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.

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.

An assay recapitulating the 3′ processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/ aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3=-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (

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...