copies/ml (10). In a phase II trial, 125 mg elvitegravir dosed once daily and coadministered with ritonavir was shown to have potent antiviral activity that was superior to a ritonavir-boosted protease inhibitor (PI) regimen (47). Elvitegravir is currently in phase III studies. HIV IN inhibitor-resistant mutants that develop clinically display cross-resistance to both raltegravir and elvitegravir (11, 43).

The IN strand transfer inhibitor L-870,812 provided the first proof of concept that antagonizing this enzyme can suppress retroviral replication in vivo (21). Subsequently, the close analog L-870,810 was shown to be efficacious in HIV-1-infected humans (13, 18, 29). Because L-870,810 can exist as two different conformers, with the higher-energy conformer being active against IN, a preorganized tricyclic pharmacophore was designed to lock the structure into the active conformation and increase binding affinity (23). GS-9160, which emerged from this effort, retains inhibitory activity against the IN strand transfer reaction and displays potent anti-HIV-1 activity (14, 24, 34). In this report, we describe the biological characterization of GS-9160 and the development of a novel pattern of viral resistance mutations to GS-9160.


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MATERIALS AND METHODS
 
Compounds. GS-9160 and capravirine (CPV) were synthesized at Gilead Sciences, Inc. L-870,810 was contract synthesized by Combi-Blocks Inc. (San Diego, CA). GS-9137 was supplied by Japan Tobacco Inc. Amprenavir (APV), lopinavir (LPV), atazanavir, and nelfinavir were isolated from commercial tablets. Tenofovir disoproxil fumarate (TDF) was synthesized in the process chemistry department of Gilead Sciences (lot no. 4331-05-XK-1). Emtricitabine (FTC) was obtained from Triangle Pharmaceuticals, Inc. (now a part of Gilead Sciences). Efavirenz (EFV) was purchased from Toronto Research Chemicals Inc. (catalog no. E425000; North York, Ontario, Canada). 3TC was purchased from Moravek Biochemicals Inc. (catalog no. M-975; Brea, CA). Zidovudine (AZT) (catalog no. A-2169), stavudine (d4T) (catalog no. D-1413), and ribavirin (RBV) (catalog no. R-9644) were purchased from Sigma (St. Louis, MO).

Cells. MT-2 cells were obtained from Stanford University, and MT-4 cells were obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD). SupT1 cells were obtained from the American Type Culture Collection (Rockville, MD). MT-2, MT-4, and SupT1 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. The SODk1 2G cell line that produces vesicular stomatitis virus G glycoprotein-pseudotyped viral particles used in single-cycle infection was licensed from the Salk Institute, La Jolla, CA. (3, 26). SODk1 2G cells were maintained in Dulbecco modified Eagle medium supplemented with 10% tetracycline-free FBS (Clontech, Mountain View, CA), 1 mM glutamine, 1 mM pyruvate, 1 µg/ml doxycycline, and antibiotics. Production of the pseudotyped particles from SODk1 2G cells has been previously described (26).

Human CD4-positive T lymphocytes (T cells) were isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Stanford, CA). Cells from these buffy coats were centrifuged over endotoxin-free Ficoll-Paque Plus (GE Healthcare Life Sciences, Piscataway, NJ), and the peripheral blood mononuclear cell layer was harvested. Residual red blood cells were removed by ammonium chloride hypotonic cell lysis. Total peripheral blood mononuclear cells were cultured in RPMI medium with Glutamax (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA), 1 µg/ml phytohemagglutinin P (Sigma-Aldrich, St. Louis, MO), and 10 U/ml interleukin-2 (Roche, Indianapolis, IN). After 48 h of culture, the nonadherent cell suspension was harvested and centrifuged. The CD4-positive T lymphocytes were isolated from the pellet using anti-CD4-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.

