Discovery of the Human Immunodeficiency Virus Type 1 (HIV-1) Attachment Inhibitor Temsavir and Its Phosphonooxymethyl Prodrug Fostemsavir.

The optimization of the 4-methoxy-6-azaindole series of HIV-1 attachment inhibitors (AIs) that originated with 1 to deliver temsavir (3, BMS-626529) is described. The most beneficial increases in potency and pharmacokinetic (PK) properties were attained by incorporating N-linked, sp2-hybridized heteroaryl rings at the 7-position of the heterocyclic nucleus. Compounds that adhered to a coplanarity model afforded targeted antiviral potency, leading to the identification of 3 with characteristics that provided for targeted exposure and PK properties in three preclinical species. However, the physical properties of 3 limited plasma exposure at higher doses, both in preclinical studies and in clinical trials as the result of dissolution- and/or solubility-limited absorption, a deficiency addressed by the preparation of the phosphonooxymethyl prodrug 4 (BMS-663068, fostemsavir). An extended-release formulation of 4 is currently in phase III clinical trials where it has shown promise as part of a drug combination therapy in highly treatment-experienced HIV-1 infected patients.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

Hepatitis C virus NS5A replication complex inhibitors: the discovery of daclatasvir.

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

Discovery and preclinical characterization of the cyclopropylindolobenzazepine BMS-791325, a potent allosteric inhibitor of the hepatitis C virus NS5B polymerase.

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 12. Structure-activity relationships associated with 4-fluoro-6-azaindole derivatives leading to the identification of 1-(4-benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1h-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248).

A series of highly potent HIV-1 attachment inhibitors with 4-fluoro-6-azaindole core heterocycles that target the viral envelope protein gp120 has been prepared. Substitution in the 7-position of the azaindole core with amides (12a,b), C-linked heterocycles (12c-l), and N-linked heterocycles (12m-u) provided compounds with subnanomolar potency in a pseudotype infectivity assay and good pharmacokinetic profiles in vivo. A predictive model was developed from the initial SAR in which the potency of the analogues correlated with the ability of the substituent in the 7-position of the azaindole to adopt a coplanar conformation by either forming internal hydrogen bonds or avoiding repulsive substitution patterns. 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248, 12m) exhibited much improved in vitro potency and pharmacokinetic properties than the previous clinical candidate BMS-488043 (1). The predicted low clearance in humans, modest protein binding, and good potency in the presence of 40% human serum for 12m led to its selection for human clinical studies.

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032).

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with K(i) values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC(50)s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC(50), 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC(50), >12 µM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC(50)s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment 6. Preclinical and human pharmacokinetic profiling of BMS-663749, a phosphonooxymethyl prodrug of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043).

BMS-663749, a phosphonooxymethyl prodrug 4 of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043) (2) was prepared and profiled in a variety of preclinical in vitro and in vivo models designed to assess its ability to deliver parent drug following oral administration. The data showed that prodrug 4 had excellent potential to significantly reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies in normal healthy subjects confirmed the potential of 4, revealing that the prodrug significantly increased both the AUC and C(max) of 2 compared to a solid capsule formulation containing the parent drug upon dose escalation. These data provided guidance for further efforts to obtain an effective HIV-1 attachment inhibitor.

Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir.

BACKGROUND: Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. METHODS/PRINCIPAL FINDINGS: To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. CONCLUSIONS: Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.

Utilization of in vitro Caco-2 permeability and liver microsomal half-life screens in discovering BMS-488043, a novel HIV-1 attachment inhibitor with improved pharmacokinetic properties.

Optimizing pharmacokinetic properties to improve oral exposure is a common theme in modern drug discovery. In the present work, in vitro Caco-2 permeability and microsomal half-life screens were utilized in an effort to guide the structure-activity relationship in order to improve the pharmacokinetic properties of novel HIV-1 attachment inhibitors. The relevance of the in vitro screens to in vivo pharmacokinetic properties was first demonstrated with a number of program compounds at the early stage of lead optimization. The Caco-2 permeability, tested at 200 microM, was quantitatively predictive of in vivo oral absorption, with complete absorption occurring at a Caco-2 permeability of 100 nm/s or higher. The liver microsomal half-life screen, conducted at 1 microM substrate concentration, can readily differentiate low-, intermediate-, and high-clearance compounds in rats, with a nearly 1:1 correlation in 12 out of 13 program compounds tested. Among the >100 compounds evaluated, BMS-488043 emerged as a lead, exhibiting a Caco-2 permeability of 178 nm/s and a microsomal half-life predictive of a low clearance (4 mL/min/kg) in humans. These in vitro characteristics translated well to the in vivo setting. The oral bioavailability of BMS-488043 in rats, dogs, and monkeys was 90%, 57%, and 60%, respectively. The clearance was low in all three species tested, with a terminal half-life ranging from 2.4 to 4.7 h. Furthermore, the oral exposure of BMS-488043 was significantly improved (6- to 12-fold in rats and monkeys) compared to the prototype compound BMS-378806 that had a suboptimal Caco-2 permeability (51 nm/s) and microsomal half-life. More importantly, the improvements in preclinical pharmacokinetics translated well to humans, leading to a >15-fold increase in the human oral exposure of BMS-488043 than BMS-378806 and enabling a clinical proof-of-concept for this novel class of anti-HIV agents. The current studies demonstrated the valuable role of in vitro ADME screens in improving oral pharmacokinetics at the lead optimization stage.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 5. An evolution from indole to azaindoles leading to the discovery of 1-(4-benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-488043), a drug candidate that demonstrates antiviral activity in HIV-1-infected subjects.

