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

Abstract

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.

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

“…Although biochemical data reveal direct binding of two inhibitor classes to F residue Y198 (21,30), we propose based on three major lines of evidence-structural insight, biochemical characterization, and functional data-that unprecedented broad cross-resistance of RSV against multiple structurally diverse entry inhibitors is based on indirect escape. First, compound docking into postfusion RSV F structures failed to provide a mechanistic explanation for the hotspot around F residues 392-401 in resistance (30).…”

“…Photoaffinity labeling assays implied physical binding of compounds representing two different inhibitor classes to F residue Y198 (21,30), which is located in the HR-A domain and was proposed to reside in the immediate vicinity of HR-B residue D489 in a hydrophobic pocket in the final 6HB fusion core (illustrated in Fig. S7).…”

“…In addition to these two classes, resistance hotspots were determined also for the remaining three scaffolds and, in analogy to our characterization of GPAR-3710, in all cases included F residues in the 400 and/or 489 microdomains (22,24). Although recognized as surprising that distinct chemical inhibitor classes supposedly target the same F microdomain with high affinity (30), previous studies concluded that all of these diverse compounds prevent RSV entry through docking into the same hydrophobic cavity around residue Y198 in the central HR-A triple helix (21,30), which emerges transiently during assembly of the 6HB structure. Curiously, none of the resistance mutations reported for any of these compounds maps to HR-A residues that surround Y198 and are implicated in forming the proposed target cavity, or any other position in the HR-A domain.…”

“…Based on a biochemical target analysis, it was proposed that BMS-433771 populates a hydrophobic pocket in the HR-A triple helix that contains residue 489, preventing assembly of the 6HB fusion core during F refolding into its postfusion conformation (30). Surprisingly, however, both resistance domains map to opposing ends of the rod-like postfusion F structure, separated by ∼100 Å from each other (Fig.…”

“…In the resulting thermodynamically highly stable six-helix bundle (6HB) structure, the fusion peptides and transmembrane domains and, thus, the target and donor membranes, are brought into close proximity (29). Based on the appearance of resistance mutations in F protein HR domains and biochemical evidence, some of the most advanced RSV entry inhibitors were suggested to prevent the creation of fusion pores through competitive interference with 6HB closure (21,30).…”