Structure-based Design of Novel Hepatitis B Virus Capsid Assembly Modulators.

Small-molecule capsid assembly modulators (CAMs) have been recently recognized as promising antiviral agents for curing chronic hepatitis B virus (HBV) infection. A target-based in silico screening study is described, aimed towards the discovery of novel HBV CAMs. Initial optimization of four weakly active screening hits was performed via focused library synthesis. Lead compound 42 and close analogues 56 and 57 exhibited in vitro potency in the sub- and micromolar range along with good physico-chemical properties and were further evaluated in molecular docking and mechanism of action studies.

Protein purification strategies must consider downstream applications and individual biological characteristics.

BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.

Discovery of substituted N-phenylbenzenesulphonamides as a novel class of non-nucleoside hepatitis C virus polymerase inhibitors.

The RNA-dependent RNA polymerase NS5B of the hepatitis C virus (HCV) has emerged as one of the key targets for antiviral drug discovery. Here we describe a novel non-nucleoside inhibitor (NNI) chemotype identified by screening: The substituted N-phenylbenzenesulphonamides (SPBS) which showed reversible inhibition of NS5B from HCV genotype 1b with IC(50) values up to 40 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M or M423T we assumed that the SPBS inhibitors bind to the thumb site II which has already been described as the allosteric binding site for the NNI carboxy thiophene. The postulated binding site was consequently confirmed by solving two co-crystal structures of NS5B in complex with SPBS analogues at 2.3 and 2.2Å resolutions. The inhibitors are hydrogen-bonded to the main chain Ser476 and Tyr477 and to the side chain of Arg501. In addition, the inhibitors displayed van der Waals interactions with several residues of the hydrophobic binding pocket Leu419, Ile482, Leu497, Met423 and Trp528. Notably, the two SPBS analogues reported here revealed significant differences in addressing the NH-group of the main chain Tyr477 by hydrogen-bonds, water-mediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the His475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using a panel of 38 individual NS5B proteins derived from different HCV genotypes, we could demonstrate inhibitory activity of the SPBS against polymerases from HCV genotypes 1a and 1b whereas the inhibitor class failed to inhibit any of the non-genotype 1 polymerases efficiently. Furthermore we demonstrated initial antiviral activity for SPBS against the subgenomic replicons of HCV genotypes 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types.

RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities.

The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1-6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg(2+) were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a>2a, 3a, 4a, 5a>1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.