1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein.

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.

Genotype dependent QSAR for HIV-1 protease inhibition.

The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs.