Respiratory syncytial virus fusion inhibitors. Part 5: Optimization of benzimidazole substitution patterns towards derivatives with improved activity.

Extensive SAR studies and optimization of ADME properties of benzimidazol-2-one derivatives led to the identification of BMS-433771 (3) as an orally active RSV fusion inhibitor. In order to extend the structure-activity relationships for this compound series, substitution of the benzimidazole ring was examined with a view to establishing additional productive interactions between the inhibitor and functionality present in the proposed binding pocket. Amongst the compounds synthesized, the 5-aminomethyl analogue 10aa demonstrated potent antiviral activity towards wild-type RSV and retained excellent inhibitory activity towards a virus that had been developed to express resistance to BMS-433771 (3), data consistent with an additional productive interaction between the inhibitor and the fusion protein target.

Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion.

Trimeric class I virus fusion proteins undergo a series of conformational rearrangements that leads to the association of C- and N-terminal heptad repeat domains in a "trimer-of-hairpins" structure, facilitating the apposition of viral and cellular membranes during fusion. This final fusion hairpin structure is sustained by protein-protein interactions, associations thought initially to be refractory to small-molecule inhibition because of the large surface area involved. By using a photoaffinity analog of a potent respiratory syncytial virus fusion inhibitor, we directly probed the interaction of the inhibitor with its fusion protein target. Studies have shown that these inhibitors bind within a hydrophobic cavity formed on the surface of the N-terminal heptad-repeat trimer. In the fusogenic state, this pocket is occupied by key amino acid residues from the C-terminal heptad repeat that stabilize the trimer-of-hairpins structure. The results indicate that a low-molecular-weight fusion inhibitor can interfere with the formation or consolidation of key structures within the hairpin moiety that are essential for membrane fusion. Because analogous cavities are present in many class I viruses, including HIV, these results demonstrate the feasibility of this approach as a strategy for drug discovery.

Orally active fusion inhibitor of respiratory syncytial virus.

BMS-433771 was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. It exhibited excellent potency against multiple laboratory and clinical isolates of both group A and B viruses, with an average 50% effective concentration of 20 nM. Mechanism-of-action studies demonstrated that BMS-433771 inhibits the fusion of lipid membranes during both the early virus entry stage and late-stage syncytium formation. After isolation of resistant viruses, resistance was mapped to a series of single amino acid mutations in the F1 subunit of the fusion protein. Upon oral administration, BMS-433771 was able to reduce viral titers in the lungs of mice infected with RSV. This new class of orally active RSV fusion inhibitors offers potential for clinical development.

Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region.

Mammalian beta-globin loci contain multiple beta-like genes that are expressed at different times during development. The murine beta-globin locus contains two genes expressed during the embryo stage, Ey and betah1, and two genes expressed at both the fetal and postnatal stages, beta-major and beta-minor. Studies of transgenic human beta-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or betah1 promoter in the endogenous murine beta-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal-adult beta-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic beta-globin gene promoters and other genes in the locus. The implication of these findings for models of beta-globin gene expression are discussed.