Discovery of fluorobenzimidazole HCV NS5A inhibitors.

Research toward a next-generation HCV NS5A inhibitor has identified fluorobenzimidazole analogs that demonstrate potent, broad-genotype in vitro activity against HCV genotypes 1-6 replicons as well as HCV NS5A variants that are orders of magnitude less susceptible to inhibition by first-generation NS5A inhibitors in comparison to wild-type replicons. The fluorobenzimidazole inhibitors have improved pharmacokinetic properties in comparison to non-fluorinated benzimidazole analogs. Discovery of these inhibitors was facilitated by exploring SAR in a structurally simplified inhibitor series.

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.

High potency improvements to weak aryl uracil HCV polymerase inhibitor leads.

Described herein is the development of a potent non-nucleoside, small molecule inhibitor of genotype 1 HCV NS5B Polymerase. A 23 µM inhibitor that was active against HCV polymerase was further elaborated into a potent single-digit nanomolar inhibitor of HCV NS5B polymerase by additional manipulation of the R and R1 substituents. Subsequent modifications to improve physical properties were made in an attempt to achieve an acceptable pharmacokinetic profile.

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.

Interviews with Indian Animal Shelter Staff: Similarities and Differences in Challenges and Resiliency Factors Compared to Western Counterparts.

Animal shelters in India are at the forefront of efforts to improve free-ranging dog welfare and tackle animal overpopulation. In terms of cultural and political context, access to resources, and public health challenges, they operate in a very different environment than Western counterparts. Despite these distinctions, current sheltering literature is largely centered around countries such as the United States. The goal of this exploratory study was to examine the experiences of Indian animal shelter staff. Researchers conducted ten semi-structured interviews, in a mix of Hindi and English, with managers, veterinary nurses, and animal caretakers from three shelters. Using thematic analysis, shelter challenges as well as resiliency factors that enable staff to cope with these challenges were identified. Key challenges were inadequate funding, community conflict, and high intake numbers. Resiliency factors included flexibility, duty of care, co-worker relationships, and understanding animal needs. The results of this qualitative study revealed that the experiences of shelter staff are shaped by social, political, and cultural factors and that there is a need for further, context specific research on Indian sheltering rather than only relying on Western perspectives.

Wild Zebrafish Sentinels: Biological Monitoring of Site Differences Using Behavior and Morphology.

Environmental change poses a devastating risk to human and environmental health. Rapid assessment of water conditions is necessary for monitoring, evaluating, and addressing this global health danger. Sentinels or biological monitors can be deployed in the field using minimal resources to detect water quality changes in real time, quickly and cheaply. Zebrafish (Danio rerio) are ideal sentinels for detecting environmental changes due to their biomedical tool kit, widespread geographic distribution, and well-characterized phenotypic responses to environmental disturbances. Here, we demonstrate the utility of zebrafish sentinels by characterizing phenotypic differences in wild zebrafish between two field sites in India. Site 1 was a rural environment with flowing water, low-hypoxic conditions, minimal human-made debris, and high iron and lead concentrations. Site 2 was an urban environment with still water, hypoxic conditions, plastic pollution, and high arsenic, iron, and chromium concentrations. We found that zebrafish from Site 2 were smaller, more cohesive, and less active than Site 1 fish. We also found sexually dimorphic body shapes within the Site 2, but not the Site 1, population. Advancing zebrafish sentinel research and development will enable rapid detection, evaluation, and response to emerging global health threats.

Temporal and environmental drivers of fish-community structure in tropical streams from two contrasting regions in India.

Environmental and anthropogenic factors are known to drive fish community structure in aquatic systems across the world. This study investigates fish assemblages in lower order streams across contrasting landscapes in central and eastern India. We documented the species diversity of these monsoon driven lower order streams in the two regions. We also investigated the potential common environmental drivers of richness and diversity and effect of season in these tropical streams. The study was based on seasonal data on abundance of fishes and environmental parameters collected between 2015-2017 from streams in states of Madhya Pradesh and West Bengal. Species diversity were compared across regions and seasons, based on their richness (SR) as well as diversity (Shannon index H'). Drivers of overall richness and diversity were analyzed using multiple linear regression methods, based on best subset selection. Analysis of data revealed high diversity in these streams in both regions. Cyprinidae, Bagridae and Channidae were the most dominant families in both regions. Despite the geographical and local ecological differences across the regions, common environmental parameters were found to influence richness and diversity across the two regions, indicating these as being key drivers of fish community structure. Water flow was a common factor driving both richness and diversity across both regions. Our study revealed a lack of seasonal effect in structuring fish communities in tropical streams. With stream and river ecosystems facing increasing threats due to habitat alterations and water quality degradation in countries such as India, a clear understanding of regional and local drivers of community structure of aquatic fauna is crucial. These results on the role of common environmental factors across ecoregions provides baseline information for understanding their ecological roles and developing management plans for important river basins and fish conservation in future.