Azaindole derivatives derived from the screening lead 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (1) were prepared and characterized to assess their potential as inhibitors of HIV-1 attachment. Systematic replacement of each of the unfused carbon atoms in the phenyl ring of the indole moiety by a nitrogen atom provided four different azaindole derivatives that displayed a clear SAR for antiviral activity and all of which displayed marked improvements in pharmaceutical properties. Optimization of these azaindole leads resulted in the identification of two compounds that were advanced to clinical studies: (R)-1-(4-benzoyl-2-methylpiperazin-1-yl)-2-(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)ethane-1,2-dione (BMS-377806, 3) and 1-(4-benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-488043, 4). In a preliminary clinical study, 4 administered as monotherapy for 8 days, reduced viremia in HIV-1-infected subjects, providing proof of concept for this mechanistic class.

Ultrasensitive genotypic detection of antiviral resistance in hepatitis B virus clinical isolates.

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.

Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naïve patients is rare through 5 years of therapy.

UNLABELLED: Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T184, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V +/- L180M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years. Monitoring included genotypic analysis of isolates from all patients at baseline and when HBV DNA was detectable by polymerase chain reaction (> or = 300 copies/mL) from Years 1 through 5. In addition, genotyping was performed on isolates from patients experiencing virologic breakthrough (> or = 1 log(10) rise in HBV DNA). In vitro phenotypic ETV susceptibility was determined for virologic breakthrough isolates, and for HBV containing novel substitutions emerging during treatment. The results over 5 years of therapy showed that in nucleoside-naïve patients, the cumulative probability of genotypic ETVr and genotypic ETVr associated with virologic breakthrough was 1.2% and 0.8%, respectively. In contrast, a reduced barrier to resistance was observed in LVD-refractory patients, as the LVD resistance substitutions, a partial requirement for ETVr, preexist, resulting in a 5-year cumulative probability of genotypic ETVr and genotypic ETVr associated with breakthrough of 51% and 43%, respectively. Importantly, only four patients who achieved < 300 copies/mL HBV DNA subsequently developed ETVr. CONCLUSION: Long-term monitoring showed low rates of resistance in nucleoside-naïve patients during 5 years of ETV therapy, corresponding with potent viral suppression and a high genetic barrier to resistance. These findings support ETV as a primary therapy that enables prolonged treatment with potent viral suppression and minimal resistance.

Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole substitution patterns.

The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore.

Comprehensive evaluation of hepatitis B virus reverse transcriptase substitutions associated with entecavir resistance.

UNLABELLED: Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV) requires three substitutions in the viral reverse transcriptase (RT), signifying a high barrier to resistance. Two of these substitutions are associated with lamivudine resistance (LVDr) in the tyrosine-methionine-aspartate-aspartate (YMDD) motif (rtM204V and rtL180M), whereas the other occurs at one or more positions specifically associated with ETV resistance (ETVr): rtT184, rtS202, or rtM250. Although a variety of substitutions at these primary ETVr positions arise during ETV therapy, only a subset give rise to clinical virologic breakthrough. To determine the phenotypic impact of observed clinical and potential new ETVr substitutions, a comprehensive panel of clones containing every possible amino acid at the three primary ETVr positions in LVDr HBV was constructed and analyzed in vitro. A range of replication capacities was observed for the panel, but none of the mutations rescued replication of the LVDr mutant to the wild-type level. More clones with residue rtS202 substitutions were severely impaired than those at rtT184 or rtM250. A wide variety of ETV susceptibilities was observed, ranging from approximately eight-fold (no increase over the LVDr parent) to greater than 400-fold over the wild-type. A correlation was identified between clinically observed substitutions and those displaying higher in vitro replication and resistance, especially those from virologic breakthrough patients. CONCLUSION: The high number of tolerated and resistant ETVr substitutions is consistent with models predicting that the mechanism for ETVr is through enhancement of LVDr changes in the RT deoxyribonucleotide triphosphate (dNTP)-binding pocket.

Hepatitis B virus quasispecies susceptibility to entecavir confirms the relationship between genotypic resistance and patient virologic response.