Viruses. HIV-1 strain IIIb was obtained from Advanced Biotechnologies Inc. (Columbia, MD). The nucleoside reverse transcriptase inhibitor (NRTI)-resistant virus containing mutation K65R in reverse transcriptase (RT) was obtained from Mark Wainberg (McGill AIDS Center, Montreal, Canada). The NRTI-resistant virus RT-M184V and nonnucleoside RT inhibitor (NNRTI)-resistant viruses RT-K103N and RT-Y181C were constructed by site-directed mutagenesis. The NRTI-resistant virus RT-6TAMs (containing the six thymidine analog mutations M41L, D67N, K70R, L210W, T215Y, and K219Q, which confer resistance to thymidine analogs) and the PI-resistant viruses PR-I84V/L90M and PR-G48V/V82A/L90M were constructed by homologous recombination in electroporated SupT1 cells.

Construction of infectious HIV-1 DNA clones with IN mutations. Mutations conferring resistance to IN inhibitors were introduced into the infectious wild-type HIV-1 DNA clone HXB2 by site-directed mutagenesis. Mutation(s) was confirmed by sequencing.

Expression and purification of HIV-1 IN. Recombinant IN containing an N-terminal six-His tag was expressed in BL21(DE3)pLysS bacteria. Bacteria were lysed with 0.5 mg/ml lysozyme and sonicated. After centrifugation, the pellet was resuspended in lysis buffer containing PIs (catalog no. P8849; Sigma, St. Louis, MO) and DNase I (catalog no. 104132; Roche, Indianapolis, IN) and incubated for 20 min on ice. Recombinant IN was solubilized by the addition of NaCl and CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} to final concentrations of 1.25 M and 10 mM, respectively. The slurry was stirred gently on ice for 30 min and centrifuged at 17,542 x g for 30 min. The supernatant was saved, and an equal volume of adjustment buffer (62.5 mM Tris-HCl [pH 7.6], 12.5 mM MgCl2, 10 mM CHAPS, 40 mM imidazole, 20% glycerol, 2.5 mM β-mercaptoethanol) was added. The supernatant was loaded at 1 ml/min onto an Ni-nitrilotriacetic acid column preequilibrated in column buffer (50 mM Tris-Cl [pH 7.6], 20 mM imidazole, 600 mM NaCl, 10 mM MgCl2, 10% glycerol, 10 mM CHAPS, 2 mM β-mercaptoethanol). After washing the column, IN was eluted with imidazole, and fractions containing IN were pooled and dialyzed to remove imidazole. The purity of IN preparations was generally >95%.

IN strand transfer assay. The IN strand transfer assay has been previously described (46). Briefly, biotinylated donor DNA was bound to streptavidin-coated plates. Unbound donor DNA was removed by washing, and IN was then added to each well to allow 3' processing of the donor DNA end for 30 min. Digoxigenin-tagged target DNA was next added to each well, and the 3'-end-joining reaction was allowed to proceed for 30 min. The wells were washed to remove unjoined target DNA, and the chemiluminescence signal was detected following addition of horseradish peroxidase-conjugated antidigoxigenin antibody. Test compounds were added immediately before the target DNA.

Antiviral and cytotoxicity assays. For the antiviral assay utilizing MT-2 and MT-4 cells, 50 µl of 2x test concentration of the fivefold serially diluted compound in culture medium with 10% FBS was added to each well of a 96-well plate (nine concentrations) in triplicate. MT-2 and MT-4 cells were infected with HIV-1 IIIb at a multiplicity of infection (MOI) of 0.01 for 3 h. Fifty microliters of infected cell suspension in culture medium with 10% FBS (~1.5 x 104 cells) was then added to each well containing 50 µl of diluted compound. The plates were then incubated at 37°C for 5 days. After 5 days of incubation, 100 µl of CellTiter-Glo reagent (catalog no. G7571; Promega Biosciences, Inc., Madison, WI) was added to each well containing MT-2 or MT-4 cells. Cell lysis was carried out by incubation at room temperature for 10 min, and chemiluminescence was read. For compound cytotoxicity assessment, the protocol was identical except that uninfected cells were used and compounds were serially diluted threefold.

For the antiviral assay utilizing phytohemagglutinin P and interleukin-2-stimulated T cells, the cells were infected in bulk culture with HIV-1 BaL at an MOI of ~0.001 for 3 h. After removal of unadsorbed virus, the cells were plated at 200,000 cells/well in a 96-well plate containing serially diluted compounds. After 5 days of incubation at 37°C, the supernatant was collected, and HIV-1 yield was quantified by p24 antigen capture enzyme-linked immunosorbent assay (Beckman Coulter, Fullerton, CA). To determine cytotoxicity, compounds were incubated with uninfected cells for 5 days, and cytopathic effect was measured by addition of the XTT reagent (Sigma, St. Louis, MO).

