Modeling the Molecular Biology of Infection & Anti-Viral Activity of Small Molecules for Ebola Virus & SARS-CoV-2 using Virus-Like Particles

Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent modern threats to global health. Although prophylactic vaccines exist for the prevention of both EBOV and SARS-CoV-2 infections, there are limited FDA-approved options for treatment of those that become infected with either. In this work, a small molecule natural product called diphyllin and a series of phenol-substituted diphyllin derivatives were evaluated for activity against authentic EBOV and SARS-CoV-2 infections. Diphyllin and its synthetic derivatives were active against both viruses, with the N-methyl piperazine substituent yielding the most potent activity against the diphyllin pharmacophore’s molecular target, vacuolar (V)-ATPase, as well as against both the authentic EBOV and SARS-CoV-2 infections.

To gain insight into the mechanism of the diphyllin derivatives against EBOV, a series of experiments modeling different stages of the viral lifecycle were performed using virus-like particles (VLPs). VLPs morphologically and functionally resemble the authentic virus; however, they lack the viral genome and are thus considered non-infectious, making them BSL-2 compatible models. VLP studies revealed that diphyllin derivatives do not inhibit virus association with cells, uptake into the endocytic system, or trafficking to lysosomes; however, diphyllin derivatives specifically inhibit pH-dependent virus fusion with lysosomes and virus escape into the cytoplasm. By this mechanism, diphyllin and derivatives prevent EBOV from gaining access to the host cell cytoplasm where viral transcription, replication, and translation would be established. As some of the most potent in-vitro inhibitors known for EBOV, this work fills an important knowledge gap in understanding the mechanism of these inhibitors.

Due to the shared use of pH-dependent fusion in lysosomes between EBOV and SARS-CoV-2, we hypothesized that diphyllin derivatives would function in the same manner against this virus as it does EBOV: by specifically inhibiting fusion. To test this hypothesis, we wanted to use VLPs; however, a functional BSL-2 compatible VLP model for SARS-CoV-2 did not yet exist. We developed and validated multiple SARS-CoV-2 VLP assays to model viral association with cells, uptake into the endocytic system, trafficking to lysosomes, fusion with lysosomes, assembly at internal membranes, and budding/release using different chemical tags accessible to confocal microscopy, electron microscopy, and fluorescent microplate readers.