Targeting the Non-catalytic RVxF Site of Protein Phosphatase-1 With Small Molecules for Ebola Virus Inhibition.

Xionghao Lin,Aykut Üren,Colette A Pietzsch,Dmytro Kovalskyy,Andrey Ivanov,Alexander Bukreyev,Tatiana Ammosova,Sergei Nekhai,Namita Kumari,Meng S Choy,Asrar Ahmad,Wolfgang Peti,Yasemin Saygideğer

doi:10.3389/fmicb.2019.02145

Abstract

Ebola virus (EBOV) is a non-segmented negative-sense RNA virus that causes a severe human disease. The ongoing EBOV outbreak in the Eastern part of Democratic Republic of the Congo has resulted to date in over 2500 confirmed cases including over 1500 deaths. Difficulties with vaccine administration indicate the necessity for development of new general drugs and therapeutic strategies against EBOV. Host Ser/Thr protein phosphatases, particularly PP1 and PP2A, facilitate EBOV transcription by dephosphorylating the EBOV VP30 protein and switching activity of the polymerase complex toward replication. Previously, we developed small molecule 1E7-03 that targeted host protein phosphatase-1 (PP1) and induces phosphorylation of EBOV VP30 protein thus shifting transcription–replication balance and inhibiting EBOV replication. Here, we developed a new EBOV inhibitor, 1E7-07, that potently inhibits EBOV replication and displays significantly improved metabolic stability when compared to previously described 1E7-03. Proteome analysis of VP30 shows that 1E7-07 increases its phosphorylation on Thr-119 and Ser-124 over 3-fold with p < 0.001, which likely contributes to EBOV inhibition. We analyzed 1E7-07 binding to PP1 using a mass spectrometry-based protein painting approach. Combined with computational docking, protein painting shows that 1E7-07 binds to several PP1 sites including the RVxF site, C-terminal groove and NIPP1-helix binding pocket. Further analysis using surface plasmon resonance and a split NanoBiT system demonstrates that 1E7-07 binds primarily to the RVxF site. Together, detailed analysis of 1E7-07 binding to PP1 and identification of the RVxF site as the main binding site opens up an opportunity for future development of PP1-targeting EBOV inhibitors.

Highlights

Ebola virus (EBOV) is a non-segmented negative-sense RNA virus that causes a severe human disease (Feldmann and Geisbert, 2011)
EBOV-eGFP was added at a multiplicity of infection (MOI) of 0.3 PFU/cell, and viral replication was measured by eGFP fluorescence at day 3 post-infection (Edwards et al, 2015)
We previously demonstrated that 1E7-03 induces phosphorylation of EBOV VP30 (Ilinykh et al, 2014)

Summary

Introduction

Ebola virus (EBOV) is a non-segmented negative-sense RNA virus that causes a severe human disease (Feldmann and Geisbert, 2011). The 2013–2016 epidemic of EBOV in West Africa was the largest in recorded history and resulted in more than 28,000 cases and over 11,000 deaths (Who Ebola Response Team et al, 2016). There are no Food and Drug Administration (FDA)-approved drugs for the treatment or prevention of EBOV infection. A cocktail of monoclonal antibodies has been used on individuals infected with EBOV and this treatment is still undergoing clinical trials (The PREVAIL II Writing Group for the Multi-National PREVAIL II Study Team et al, 2016). A recent Merck vaccine showed good protection against EBOV during a phase III trial (Henao-Restrepo et al, 2017), but is not expected to provide protection against additional Ebolaviruses such as Bundibugyo and Sudan, which are important human pathogens. Small molecules favipiravir and GS5734 are yet to demonstrate success in preventing EBOV infection in their clinical trials (Hayden, 2018). It is imperative to develop new general drugs and therapeutic strategies against EBOV

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Conclusion