To study the effect of serum proteins on the antiviral activity of IN inhibitors, compounds were tested in the presence of either 35 mg/ml human serum albumin (HSA) (catalog no. A-1653; Sigma, St. Louis, MO) or 1.5 mg/ml {alpha}1-acid glycoprotein ({alpha}1-AGP) (catalog no. G-9885, Sigma, St. Louis, MO). To assess the effect of human serum on compound potency, the assay was performed in the presence of 10%, 20%, 35%, and 50% human serum from clotted whole blood (catalog no. 14-498E; Cambrex Bio Science, Walkersville, MD) at the following three different MOIs: 0.01, 0.02, and 0.04. The serum-adjusted 50% effective concentration (EC50) versus the fractional serum concentration plot was analyzed by curve fitting using the following equation:

Formula
where f is the fractional concentration of serum used; S is the protein concentration in 100% serum (pg/µl); M is the mean molecular mass of a serum protein (pg/pmol); n is the Hill coefficient, a lower estimate for the average number of binding sites for the inhibitor per protein molecule; EC50 is the 50% inhibitory concentration (IC50) in the absence of serum (µM); EC50x is the IC50 in the presence of serum (µM); KD is the apparent equilibrium dissociation constant of the inhibitor from the mean binding site (µM); and m is the relative MOI, where m = 1 represents an MOI of 0.01. See the derivation of this equation in the supplemental material.

Drug combination studies. The effect of combining any two drugs in the MT-2 antiviral assay was analyzed by two different methods, the Prichard and Shipman method using MacSynergy II software (38) and the combination index (CI) method (6) using CalcuSyn software (Biosoft, Ferguson, MO). MacSynergy II and CalcuSyn provide certain guidelines for the interpretation of the drug combination results, as shown in Fig. 1.


Figure 1
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  		FIG. 1. Interpretation of difference volumes and CI. The ranges of values that are equivalent between the two methods (MacSynergy II and CalcuSyn) are shaded similarly.

2-LTR circle accumulation assay. The assay for two-long terminal repeat (2-LTR) circle accumulation was adapted from a previously described method (3). SupT1 cells were infected in bulk culture with HIV-1 IIIb at an MOI of 10 and at a cell density of 4 x 106 cells/ml for 3 h at 37°C. Both the 2-LTR junction (2-LTR circles) and the host globin gene (used to normalize for cell number) were quantified using TaqMan real-time PCR. The copy number of 2-LTR circles was normalized to that of the globin gene and plotted against drug concentration for EC50 determination.

Alu-PCR assays. The Alu-PCR assay has been previously described (3). SupT1 cells were infected as a bulk culture with pseudotyped HIV-1 vector particles produced from the SODk1 2G cell line (3, 26) at an MOI of 10 and a cell density of 4 x 106 cells/ml for 3 h at 37°C. The plates were incubated at 37°C for 12 h for late RT product quantification and for 48 h for Alu-PCR product quantification. The levels of the late RT sequences and integration junctions quantified at 12 and 48 h postinfection, respectively, were normalized to the level of globin gene present in the sample at these time points. Quantification by PCR was performed by the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA).

Viral resistance selections. Resistance selections were carried out in 6-well tissue culture plates. MT-2 cells were seeded at a density of 0.5 x 106 cells per well in 5 ml of culture medium. Compounds were added at final concentrations corresponding to their antiviral EC50 or twice the EC50. HIV-1 IIIb was used at an MOI of ~0.01. The cultures were incubated at 37°C and split one-half to one-third once or twice a week depending on the growth status of the cells. The cytopathic effect manifested as syncytium formation was used to follow the progression of infections. Virus was harvested and transferred to fresh MT-2 cells in the presence of the same compound but at a twofold-higher concentration. Successive viral passages were obtained by repeating this procedure. The duration of each passage ranged from 10 to 15 days.

