High throughput screening and drug discovery for antagonists of the Ebola VP40 protein assembly

PROJECT SUMMARY
This R43 proposal answers the call of the RFA PA-22-176 for assay development and
chemical probe screening by addressing the unmet need for development of antiviral
treatments for Ebola patients through an innovative high-content small molecule screen
for antagonists of the Ebola VP40 matrix protein. There is a need for a fast-acting therapy
that is independent of the immune system and targets essential viral proteins. Such a
novel therapeutic is anticipated to enhance the survival probability for infected people in
hot zones of an Ebola outbreak. We chose to target EBOV VP40 because it is absolutely
required for EBOV particle assembly at the cell membrane, is capable of budding virus-
like particles (VLPs) when expressed in isolation and VP40 protein-protein interaction
domains have been structurally determined. Further guiding this application is our
published pilot screen which established proof-of-concept by demonstrating the
accessibility of Ebola VP40 protein-protein interactions to sangivamycin, a dual acting
small molecule antagonist of both EBOV VP40 assembly of virions and the viral
replication machinery. Given this success and due to anticipated complications with
efficacy and MOA studies inherent to compounds with dual targets, our Specific Aims
propose to screen a library of ~123,000 small molecule compounds to identify antagonists
of VP40 accumulation at the cell membrane for VLP formation and release from cells
through a fully automated, quantitative, and high-content assay. The assay has been
vetted to quantify the effect of small molecules on the cellular distribution of a fluorescent
VP40 expressed in 293T cells. Hits validated as dose-dependent by qHTS and displaying
low cytotoxicity will be further prioritized based on their absolute requirement for VP40 in
antiviral mechanism of action through counter screening with the VP40- independent
minigenome assay. Lead compounds also will be prioritized by their favorable ADMET
profiles. Our proposed critical path anticipates identifying 2-4 dose-dependent, VP40-
selective antagonists with potent antiviral activity that display low cytotoxicity for future
medicinal chemistry and preclinical development.

Grant Number: 5R43AI179320-02
NIH Institute/Center: NIH
Principal Investigator: Ryan Bennett