In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.

Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

In vitro and in vivo antiviral activity and resistance profile of the hepatitis C virus NS3/4A protease inhibitor ABT-450.

The development of direct-acting antiviral agents is a promising therapeutic advance in the treatment of hepatitis C virus (HCV) infection. However, rapid emergence of drug resistance can limit efficacy and lead to cross-resistance among members of the same drug class. ABT-450 is an efficacious inhibitor of HCV NS3/4A protease, with 50% effective concentration values of 1.0, 0.21, 5.3, 19, 0.09, and 0.69 nM against stable HCV replicons with NS3 protease from genotypes 1a, 1b, 2a, 3a, 4a, and 6a, respectively. In vitro, the most common amino acid variants selected by ABT-450 in genotype 1 were located in NS3 at positions 155, 156, and 168, with the D168Y variant conferring the highest level of resistance to ABT-450 in both genotype 1a and 1b replicons (219- and 337-fold, respectively). In a 3-day monotherapy study with HCV genotype 1-infected patients, ABT-450 was coadministered with ritonavir, a cytochrome P450 3A4 inhibitor shown previously to markedly increase peak, trough, and overall drug exposures of ABT-450. A mean maximum HCV RNA decline of 4.02 log10 was observed at the end of the 3-day dosing period across all doses. The most common variants selected in these patients were R155K and D168V in genotype 1a and D168V in genotype 1b. However, selection of resistant variants was significantly reduced at the highest ABT-450 dose compared to lower doses. These findings were informative for the subsequent evaluation of ABT-450 in combination with additional drug classes in clinical trials in HCV-infected patients. (Study M11-602 is registered at ClinicalTrials.gov under registration no. NCT01074008.).

In vitro and in vivo antiviral activity and resistance profile of ombitasvir, an inhibitor of hepatitis C virus NS5A.

Ombitasvir (ABT-267) is a hepatitis C virus (HCV) NS5A inhibitor with picomolar potency, pan-genotypic activity, and 50% effective concentrations (EC50s) of 0.82 to 19.3 pM against HCV genotypes 1 to 5 and 366 pM against genotype 6a. Ombitasvir retained these levels of potency against a panel of 69 genotype 1 to 6 chimeric replicons containing the NS5A gene derived from HCV-infected patients, despite the existence of natural sequence diversity within NS5A. In vitro resistance selection identified variants that conferred resistance to ombitasvir in the HCV NS5A gene at amino acid positions 28, 30, 31, 58, and 93 in genotypes 1 to 6. Ombitasvir was evaluated in vivo in a 3-day monotherapy study in 12 HCV genotype 1-infected patients at 5, 25, 50, or 200 mg dosed once daily. All patients in the study were HCV genotype 1a infected and were without preexisting resistant variants at baseline as determined by clonal sequencing. Decreases in HCV RNA up to 3.1 log10 IU/ml were observed. Resistance-associated variants at position 28, 30, or 93 in NS5A were detected in patient samples 48 hours after the first dose. Clonal sequencing analysis indicated that wild-type virus was largely suppressed by ombitasvir during 3-day monotherapy, and at doses higher than 5 mg, resistant variant M28V was also suppressed. Ombitasvir was well tolerated at all doses, and there were no serious or severe adverse events. These data support clinical development of ombitasvir in combination with inhibitors targeting HCV NS3/4A protease (ABT-450 with ritonavir) and HCV NS5B polymerase (ABT-333, dasabuvir) for the treatment of chronic HCV genotype 1 infection. (Study M12-116 is registered at ClinicalTrials.gov under registration no. NCT01181427.).

Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A.

Efforts to improve the genotype 1a potency and pharmacokinetics of earlier naphthyridine-based HCV NS5A inhibitors resulted in the discovery of a novel series of pyrido[2,3-d]pyrimidine compounds, which displayed potent inhibition of HCV genotypes 1a and 1b in the replicon assay. SAR in this system revealed that the introduction of amides bearing an additional 'E' ring provided compounds with improved potency and pharmacokinetics. Introduction of a chiral center on the amide portion resulted in the observation of a stereochemical dependence for replicon potency and provided a site for the attachment of functional groups useful for improving the solubility of the series. Compound 21 was selected for administration in an HCV-infected chimpanzee. Observation of a robust viral load decline provided positive proof of concept for inhibition of HCV replication in vivo for the compound series.