Clonal sequencing of viral DNA. For clonal sequencing of viral passages, total DNA was extracted from infected cells using the QIAamp DNA blood minikit (Qiagen, Valencia, CA). A 2,765-bp viral DNA fragment spanning from nucleotide 372 of the RT gene to nucleotide 127 of the Vpr gene was amplified by PCR and cloned into the pCR-XLTopo plasmid (Invitrogen, Carlsbad, CA). The IN gene from 24 individual plasmid clones was sequenced using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing reactions were analyzed by the ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA).


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RESULTS
 
GS-9160 inhibits HIV-1 IN function. The inhibitory activity of GS-9160 was evaluated in a biochemical assay that measures the capacity of IN to mediate strand transfer of viral donor DNA into host or target DNA. GS-9160 produced inhibitory activity (IC50 = 28 nM) that was comparable to those of L-870,810 (IC50 = 40 nM), elvitegravir (GS-9137) (IC50 = 44 nM), and raltegravir (MK-0518) (IC50 = 34 nM). These latter three HIV-1 IN strand transfer inhibitors were demonstrated to be efficacious in HIV-1-infected individuals (10, 13, 18, 29, 31) (Table 1).


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  		TABLE 1. Inhibition of integrase strand transfer activity

GS-9160 has potent antiviral activity. The antiviral activity of GS-9160 was evaluated in HIV-1-infected MT-2 and MT-4 cell lines as well as in primary human T lymphocytes (Table 2). The potency of GS-9160 was comparable in all three cell types, with an EC50 range of 0.7 to 2 nM, and the selectivity indices (SI; 50% cytotoxic concentration [CC50]/EC50) for GS-9160 were ~2,000 in MT-2 cells and ~2,600 in primary human T cells. The potency of GS-9160 was generally comparable to those of L-870,810 and MK-0518 but was lower than that of GS-9137 in these three cell types.


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  		TABLE 2. Antiviral activity of GS-9160 in MT-2 and MT-4 cells and human T lymphocytes

To assess the impact of human serum protein binding on the antiviral potency of GS-9160 and to provide a target trough concentration that would need to be attained in humans to achieve efficacy, the antiviral activity of GS-9160 was determined in the presence of human serum and the human serum components HSA and {alpha}1-AGP (Table 2). The EC50 of GS-9160 in 100% human serum was extrapolated from the EC50s determined in the presence of 10, 20, 35, and 50% human serum. The combined effect of both serum proteins, HSA and {alpha}1-AGP, increased the EC50 of GS-9160 by 7.1-fold to 15 nM in MT-2 cells, while the EC50s of L-870,810, MK-0518, and GS-9137 were increased by 22-, 6-, and 40-fold, respectively. Whole human serum had an effect similar to that of the combined serum proteins in MT-2 cells and increased the EC50 of GS-9160 by sixfold to 12 nM (Fig. 2 and Table 2). In primary human T cells, the combined effect of HSA and {alpha}1-AGP increased the EC50 of GS-9160 by 10-fold to 15 nM, while the EC50s of L-870,810 and GS-9137 were increased by 20- and 80-fold, respectively. Therefore, we conclude that both L-870,810 and GS-9137 display higher levels of binding to human serum proteins than those of GS-9160 and MK-0518.


Figure 2
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  		FIG. 2. Extrapolation of antiviral EC50 to 100% human serum. For each compound, the EC50 was determined at 10%, 20%, 35%, and 50% human serum using the following three different MOIs: 0.01, 0.02, and 0.04; 0.01 is the standard MOI for antiviral assays. The serum-adjusted EC50 versus the fractional serum concentration plot was analyzed by curve fitting using the equation in Materials and Methods to extrapolate the EC50 in 100% human serum. The error bars represent the standard deviation of the mean EC50 from at least two separate determinations in triplicate.

GS-9160 is an authentic inhibitor of HIV-1 integration. Two assays were used to verify that the mechanism of action for GS-9160 is inhibition of HIV-1 viral DNA integration in cells. Circular HIV-1 DNA species containing 1-LTR or 2-LTR circles accumulate when viral integration fails. Thus, an elevation in the levels of these viral nucleic acid species following treatment of infected cells with a small molecule can provide key evidence that HIV-1 integration has been inhibited (2). Treatment of HIV-1-infected SupT1 cells with GS-9160 caused an approximate twofold increase in 2-LTR circles (Table 3). Similar results were obtained with the clinically validated IN strand transfer inhibitors L-870,810 and GS-9137. This result provided initial evidence that GS-9160 can block HIV-1 integration in a cell.