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.

Highlights of the Structure-Activity Relationships of Benzimidazole Linked Pyrrolidines Leading to the Discovery of the Hepatitis C Virus NS5A Inhibitor Pibrentasvir (ABT-530).

Curative interferon and ribavirin sparing treatments for hepatitis C virus (HCV)-infected patients require a combination of mechanistically orthogonal direct acting antivirals. A shared component of these treatments is usually an HCV NS5A inhibitor. First generation FDA approved treatments, including the component NS5A inhibitors, do not exhibit equivalent efficacy against HCV virus genotypes 1-6. In particular, these first generation NS5A inhibitors tend to select for viral drug resistance. Ombitasvir is a first generation HCV NS5A inhibitor included as a key component of Viekira Pak for the treatment of patients with HCV genotype 1 infection. Since the launch of next generation HCV treatments, functional cure for genotype 1-6 HCV infections has been achieved, as well as shortened treatment duration across a wider spectrum of genotypes. In this paper, we show how we have modified the anchor, linker, and end-cap architecture of our NS5A inhibitor design template to discover a next generation NS5A inhibitor pibrentasvir (ABT-530), which exhibits potent inhibition of the replication of wild-type genotype 1-6 HCV replicons, as well as improved activity against replicon variants demonstrating resistance against first generation NS5A 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.

Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A.

We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5R analogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency. Substitution with tert-butyl, as in compound 38 (ABT-267), provided compounds with low-picomolar EC50 values and superior pharmacokinetics. It was discovered that compound 38 was a pan-genotypic HCV inhibitor, with an EC50 range of 1.7-19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound 38 decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin.

Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well.

The Hepatitis C (HCV) replicon system is a useful tool for the high-volume screening of inhibitors of HCV replication. In this report, a cell-based assay has been described, which monitors the inhibition of HCV genotypes 1a and 1b as well as cytotoxicity, from a single well of a 96-well plate. A mixture of two stable replicon cell lines was used: one containing a 1a-H77 replicon expressing a firefly luciferase reporter, and the other one containing a 1b-N replicon with a secreted alkaline phosphatase reporter, thus allowing us to monitor replication of two HCV genotypes in the same well. Cytotoxicity was measured using the Resazurin cytotoxicity assay. The assay was validated with known HCV inhibitors and showed that the antiviral activity and cytotoxicity of compounds were reproducibly measured under screening conditions. It was also showed that the assay's signal-to-noise ratio and Z' coefficient were suitable for high-throughput screening. A panel of HCV inhibitors showed a good correlation between EC(50) and TD(50) values for 1a and 1b replicon activity and cytotoxicity measured using either a single replicon format or mixed replicon format. Thus, the use of this mixed replicon format provides an economical method for simultaneous measurement of compound activity against two HCV genotypes as well as cytotoxicity, thereby reducing cost of reagents and labor as well as improving throughput.

Synthesis and biological characterization of B-ring amino analogues of potent benzothiadiazine hepatitis C virus polymerase inhibitors.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology.

UNLABELLED: Hepatitis C virus (HCV) replication is highly dependent on host cell factors. Identification of these host factors not only facilitates understanding of the biology of HCV infection but also enables the discovery of novel targets for anti-HCV therapy. To identify host genes important for HCV RNA replication, we screened a library of small interfering RNA (siRNA) that targets approximately 4,000 human genes in Huh7-derived EN5-3 cells harboring an HCV subgenomic replicon with the nonstructural region NS3-NS5B from the 1b-N strain. Nine cellular genes that potentially regulate HCV replication were identified in this screen. Silencing of these genes resulted in inhibition of HCV replication by more than 60% and exhibited minimal toxicity. Knockdown of host gene expression by these siRNAs was confirmed at the RNA level and, in some instances, at the protein level. The level of siRNA silencing of these host genes correlated well with inhibition of HCV. These genes included those that encoded a G-protein coupled receptor (TBXA2R), a membrane protein (LTbeta), an adapter protein (TRAF2), 2 transcription factors (RelA and NFkappaB2), 2 protein kinases (MKK7 and SNARK), and 2 closely related transporter proteins (SLC12A4 and SLC12A5). Of interest, some of these genes are members of the tumor necrosis factor/lymphotoxin signaling pathway. CONCLUSION: Findings of this study may provide important information for understanding HCV replication. In addition, these cellular genes may constitute a novel set of targets for HCV antiviral therapy.

Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro.

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.