Small Molecules VP-14637 and JNJ-2408068 Inhibit Respiratory Syncytial Virus Fusion by Similar Mechanisms

Abstract

Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC 50 s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC 50 ‫؍‬ 5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [ 3 H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration ‫؍‬ 2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [ 3 H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

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

“…Characterization of the hit compound in infection and subinfection reporter assays and tracing of resistance to point mutations in the viral F protein confirmed interference with an F-mediated membrane merger as the underlying mechanism of anti-RSV activity. The RSV entry machinery emerges as highly susceptible to inhibition by small-molecule inhibitors, because structurally distinct lead compounds identified in several independent drug discovery campaigns all block membrane fusion (21)(22)(23)(24). Possible reasons for this prevalence may include that entry inhibition poses lower demands on the compound than other inhibition strategies, because it does not mandate plasma membrane permeability of the inhibitor.…”

“…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.…”

“…Five of the previously identified RSV entry inhibitors were synthetically developed to therapeutic candidate level and are at different stages of preclinical and clinical assessment (21)(22)(23)(24)31). 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.…”

“…Although G acts as a major virulence factor (18), the F protein alone is capable of mediating particle docking to target cells and membrane fusion (19,20). Consistent with this central role of F in initiating RSV infection, the protein emerged as a major target of small-molecule RSV inhibitors, since recent open screens identified several structurally distinct classes of RSV entry blockers (21)(22)(23)(24). Spotlighted by the benefit of the…”

Esterification of the Free Carboxylic Group in 3,4-di-O-caffeoylquinic Acid Enhances the Inhibition Activity against Respiratory Syncytial Virus (RSV)

“…Nowadays, it is recognized that RSV-F is a protein that mediates the fusion of viral envelope with host cell membrane and is regarded as a prominent target for therapeutic intervention [26][27][28]. A previous molecular docking study showed that 3,4-DCQA binds RSV-F well.…”