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  		TABLE 3. Effect of antiviral compounds on the accumulation of 2-LTR circle, late RT, and Alu-PCR products in HIV-1-infected SupT1 cellsa

Another method to assess whether HIV-1 integration is impeded in infected cells is by the direct measurement of integration junctions in the host cell DNA (3). Detection by PCR of nucleic acid products containing Alu repeat sequences and portions of HIV-1 DNA represents evidence of successful HIV-1 integration. These products typically peak at 48 h postinfection. In the presence of GS-9160, these products decreased in a dose-dependent manner, with an EC50 of 0.9 nM, which was a potency comparable to those observed with GS-9137 and L-870,810 in this assay (Table 3). To ensure that this reduced integration was not attributable to an impairment of the upstream process of reverse transcription, accumulation of late RT products was assessed in the presence of GS-9160. GS-9160, like the other two strand transfer inhibitors, GS-9137 and L-870,810, did not affect the accumulation of late reverse transcription products, which was in sharp contrast to the inhibitory effect noted with the NNRTI EFV (Table 3). This result provides further evidence that GS-9160 is an authentic inhibitor of integration in HIV-1-infected cells.

GS-9160 is synergistic in combination with approved HIV-1 antiviral drugs. To determine the effect of combining GS-9160 with clinically approved HIV-1 antiviral drugs on antiviral activity, GS-9160 was tested in pairwise combinations with a panel of drugs composed of NRTIs, NNRTIs, and PIs. Specifically, the antiviral activity of GS-9160 was evaluated in combination with eight approved HIV-1 antiviral drugs, the PIs LPV, atazanavir, and nelfinavir; the NNRTI EFV; the nucleotide reverse transcriptase inhibitor TDF; and the NRTIs AZT, FTC, and 3TC in HIV-1-infected MT-2 cells. The effect of combining any two drugs was analyzed by two different methods, the Prichard and Shipman method using MacSynergy II software (37, 38) and the CI method using CalcuSyn software (5, 6).

Using MacSynergy II, the results of the combination studies were expressed as the mean synergy/antagonism volumes (nM2·%) calculated at the 95% confidence level from at least two separate experiments performed in triplicate. With CalcuSyn, the results of the combination studies were expressed as the mean CI of at least two separate experiments performed in triplicate. The two analytical methods gave similar results for all combinations tested, and results were consistent with previous drug-drug interaction studies (12). Three pairs of drugs, EFV+TDF, TDF+FTC, and AZT+3TC, served as examples of synergistic combinations (Table 4). The RBV+d4T combination was tested to ensure that antagonism can be identified (30). In this particular case, antagonism results from RBV-mediated inhibition of the phosphorylation of d4T (1). The AZT+d4T combination was tested as an example of a suboptimal pair of drugs, since clinically, the combination of AZT+d4T results in antagonism due to the effective competition of AZT-monophosphate for thymidine kinase, which is also necessary for the phosphorylation of d4T. However, with in vitro studies, evidence of antagonism between d4T and AZT has been inconsistent (12). GS-9160 was synergistic when tested in combination with all eight of these clinically approved HIV-1 antiviral drugs (Table 4).


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  		TABLE 4. Effect of GS-9160 in combination with other HIV antiviral drugs in HIV-1 IIIb-infected MT-2 cells

GS-9160 is active against drug-resistant mutants of HIV-1. The antiviral activity (EC50) of GS-9160 was determined against a panel of drug-resistant mutants of HIV-1. The panel included mutants that were resistant to the nucleotide reverse transcriptase inhibitor TDF (K65R), the NRTI FTC (M184V), and thymidine analogs. The panel also included viral mutants that were resistant to the NNRTI (K103N and Y181C) and PI (I84V/L90M and G48V/V82A/L90M) classes of drugs. The resistance profile of GS-9160 was compared to those of the two other IN inhibitors, L-870,810 and GS-9137 (Table 5). GS-9160, like L-870,810 and GS-9137, retained activity against NRTI-, NNRTI-, and PI-resistant HIV-1 mutants. The antiviral activity of GS-9160 against these drug-resistant mutants was comparable to its activity against the wild-type reference virus HIV-1 IIIb (Table 5).