Aryl uracil inhibitors of hepatitis C virus NS5B polymerase: synthesis and characterization of analogs with a fused 5,6-bicyclic ring motif.

The synthesis and structure-activity relationships of a novel aryl uracil series which contains a fused 5,6-bicyclic ring unit for HCV NS5B inhibition is described. Several analogs display replicon cell culture potencies in the low nanomolar range along with excellent rat pharmacokinetic values.

Identification of aryl dihydrouracil derivatives as palm initiation site inhibitors of HCV NS5B polymerase.

Aryl dihydrouracil derivatives were identified from high throughput screening as potent inhibitors of HCV NS5B polymerase. The aryl dihydrouracil derivatives were shown to be non-competitive with respect to template RNA and elongation nucleotide substrates. They demonstrated genotype 1 specific activity towards HCV NS5B polymerases. Structure activity relationships and genotype specific activities of aryl dihydrouracil derivatives suggested that they bind to the palm initiation nucleotide pocket, a hypothesis which was confirmed by studies with polymerases containing mutations in various inhibitor binding sites. Therefore, aryl dihydrouracil derivatives represent a novel class of palm initiation site inhibitors of HCV NS5B polymerase.

Novel hepatitis C virus replicon inhibitors: synthesis and structure-activity relationships of fused pyrimidine derivatives.

The synthesis of several pyrido[2,3-d]pyrimidine and pyrimido[4,5-d]pyrimidine analogs is described with one such analog possessing subnanomolar potency in both genotype 1a and 1b cell culture HCV replicon assays.

Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety.

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.

Synthesis of potent pyrrolidine influenza neuraminidase inhibitors.

The synthesis of several pyrrolidine inhibitor analogs is described that possess nanomolar in vitro potencies against the neuraminidase enzymes expressed by the B/Memphis/3/89 and A/N1/PR/8/34 influenza strains.

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase.

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.

Hepatitis C NS5B polymerase inhibitors: 4,4-Dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-3-yl benzothiadiazine derivatives.

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.

Synthesis and Biological Characterization of Aryl Uracil Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of ABT-072, a trans-Stilbene Analog with Good Oral Bioavailability.

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species. Replacement of the dihydrouracil in 1 with an N-linked uracil provided better potency in the genotype 1 replicon assay. Results from phase 1 clinical studies supported once-daily oral dosing with ABT-072 in HCV infected patients. A phase 2 clinical study that combined ABT-072 with the HCV protease inhibitor ABT-450 provided a sustained virologic response at 24 weeks after dosing (SVR24) in 10 of 11 patients who received treatment.

Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro.

The combinations of Abbott Hepatitis C virus (HCV) polymerase A-782759 with either Boehringer Ingelheim HCV NS3 protease inhibitor BILN-2061 or interferon (IFN) displayed additive to synergistic relationships over a range of concentrations of two-drug combination. Treatment of HCV replicon with A-782759, IFN or BILN-2061 for about 16 days resulted in dramatic reductions in HCV RNA (5.1, 3.0 and 3.9 log10 RNA copies, respectively). However, none of the compounds tested alone lead to replicon RNA reduction to undetectable levels. Ongoing replication in the presence of A-782759 or BILN-2061 was associated with the appearance of resistant mutations M414T in NS5B and D168V in NS3, respectively. In contrast, a combination of A-782759 with BILN-2061 resulted in greater than 7 logs RNA reduction leading to undetectable replicon RNA after 16 days of treatment. Our findings suggest that a monotherapy with either drug alone is likely to result in development of resistant mutants. However, a combination therapy with polymerase inhibitor has the potential to improve the efficacy of IFN or a protease inhibitor alone in vivo, due to the lower likelihood of resistance development.

Replication efficiency of chimeric replicon containing NS5A-5B genes derived from HCV-infected patient sera.

