Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

The pharmaceutical industry has recognized that many drug-like molecules can self-aggregate in aqueous media and have physicochemical properties that skew experimental results and decisions. Herein, we introduce the use of a simple NMR strategy for detecting the formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that (1)H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation and for determining the concentrations of single molecule, aggregate entities, and precipitate. The critical concentrations above which aggregation begins can be found and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzim- More than 2% of the world population are chronically infected with hepatitis C virus (HCV), 2 a flavivirus that is the etiological agent of non-A non-B hepatitis (1, 2). A large proportion of patients fail to achieve a sustained response to current therapies consisting of a combination of pegylated interferon and ribavirin. The discovery and development of specific anti-HCV chemotherapies aims to address this unmet clinical need and has focused on inhibitors of virally encoded functions. HCV encodes a linear polyprotein of ϳ3010 amino acids that is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins (for review, see Ref.3). One of the non-structural proteins, NS5B, catalyzes the RNA-dependent RNA polymerization of a negative strand intermediate and the subsequent generation of multiple copies of the plus strand viral genome; this enzyme has emerged as a principal target for chemotherapeutic inhibition of HCV replication (4).The three-dimensional structure of the NS5B polymerase reveals an organization comparable with other nucleic acid polymerases with the familiar features of fingers, palm, and thumb domains that are organized in a "right-hand" motif (5-7). A distinct feature of the HCV polymerase (and closely related RNA-dependent RNA polymerase) active site cavity is the protrusion of a unique ␤-hairpin from the thumb subdomain that apparently plays a role in the initiation of de novo RNA synthesis as demonstrated by both structural and biochemical studies (8 -11). Another additional feature of the HCV polymerase is two loops that bridge the fingers and thumb subdomain and result in an encircled active site. This feature is now known to be shared by other RNA-dependent RNA polymerase from rhinovirus, bacteriophage 6, rabbit hemorrhagic disease virus, bovine viral diarrhea virus, Norwalk virus,. Interestingly, the interface between the HCV polymerase N-terminal 1 loop and the thumb subdomain is the location of a GTP binding site (8), although its precise biological role is unsolved.A number of different HCV polymerase inhibitors have emerged that can be broadly di...

Addition of oxone ® to a mixture of a 1,2-phenylenediamine and an aldehyde in wet DMF at room temperature results in rapid formation of benzimidazoles under very mild conditions. The reaction is applicable to a wide range of substrates including aliphatic, aromatic and heteroaromatic aldehydes, and is not significantly affected by steric or electronic effects. In most cases, crude products are isolated in good to excellent yields (59-95%) and homogeneities (86-99%) by simple precipitation or extraction from the reaction mixture and do not require additional purification. Limitations to the scope of this methodology were encountered in cases where aldehydes were sensitive to oxone ® under the acidic reaction conditions. The features of this methodology make it particularly well suited for the high-throughput, solution-phase synthesis of benzimidazole libraries. The low cost and simplicity of this procedure makes it equally attractive for preparative-scale syntheses where safety and environmental issues are of greater concern. Scheme 1 Table 1 1,2-Phenylenediamine Starting Materials 1 Starting material R 2 NH 2 Yield (%) Yield (%) 98 99 5 100 98 6 99 70 7 98 95 8

Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.