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  		TABLE 5. Activity of GS-9160 against NRTI-, NNRTI-, and PI-resistant viruses

Phenotypic resistance to GS-9160. Viral resistance selections with GS-9160 were performed in tissue culture to identify mutations that diminish susceptibility to the antiviral effects of GS-9160. Parallel resistance selections were performed with several known anti-HIV-1 drugs (3TC, EFV, APV) (Table 6). The change in antiviral EC50s for the drug-selected viral pools compared to wild-type EC50 served as an indication of the enrichment of drug-resistant strains in the viral pool. For 3TC, phenotypic resistance was >272-fold at day 33 of selection, while phenotypic resistance to EFV was 35-fold at a comparable time of 31 days and reached 281-fold 43 days later. APV resistance selections yielded 8.7-fold resistance at day 48. Selection with GS-9160 led to the emergence of a virus pool showing 4.3-fold resistance by passage 5 and 51-fold resistance by passage 9 (Table 6). Phenotypic resistance to GS-9160 developed in a time frame comparable to that of the development of phenotypic resistance to APV. The viruses selected with GS-9160 displayed levels of cross-resistance similar to those of L-870,810 and MK-0158 but higher levels of resistance to GS-9137 at every passage (passage 5 to 9).


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  		TABLE 6. Comparative resistance selection with GS-9160 and other HIV-1 antiviral drugs

GS-9160 selected a novel pattern of IN inhibitor resistance mutations. Clonal sequencing of GS-9160-selected viruses from passages 5, 6, 8, and 9 revealed the successive emergence of mutations E92V and L74M in the catalytic core domain of HIV-1 IN (Table 7). Mutation E92V emerged first at passage 5, followed by the emergence of L74M at passage 6. Both E92V and L74M were present in 100% of the clones sequenced at passage 6 and were maintained through passage 9. Since the level of resistance progressively increased from 26-fold to ~51-fold between passages 6 and 9, additional mutations could have emerged in other HIV-1 genes to further increase the resistance level. To determine whether IN mutations E92V and L74M can recapitulate resistance to GS-9160, these mutations were introduced either individually or together into an infectious molecular clone of HIV-1. Interestingly, E92V alone confers 12-fold resistance to GS-9160, but L74M alone had no effect (Table 8). However, when combined, these mutations conferred 67-fold resistance to GS-9160, suggesting that L74M may potentiate resistance to GS-9160 conferred by E92V. E92V displayed cross-resistance to GS-9137 (44-fold), L-870,810 (8-fold), and MK-0518 (6-fold), while L74M had no effect on the potency of these IN inhibitors (Table 8). The double mutant E92V/L74M was also cross resistant to GS-9137, L-870,810, and MK-0518. Thus, the IN mutation L74M acted as a potentiator of E92V resistance against L-870,810, MK-0518, and GS-9137 (Table 8). It is noteworthy that L74M has been selected previously using other IN inhibitors such as L-708,906, a diketo acid (16); S-1360, a diketo triazole (15); and L-870,810, a naphthyridine analog (22). In each case, L74M as a single mutant showed no more than ~1.7-fold resistance against various IN inhibitors tested.


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  		TABLE 7. Mutations in the IN gene of resistant viruses selected with GS-9160


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  		TABLE 8. Resistance profile of GS-9160

Activity of GS-9160 against mutations conferring resistance to other IN inhibitors. Several other IN strand transfer inhibitors have been used to select for viral resistance in tissue culture. For instance, mutation T66I was previously selected with the diketo acid IN inhibitor L-708,906 (16, 19), with S-1360, a diketo triazole IN inhibitor (15, 45), and with GS-9137 (25). The mutation E92Q was selected by GS-9137 and L-870,810 (22, 28, 39), and E138K was selected with S-1360 (15). The mutation Q148K was selected by S-1360 (45) and MK-0518 (43, 44). The mutation G140S was selected by L-chicoric acid (27), and N155S was selected by S-1360 (45). The mutation V151A was selected with GS-278012, a prototype tricyclic compound and an analog of GS-9160 (data not shown). Mutation N155H developed in simian-human immunodeficiency virus SHIV-89.6P-infected rhesus macaques treated with L-870,812, an analog of L-870,810 (21). Several of these mutations, including L74M, E92Q, E138K, G140S, Q148K, and N155H, also developed in HIV-1-infected patients that were administered MK-0518 (7, 20, 40), and T66I, E92Q, E138K, G140S, and N155H were identified in patients receiving GS-9137 (33).

