Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the ␤-flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 Å away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).

HCV NS5B polymerase is a validated target for the treatment of hepatitis C, known to be one of the most challenging enzymes for docking programs. In order to improve the low accuracy of existing docking methods observed with this challenging enzyme, we have significantly modified and updated F itted 1.0, a recently reported docking program, into F itted 1.5. This enhanced version is now applicable to the virtual screening of compound libraries and includes new features such as filters and pharmacophore- or interaction-site-oriented docking. As a first validation, F itted 1.5 was applied to the testing set previously developed for F itted 1.0 and extended to include hepatitis C virus (HCV) polymerase inhibitors. This first validation showed an increased accuracy as well as an increase in speed. It also shows that the accuracy toward HCV polymerase is better than previously observed with other programs. Next, application of F itted 1.5 to the virtual screening of the Maybridge library seeded with known HCV polymerase inhibitors revealed its ability to recover most of these actives in the top 5% of the hit list. As a third validation, further biological assays uncovered HCV polymerase inhibition for selected Maybridge compounds ranked in the top of the hit list.

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The HCV NS5B RNA dependent RNA polymerase plays an essential role in viral replication. The discovery of a novel class of inhibitors based on an N,N-disubstituted phenylalanine scaffold and structure-activity relationships studies to improve potency are described.

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.