The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC 50 of ϳ0.5 M, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.Hepatitis C virus (HCV), 1 a positive strand RNA virus of the Flaviviridae family, is the major etiological agent of post-transfusion and sporadic non-A, non-B hepatitis (1). An estimated 2-3% of the world population is chronically infected with HCV, which causes significant liver disease, cirrhosis, and can eventually lead to the development of hepatocellular carcinoma. In infected cells, translation of the viral RNA yields a 3011-residue polyprotein chain (2-4), which is subsequently cleaved to generate envelope and core proteins, for assembly of new virus particles and nonstructural enzymes essential for viral replication (5-7). Studies using recombinant NS5B polymerase have provided direct evidence for RNA-dependent RNA polymerase activity (8, 9), and this catalytic activity has been confirmed to be required for infectivity in chimpanzees (10).NS5B polymerase contains a hydrophobic C-terminal domain thought to be responsible for anchoring the protein to mammalian cell membranes. Removal of the C-terminal 21 residues has been reported to facilitate protein isolation from Escherichia coli without compromising RdRp activity (11). The HCV RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12, 13-15) but lacks specificity for HCV RNA in vitro, because it readily utilizes heterologous nonviral templates (8). Based on crystallographic studies of the enzyme containing C-terminal truncations (16, 17), the hydrophobic tail present in the full-length ...
Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.
Germ-line mutations in DNA mismatch-repair genes impart a markedly elevated cancer risk, often presenting as autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC). However, there are no pathognomonic features of HNPCC, not all gene carriers have a family history of the disease, and families fulfilling the Amsterdam criteria are relatively uncommon. Genetic testing of probands with early-onset colorectal cancer, irrespective of family history, is one approach that would allow predictive genetic testing of at-risk relatives. We cloned and sequenced hMSH2 and hMLH1 introns, to optimize genomic sequencing. We then systematically analyzed the entire hMSH2 and hMLH1 genes, by genomic sequencing and in vitro synthesized-protein-truncation assay (IVSP), in 50 colorectal cancer patients <30 years of age at diagnosis. To determine polymorphic variants, 26 anonymous donors also were sequenced. All subjects analyzed had at least 1 of 37 different polymorphic or pathogenic variants. IVSP complemented genomic sequencing, by detection of mutations not identified by genomic analysis. Fourteen cancer patients (28%) had pathogenic mutations, and a number of other variants also may have had a pathogenic significance that remains to be elucidated. Tumor replication-error status was useful in targeting sequencing efforts for this cohort of young patients: sensitivity was 86%, specificity 73%, and positive and negative predictive values 63% and 90%, respectively. These data indicate that an appreciable proportion of young colon cancer probands carry a germ-line mutation in a DNA mismatch-repair gene.
Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.The Flaviviridae family is comprised of three genera, Flavivirus (such as Yellow fever virus and Dengue virus types 1 to 4), Hepacivirus (such as Hepatitis C virus [HCV]), and Pestivirus (such as Bovine viral diarrhea virus [BVDV]) (28). BVDV infection represents an economically important disease of cattle, and BVDV has been identified as the causative agent of viral diarrhea-mucosal disease (reviewed in references 1, 12, 25, and 37). Like the other members of the Flaviviridae, BVDV is an enveloped, plus-stranded RNA virus whose genome consists of a nonsegmented single-stranded RNA molecule. BVDV genomic RNA is approximately 12.5 kb and encodes a single open reading frame of approximately 3,900 amino acids (7-9, 24). The polyprotein translated from the open reading frame is subsequently processed by virally encoded and cellular proteases into 12 individual proteins (13,30,31,35,44). These individual proteins function either as structural components of the virion or presumably, at least in part, as components of the viral RNA replicase complex as described for other Flaviviridae family members (2, 6, 17). RNA replicons derived from defective interfering particles have shown that the 5Ј and 3Ј nontranslated regions (NTRs) along with the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B can support RNA replication (4, 45). However, the essentiality of the individual nonstructural proteins has not yet been tested.The NS5B proteins of both BVDV (46) and the related HCV (3, 23) display RNA...
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