These various IN inhibitor-selected mutations were introduced into a wild-type HIV-1 infectious molecular clone to determine if they are cross resistant to GS-9160 (Table 8). The T66I mutant virus showed no cross-resistance against L-870,810, MK-0518, and GS-9160 but displayed 28-fold resistance against GS-9137. E92Q displayed comparable resistance to GS-9160 and L-870,810 (~30- to 40-fold), 153-fold resistance to GS-9137, and 7-fold resistance to MK-0518. Similarly, Q148K and N155H conferred a comparable degree of resistance to GS-9160 (23- and 50-fold, respectively) and L-870,810 (35- and 65-fold, respectively) and higher resistance to GS-9137 (600- and 100-fold, respectively). N155S also displays comparable levels of resistance, albeit lower than N155H, to GS-9160 (12-fold) and L-870,810 (21-fold) and higher levels of resistance to GS-9137 (133-fold). In summary, IN mutations E92Q, Q148K, N155H, and N155S appear to be cross resistant to the four IN inhibitors, GS-9160, L-870,810, MK-0518, and GS-9137.


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DISCUSSION
 
In this report, we describe the biological characterization of GS-9160, an antiviral inhibitor of the HIV-1 IN strand transfer reaction. GS-9160 is a prototype from a novel structural class, the N-benzyl-pyrrolidinone-hydroxyquinoline, which has potent antiviral activity in both T-lymphoblastoid cell lines and primary human T lymphocytes. GS-9160 is an authentic inhibitor of HIV-1 integration in tissue culture as measured by both an elevation of 2-LTR circles and a decrease of integration junctions in HIV-1-infected cells. GS-9160 remained active against various NRTI-, NNRTI-, and PI-resistant HIV-1 mutants and was synergistic with various clinically approved anti-HIV-1 drugs. Because the pharmacokinetics of GS-9160 in healthy human volunteers revealed that once-daily dosing was unlikely, clinical development of this compound was discontinued.

It was previously reported that different resistance mutations emerged in cell culture when virus selections were carried out with two structurally distinct strand transfer inhibitors, the diketo acid L-841,411 and the naphthyridine carboxamide L-870,810. Only one mutation selected by the diketo acid (N155S) conferred cross-resistance to L-870,810 (18). In this report, we have performed viral resistance selections with the novel tricyclic IN strand transfer inhibitor GS-9160 and discovered a distinct resistance pattern, E92V and L74M. These mutations confer cross-resistance to the structurally distinct strand transfer inhibitors L-870,810 and GS-9137.

The E92V resistance mutation in the IN catalytic core has not been previously selected with IN inhibitors. The second mutation selected by GS-9160, L74M, appeared later and appeared to potentiate resistance to GS-9160, as well as L-870,810, MK-0518, and GS-9137, by the primary mutation E92V. Although mutation of E92 has been previously observed with in vitro selections using GS-9137 (28, 39) and with patients experiencing virological failure with MK-0518 (7, 40), the mutation was a conversion to glutamine. Resistance selections performed with GS-278012, a close analog of GS-9160, also yielded E92V (data not shown). Because E92V was selected with GS-9160 and GS-278012, both containing a tricyclic pharmacophore, and was never previously observed with other IN inhibitors belonging to different chemical classes, it is possible that selection of E92V is specific to this novel tricyclic IN inhibitor. The other mutation selected by GS-9160, L74M, has been previously observed in viral selections using other IN inhibitors, yet interestingly, this mutation on its own does not confer resistance to IN strand transfer inhibitors. A more-recent resistance selection using L-870,810 generated a resistance pattern in IN consisting of the mutations L74M, E92Q, and S230N (22). The emergence of mutations at L74 and E92 is consistent with our findings that phenotypically resistant virus pools selected with GS-9160 were cross resistant to L-870,810 and suggest that GS-9160 and L-870,810 may interact similarly with the IN active site.

