NS5B Enzyme Research Articles

Identification of [(Naphthalene‐1‐carbonyl)‐amino]‐acetic Acid Derivatives as Nonnucleoside Inhibitors of HCV NS5B RNA Dependent RNA Polymerase.

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Identification of [(naphthalene-1-carbonyl)-amino]-acetic acid derivatives as nonnucleoside inhibitors of HCV NS5B RNA dependent RNA polymerase

A novel series of HCV NS5B RNA dependent RNA polymerase inhibitors containing a naphthalene carboxamide scaffold were identified by high throughput screening. Optimization of substituents by parallel synthesis and the iterative design towards understanding structure–activity relationship to improve potency are described. Tetra substituted naphthalene 31 displayed potent activity with IC 50 of 120 nM against HCV NS5B enzyme and was selective over a panel of polymerases.

Solid‐Phase Synthesis and Screening of Macrocyclic Nucleotide‐Hybrid Compounds Targeted to Hepatitis C NS5B

A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modified ribonucleosides (such as 2'-OMe-ribonucleotides), as well as threoninol derivatives. This methodology allows a fully automated assembly by means of standard phosphoramidite chemistry and is based on a recently published procedure for the preparation of cyclic oligodinucleotides in the DNA series (M. Smietana, E. T. Kool, Angew. Chem. 2002, 114, 3856-3859; Angew. Chem. Int. Ed. Engl. 2002, 41, 3704-3707). A library of potential cyclic hybrid inhibitor compounds targeting hepatitis C virus NS5B enzyme (the replicating polymerase of HCV) was generated by means of the parallel-pool strategy. Screening of the library revealed that cyclic hybrid c(C(OME)EthenodA) was a significant inhibitor of NS5B, with an IC(50) of 40 microM. Preliminary structure-activity studies of this lead compound are described.

Modulation of Hepatitis C Virus RNA-dependent RNA Polymerase Activity by Structure-based Site-directed Mutagenesis

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.