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.

One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1). In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro. Sequence comparison reveals that residues 173-357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.Promoter-dependent transcription by RNA polymerase II (RPII) requires six general transcription factors (reviewed in ref.

TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxylterminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex. Initiation of transcription by RNA polymerase (RNAP)1 II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Ref. 1). Beginning with TFIID, whose TATA box-binding protein subunit recognizes the TATA box present in many promoters, these factors can assemble in an ordered pathway in vitro onto a promoter (2, 3), resulting in the formation of a preinitiation complex containing more than 40 polypeptides. Subsequently, however, yeast and mammalian RNAP II holoenzymes that contain several or all of the general transcription factors and other polypeptides were discovered (4 -9). There is evidence that transcription by RNAP II in Saccharomyces cerevisiae generally depends on such a holoenzyme (10) and that recruitment of yeast holoenzyme to a promoter would lead to a high rate of transcription (11).During or shortly after initiation by RNAP II, the carboxylterminal domain (CTD) of its largest subunit becomes heavily phosphorylated and remains so during transcript elongation (12). The phosphorylated form of RNAP II is designated RNAP IIO, whereas the unphosphorylated form is designated RNAP IIA. One subunit of TFIIH is a protein kinase that can phosphorylate the CTD (13). Phosphorylation of the CTD by PTEFb, a different Drosophila CTD kinase, has been shown to enhance the processivity of chain elongation by RNAP II in vitro (14). Concomitant with or following the termination of transcription, the CTD must be dephosphorylated by a protein phosphatase, since RNAP IIO cannot assemble directly into a preinitiation complex on either the adenovirus-2 major late or murine dihydrofolate reductase promoter in vitro (15-17). Accordingly, CTD phosphatase may function as a global regulator of gene expression by controlling the pool of RNAP IIA available for initiation. A phosphatase whose activity is stimulated by RAP74 and dephosphorylates the CTD in a processive manner has been purified from HeLa cell extracts (18,19).Certain activator proteins increase the efficiency of RNA chain elongation downstream from the promoter. For example, RNAP II pauses with an unphosphorylated CTD about 25-40 nucleotides downstream from the initiation site of Drosophila hsp70 genes and is stimulated by heat shock and the heat shock factor to become phosphorylated and leave these pause sites (Ref. 20 and references therein). Increasing evidence suppo...

A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

Nosiheptide (9671 R.P.) isolated from Streptomyces actuosus 40037 (NRRL 2954) is a sulfur-containing polypeptidic antibiotic, quite different from all the other members of this family. Very active in vitro against gram-positive bacteria, it is inactive in vivo in experimentally infected mice. Not toxic, even at high dose, it may be used as a feed additive for chickens and pigs and it shows a favourable effect on the growth and conversion index.

Phosphatidylinositol-4-kinase III␣ (PI4KIII␣) is an essential host cell factor for hepatitis C virus (HCV)replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for smallmolecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIII␣ inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIII␣ inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4III␣-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4III␣ inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIII␣ as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIII␣, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIII␣ inhibitors for treatment of chronic HCV infection.

The murine Elo (eye lens obsolescence) mutation confers a dominant phenotype characterized by malformation of the eye lens. The mutation maps to chromosome 1, in close proximity to the gamma E-crystallin gene which is the 3'-most member of the gamma-crystallin gene cluster. We have analysed the sequence of this gene from the Elo mouse and identified a single nucleotide deletion which destroys the fourth and last "Greek key" motif of the protein. This mutation is tightly associated with the phenotype, as no recombination was detected in 274 meioses. In addition, the mutant mRNA is present in the affected lens, providing further support for our hypothesis that the deletion is responsible for the dominant Elo phenotype.