Formation of a triple complex of viral capsid protein 1 and two capsid-binding inhibitors explains synergistic interactions observed in combination studies with rhinoviruses

Capsid-binding inhibitors, such as pleconaril and OBR-5-340, which prevent the attachment and/or uncoating of most rhinovirus (RV) types, were considered promising candidates for effective anti-RV drug development. Both inhibitors target viral protein 1 (VP1), but their efficacy, spectrum of anti-RV activity, and binding mechanism are different. We hypothesized that combinations of pleconaril and OBR-5-340 might be a promising approach to improve treatment strategies for RV infections. To validate our hypothesis, we analyzed the anti-RV effects of a 6 × 6 concentration matrix compared to monotherapy in vitro. Antiviral studies included multiple RV types, some of which are sensitive or insensitive to pleconaril and OBR-5-340, to consider the diversity of VP1. Synergy analysis was performed with Loewe additivity, Bliss independence, highest single agent, and zero interaction potency models. The results indicate a significant synergistic effect at certain concentrations. Molecular dynamic simulations investigated the molecular basis of the observed synergy. Intriguingly, VP1, pleconaril and OBR-5-340 can bind simultaneously to form a triple complex with additional stabilizing hydrophobic interactions and hydrogen bonds. Consequently, pleconaril-OBR-5-340 combination surpassed the efficacy of monotherapy and inhibited a broader RV spectrum compared to monotherapy. In conclusion, this study contributes to the development of broader-spectrum anti-RV treatments and provides insights into the mechanism behind. This strategy could also be important for treatment of diseases caused by other enteroviruses. The identified strong synergism warrants further preclinical studies for example with, ex vivo or human viral challenge models to translate this synergy into oral or inhaled/topical use for rhinovirus treatment.