Potent in vitro synergistic antiviral effects of the pan-coronavirus fusion inhibitor EK1 in combination with RBD-specific antibodies or M pro inhibitors

ABSTRACT The continuous evolution of coronaviruses compromises the efficacy of existing therapeutic antibodies and replication inhibitors, necessitating antiviral strategies that are resilient to viral escape. Distinct from conventional antibody cocktails that remain vulnerable to spike protein mutations, we evaluated a multi-mechanistic therapy targeting three discrete stages of the viral life cycle: receptor binding, membrane fusion, and viral replication. Using pseudotyped and authentic human coronaviruses (HCoVs), including SARS-CoV-2 and HCoV-OC43, in cell culture models, we demonstrated that combining the pan-coronavirus fusion inhibitor EK1 with a receptor-binding domain-specific broadly neutralizing antibody (bnAb) and/or a main protease (M pro ) inhibitor yields synergistic inhibition. Collectively, our findings establish a conceptual framework for a trident antiviral regimen that theoretically restricts the evolutionary space for viral escape, although clinical translation will require optimizing delivery strategies to reconcile the distinct pharmacokinetic profiles of these agents. IMPORTANCE The continuous emergence of highly transmissible and immune-evasive SARS-CoV-2 variants highlights an urgent need for broad-spectrum antiviral approaches capable of countering rapid viral evolution and future coronavirus outbreaks. Here, we designed a dual- or triple-stage antiviral strategy centered on the membrane fusion inhibitor EK1, in combination with a receptor-binding domain-targeting antibody and/or a small-molecule viral replication inhibitor (M pro inhibitor). In cell culture models, our results demonstrate marked synergy between EK1 and the antibody and/or the replication inhibitor. These combinations not only enhance antiviral potency but are also anticipated to maintain efficacy against emerging variants. By targeting non-overlapping stages of the viral life cycle, this approach theoretically raises the genetic barrier to resistance. While clinical translation will require reconciling the distinct pharmacokinetic profiles of these components, our study provides a conceptual framework for developing a trident anti-coronavirus therapy.