BACKGROUND/AIMS: The efficacy of anti-viral therapy for chronic hepatitis B virus (HBV) is lost upon the emergence of resistant virus. Using >500 patient HBV isolates from several entecavir clinical trials, we show that phenotypic susceptibility correlates with genotypic resistance and patient virologic responses. METHODS: The full-length HBV or reverse transcriptase gene was amplified from patient sera, sequenced, and cloned into an HBV expression vector. Entecavir susceptibilities of individual virus clones and patient quasispecies populations were analyzed in conjunction with the sequenced resistance genotype and the patient's virologic response. RESULTS: Entecavir susceptibility decreased approximately 8-fold for isolates with various constellations of lamivudine resistance substitutions. The spectrum of additional substitutions that emerged during therapy at residues rtT184, rtS202, or rtM250 displayed varying levels of entecavir susceptibility according to the specific resistance substitutions and the proportion of resistant variants in the quasispecies. Phenotypic analyses of samples associated with virologic breakthrough confirmed the role of these residue changes in entecavir resistance. Additional longitudinal phenotypic analyses showed that decreased susceptibility correlated with both genotypic resistance and increased circulating HBV DNA. CONCLUSIONS: HBV phenotypic analysis provides additional insight as part of a resistance monitoring program that includes genotypic analysis and quantification of circulating virus.

Entecavir for treatment of hepatitis B virus displays no in vitro mitochondrial toxicity or DNA polymerase gamma inhibition.

Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Entecavir (ETV) is an approved deoxyguanosine nucleoside for the treatment of chronic hepatitis B virus (HBV) infection that exhibits potent activity against viral reverse transcriptase. We assessed the potential for mitochondrial toxicity of ETV in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and levels of mitochondrial proteins COX II and COX IV. Furthermore, we tested the activity of ETV-triphosphate (ETV-TP) against mitochondrial DNA polymerase gamma (Pol gamma) in vitro. ETV concentrations as high as 100 times the maximal clinical exposure (C(max)) did not affect cell proliferation, levels of lactate, mitochondrial DNA, or mitochondrial proteins throughout the 15-day culture. The lack of mitochondrial toxicity was consistent with the finding that ETV-TP was not recognized by mitochondrial DNA Pol gamma and failed to be incorporated into DNA or inhibit the polymerase assay at the highest levels tested, 300 microM. Combinations of ETV with each of the other HBV NRTI antivirals, adefovir, tenofovir, and lamivudine at 10 times their respective C(max) levels also failed to result in cellular or mitochondrial toxicity. In summary, cell culture and enzymatic studies yielded no evidence that would predict mitochondrial toxicity of ETV at exposure levels in excess of those expected to be achieved clinically.

Inhibition of hepatitis B virus polymerase by entecavir.

Entecavir (ETV; Baraclude) is a novel deoxyguanosine analog with activity against hepatitis B virus (HBV). ETV differs from the other nucleoside/tide reverse transcriptase inhibitors approved for HBV therapy, lamivudine (LVD) and adefovir (ADV), in several ways: ETV is >100-fold more potent against HBV in culture and, at concentrations below 1 microM, displays no significant activity against human immunodeficiency virus (HIV). Additionally, while LVD and ADV are obligate DNA chain terminators, ETV halts HBV DNA elongation after incorporating a few additional bases. Three-dimensional homology models of the catalytic center of the HBV reverse transcriptase (RT)-DNA-deoxynucleoside triphosphate (dNTP) complex, based on the HIV RT-DNA structure, were used with in vitro enzyme kinetic studies to examine the mechanism of action of ETV against HBV RT. A novel hydrophobic pocket in the rear of the RT dNTP binding site that accommodates the exocyclic alkene moiety of ETV was predicted, establishing a basis for the superior potency observed experimentally. HBV DNA chain termination by ETV was accomplished through disfavored energy requirements as well as steric constraints during subsequent nucleotide addition. Validation of the model was accomplished through modeling of LVD resistance substitutions, which caused an eightfold decrease in ETV susceptibility and were predicted to reduce, but not eliminate, the ETV-binding pocket, in agreement with experimental observations. ADV resistance changes did not affect the ETV docking model, also agreeing with experimental results. Overall, these studies explain the potency, mechanism, and cross-resistance profile of ETV against HBV and account for the successful treatment of naive and LVD- or ADV-experienced chronic HBV patients.

Respiratory syncytial virus fusion inhibitors. Part 4: optimization for oral bioavailability.

A series of benzimidazole-based inhibitors of respiratory syncytial virus (RSV) fusion were optimized for antiviral potency, membrane permeability and metabolic stability in human liver microsomes. 1-Cyclopropyl-1,3-dihydro-3-[[1-(4-hydroxybutyl)-1H-benzimidazol-2-yl]methyl]-2H-imidazo[4,5-c]pyridin-2-one (6m, BMS-433771) was identified as a potent RSV inhibitor demonstrating good bioavailability in the mouse, rat, dog and cynomolgus monkey that demonstrated antiviral activity in the BALB/c and cotton rat models of infection following oral administration.