A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from "quasispecies" pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A-5B genes were constructed with the S2204I "adaptive" mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A-5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-alpha (IFN-alpha) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC(50) values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A-5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-alpha and to small molecule inhibitors.

A replicon-based shuttle vector system for assessing the phenotype of HCV NS5B polymerase genes isolated from patient populations.

Hepatitis C virus (HCV) replicon-based shuttle vectors that permit phenotypes of NS5B polymerase genes from a large number of patient isolates to be rapidly assessed when transiently expressed in cultured cells were designed. When used to test responses to an inhibitor of HCV RNA-dependent RNA polymerase, IC(50) values for inhibition covered a several hundred-fold range among 47 patient samples tested. This observation highlights the variability that can be found by testing isolates derived from HCV-infected subjects. Partial suppression with a polymerase inhibitor of the most sensitive species permitted detection of minor quasispecies that were 7-200-fold more resistant than the bulk population in approximately half of the samples. Sequence analysis showed a wide range of amino acid changes not detected by conventional selection methods using laboratory-derived strains. This approach provides a means to assess variation in antiviral efficacy, and to predict possible responses in a clinical setting.

Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors.

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp(2) hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable pi-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat.

Evolving therapeutic paradigms for HIV and HCV.

The continued development of enabling molecular technologies that can be employed to better understand viral replication and the action of currently available therapeutic agents promises to lead to the development of the next generation of drugs with improved therapeutic utility against human immunodeficiency virus (HIV) and hepatitis C virus (HCV). New therapeutic approaches have been developed for treating HIV and HCV infections, but key technical and therapeutic challenges must be addressed to further advance treatment options in each of these areas.

Characterization of resistant HIV variants generated by in vitro passage with lopinavir/ritonavir.

Lopinavir (LPV, formerly ABT-378) is an HIV protease inhibitor (PI) that is co-administered with a small amount of ritonavir (RTV), which greatly increases and sustains the plasma levels of LPV. Lopinavir/ritonavir (LPV/r) has shown potent antiviral activity in both therapy-nai;ve and PI-experienced patients. To assess the effect of pharmacologically relevant ratios of LPV/RTV (LPV/r) on the emergence of resistant HIV in vitro, HIV-1 pNL4-3 was passaged in the presence of increasing concentrations of LPV alone and LPV/r. Passages with fixed 5/1 and 15/1 concentration ratios of LPV/r initially selected I84V and I50V/M46I mutants, respectively. Selection with LPV alone also generated the same initial mutants (I50V/M46I) as the 15/1 LPV/r passage. Further passage produced other mutations previously found to be associated with PI-resistance. Phenotypic susceptibility to both LPV and RTV decreased with successive passages, irrespective of whether RTV was present in the selection experiment. Furthermore, in the two selection experiments that included RTV (at either 5/1 or 15/1 LPV/r ratio), the IC(50) of RTV at each passage evaluated was at least five-fold higher than the concentration of RTV employed at that passage, while the IC(50) of LPV toward the passaged virus was similar to the concentration of LPV used at that passage, indicating that the selective pressure was attributable to LPV and not RTV.

Inhibition of human immunodeficiency virus type I integrase by naphthamidines and 2-aminobenzimidazoles.

Retroviral integrases catalyze two of the steps of insertion of proviral DNA into the host genomic DNA. Inhibitors that target the second step, strand transfer into the host DNA, have been demonstrated to have antiviral activity in cell culture. We describe two classes of HIV-1 integrase inhibitors that block strand transfer, one based on a naphthamidine core and one on a benzimidazole core. While the naphthamidine compounds showed some propensity to interact with the DNA substrate, both classes were shown to bind directly to integrase. The naphthamidine compounds showed activity in cell culture, and a direct effect on integrase was indicated by an increase in 2-LTR products in the presence of a naphthamidine compound. These two classes of compounds represent potential starting points for the development of new classes of integrase inhibitors.

P1-substituted symmetry-based human immunodeficiency virus protease inhibitors with potent antiviral activity against drug-resistant viruses.

Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.