We have developed an active site model of HIV-1 IN with one 3'-processed donor DNA end interacting with the active site and a tricyclic compound bound in an active site pocket formed by IN and the 3'-processed donor DNA end (Fig. 3) (4). This active site model features three sites of interaction with GS-9160, as follows: a hydrophobic pocket accommodating the benzyl group of the compound (site 1), a metal-chelating site where a metal can interact with the carboxy and hydroxy groups of the compound (site 2), and a site interacting with the quinoline nitrogen through either a metal or a water molecule (site 3). Q148 and V151 are located in the benzyl binding pocket and in direct contact with the benzyl group of the tricyclic scaffold (Fig. 3). Our previous finding that mutagenesis of these two residues decreased the susceptibility of IN to inhibitors with either a tricyclic, a quinolone carboxylate, or a naphthyridine carboxamide pharmacophore (4) is consistent with Q148K and V151A mutant viruses being cross resistant to GS-9160, GS-9137, and L-870,810, respectively. Individually, L74M, E138K, and G140S do not confer much resistance to GS-9160 but when combined with E92V, Q148K, and E92V/V151A, respectively, they enhanced resistance to GS-9160 (Table 8). In our model, L74, E138, and G140 are in the proximity of the bound compound but do not make direct contact with the compound, suggesting that the L74M, E138K, and G140S mutations may induce a slight conformational change in IN which, in itself, will not decrease susceptibility but may magnify the resistance conferred by E92V, Q148K, and V151A. According to our model, the carboxylic side chain of residue E92 could interact with the quinoline nitrogen of GS-9160 through a water molecule. The E92V mutation would eliminate this site 3 interaction and weaken the binding of GS-9160. In the case of the E92Q mutation, substitution of the carboxylic acid group by an amide group could make hydrogen bonding less favorable with the water molecule because of the diminished hydrogen bonding flexibility of the amide group, which is planar.


Figure 3
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  		FIG. 3. Locations of resistance mutations in the IN catalytic core domain and a potential binding site for GS-9160. GS-9160 is illustrated in orange, and the IN catalytic triad residues are shown in red. Residues in IN making direct contact with GS-9160 are shown in green, while residues making indirect contact with GS-9160 are shown in blue. Metal ions are shown in magenta. GS-9160 is shown with the benzyl group on the right pointing toward the benzyl binding pocket delimited by V151 and Q148 and with the C5 sulfonamide group pointing toward the reader. A metal ion (1) interacts with N155 and stabilizes the 3'-processed end of donor DNA (not shown here). A metal ion (2) interacts with catalytic residue D116 and the carbonyl and C9 hydroxyl of GS-9160. E92V and L74M were mutations selected after serial passages of HIV-1 in the presence of increasing concentrations of GS-9160 and are shown to confer resistance to GS-9160.

Our model suggests that a single binding mode would exist for most current strand transfer inhibitors, including diketo acids, L-870,810, GS-9137, and GS-9160, with the benzyl groups shared by all these compounds buried deep into a benzyl binding pocket (4). This binding model provides some insights into the mutations in the IN active site that were selected by various compounds, including diketo acids or diketo acid analogs and our tricyclic compound GS-9160. With a better understanding of how certain resistance mutations may weaken the affinity of IN inhibitors, the rational design of second generation IN inhibitors that retain activity against drug-resistant mutants may be possible.


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ACKNOWLEDGMENTS
 
We thank Richard Pastor, Sammy Metobo, Rachael Lansdown, and Salman Jabri for the synthesis of GS-9160 and You-Chul Choi for the synthesis of CPV.


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FOOTNOTES
 
* Corresponding author. Mailing address: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404. Phone: (650) 522-5860. Fax: (650) 522-5143. E-mail: MTsiang{at}gilead.com Back

{triangledown} Published ahead of print on 22 December 2008. Back

{dagger} Supplemental material for this article may be found at http://aac.asm.org/. Back


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Antimicrobial Agents and Chemotherapy, March 2009, p. 1194-1203, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.00984-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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