Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry.

Adam Schafer,Hyun Lee,Norton P Peet,Benjamin E Ramirez,Yangfeng Li,Han Cheng,Erica Ollmann Saphire,Raghad Nowar,Lijun Rong,Michael Caffrey,Laura Cooper,Rui Xiong,Gregory R J Thatcher

doi:10.1371/journal.ppat.1009312

Abstract

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

Highlights

Ebola virus (EBOV) and Marburg virus (MARV), are single-stranded, non-segmented RNA viruses belonging to the family Filoviridae [1,2]
We identify the heptad repeat 2 (HR2) domain as a potential secondary binding site for small molecule EBOV GP inhibitors and the potential primary GP binding site for MARV GP inhibitors
In the co-crystal structure of toremifene and the EBOV GP (PDB:5JQ7), tyrosine 517 (Y517) is positioned to form a T-shaped pi stacking interaction with all three phenyl rings (A-C) of toremifene at distances of 5.4 Å, 5.7 Å, and 5.4 Å, respectively (Fig 1D), suggesting that Y517 could be a critical residue for stabilization of drug binding

Summary

Introduction

Ebola virus (EBOV) and Marburg virus (MARV), are single-stranded, non-segmented RNA viruses belonging to the family Filoviridae [1,2]. Due to their stability in aerosolized form and high case-fatality rates, EBOV and MARV are classified as risk group 4, category A priority pathogens in the NIAID Biodefense Research Agenda. The host proteolytically processes the GP resulting in the removal of the mucin-domain and the glycan cap of the GP1 subunit [15,16,17]. Developing broad-spectrum small molecule GP inhibitors with a long shelf-life and oral bioavailability is an attractive and complementary therapeutic strategy for the treatment and prevention of filovirus infections

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Discussion

Conclusion