ABSTRACT
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Subject(s)
Amides/chemistry , Antiviral Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Hepacivirus/drug effects , Thiophenes/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Viral Nonstructural Proteins/metabolismABSTRACT
Benzothiazine-substituted tetramic acids were discovered as highly potent non-nucleoside inhibitors of HCV NS5B polymerase. X-ray crystallography studies confirmed the binding mode of these inhibitors with HCV NS5B polymerase. Rational optimization of time dependent inactivation of CYP 3A4 and clearance was accomplished by incorporation of electron-withdrawing groups to the benzothiazine core.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Pyrrolidinones/chemistry , Thiazines/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Binding Sites , Crystallography, X-Ray , Pyrrolidinones/chemical synthesis , Pyrrolidinones/pharmacokinetics , Rats , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
A series of benzo[d]isothiazole-1,1-dioxides were designed and evaluated as inhibitors of HCV polymerase NS5B. Structure-based design led to the incorporation of a high affinity methyl sulfonamide group. Structure-activity relationship (SAR) studies of this series revealed analogues with submicromolar potencies in the HCV replicon assay and moderate pharmacokinetic properties. SAR studies combined with structure based drug design focused on the sulfonamide region led to a novel and potent cyclic analogue.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Thiazoles/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Binding Sites , Crystallography, X-Ray , Haplorhini , Rats , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/pharmacokinetics , Viral Nonstructural Proteins/metabolismABSTRACT
The importance of internal hydrogen bonding in a series of benzothiadiazine and 1,4-benzothiazine NS5b inhibitors has been explored. Computational analysis has been used to compare the protonated vs. anionic forms of each series and we demonstrate that activity against HCV NS5b polymerase is best explained using the anionic forms. The syntheses and structure-activity relationships for a variety of new analogs are also discussed.
Subject(s)
Antiviral Agents/chemical synthesis , Benzothiadiazines/chemical synthesis , DNA-Directed RNA Polymerases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Hepacivirus/drug effects , Thiazines/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Benzothiadiazines/chemistry , Benzothiadiazines/pharmacology , Computational Biology , Computer Simulation , Crystallography, X-Ray , DNA-Directed RNA Polymerases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Protein Binding , Structure-Activity Relationship , Thiazines/chemistry , Thiazines/pharmacology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
The discovery of 4'-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett. 2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC(50) = 1.28 microM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of several monofluoro and difluoro derivatives of 4'-azidocytidine. The most potent compounds in this series were 4'-azido-2'-deoxy-2',2'-difluorocytidine and 4'-azido-2'-deoxy-2'-fluoroarabinocytidine with antiviral EC(50) of 66 nM and 24 nM in the HCV replicon system, respectively. The structure-activity relationships within this series were discussed, which led to the discovery of these novel nucleoside analogues with the most potent compound, showing more than a 50-fold increase in antiviral potency as compared to 4'-azidocytidine (3).
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Azides/chemical synthesis , Azides/pharmacology , Deoxycytidine/analogs & derivatives , Drug Design , Hepacivirus/physiology , Virus Replication/drug effects , Antiviral Agents/chemistry , Azides/chemistry , Cell Line, Tumor , Deoxycytidine/chemical synthesis , Deoxycytidine/chemistry , Deoxycytidine/pharmacology , Hepacivirus/drug effects , HumansABSTRACT
Isoquinoline-based non-nucleoside inhibitors of HCV NS5b RNA-dependent RNA-polymerase are described. The synthesis and structure-activity relationships are detailed, along with enzyme and cellular activity.
Subject(s)
Enzyme Inhibitors/pharmacology , Isoquinolines/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Isoquinolines/chemistry , Models, Molecular , Structure-Activity RelationshipABSTRACT
The NS5B encoded by the hepatitis C virus genome is a RNA-dependent RNA polymerase essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. Reported here is the molecular cloning and expression of the full-length NS5B polymerase (NS5B-FL) in bacterial cells as a non-fusion protein. The non-tagged NS5B-FL was purified to homogeneity using sequential chromatographic columns and its identity was confirmed using anti-NS5B peptide antibodies and amino acid sequencing. Purified NS5B-FL demonstrated RNA-dependent RNA polymerase activity and was able to replicate a HCV RNA genome fragment through both copy-back and de novo mechanisms. Its biochemical properties were further characterized in comparison with a truncated form of NS5B polymerase with a deletion of 51 residues from its C-terminus.
Subject(s)
Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , Base Sequence , Cloning, Molecular , DNA Primers , Substrate Specificity , Viral Nonstructural Proteins/metabolismABSTRACT
The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.