Fragment screening targeting Ebola virus nucleoprotein C-terminal domain identifies lead candidates.
David J Aceti, Hamza Ahmed, William M Westler, Chao Wu, Hesam Dashti, Marco Tonelli, Hamid Eghbalnia, Gaya K Amarasinghe, John L Markley
Author Information
David J Aceti: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA. Electronic address: djaceti@wisc.edu.
Hamza Ahmed: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA. Electronic address: hahmed@wustl.edu.
William M Westler: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA. Electronic address: milo@nmrfam.wisc.edu.
Chao Wu: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA. Electronic address: chaowu@wustl.edu.
Hesam Dashti: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 02215, USA. Electronic address: hdashti@bwh.harvard.edu.
Marco Tonelli: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA. Electronic address: tonelli@nmrfam.wisc.edu.
Hamid Eghbalnia: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA. Electronic address: eghbalni@nmrfam.wisc.edu.
Gaya K Amarasinghe: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA. Electronic address: gamarasinghe@wustl.edu.
John L Markley: Biochemistry Department and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, 53706, USA. Electronic address: jmarkley@wisc.edu.
The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013-2016) led to 11,000+ deaths and 30,000+ Ebola infected individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3000+ confirmed cases and 2000+ deaths attributed to Ebola virus infections provides a reminder that innovative countermeasures are still needed. Ebola virus encodes 7 open reading frames (ORFs). Of these, the nucleocapsid protein (eNP) encoded by the first ORF plays many significant roles, including a role in viral RNA synthesis. Here we describe efforts to target the C-terminal domain of eNP (eNP-CTD) that contains highly conserved residues 641-739 as a pan-Ebola antiviral target. Interactions of eNP-CTD with Ebola Viral Protein 30 (eVP30) and Viral Protein 40 (eVP40) have been shown to be crucial for viral RNA synthesis, virion formation, and virion transport. We used nuclear magnetic response (NMR)-based methods to screened the eNP-CTD against a fragment library. Perturbations of 1D H NMR spectra identified of 48 of the 439 compounds screened as potential eNP CTD interactors. Subsequent analysis of these compounds to measure chemical shift perturbations in 2D H,N NMR spectra of N-labeled protein identified six with low millimolar affinities. All six perturbed an area consisting mainly of residues at or near the extreme C-terminus that we named "Site 1" while three other sites were perturbed by other compounds. Our findings here demonstrate the potential utility of eNP as a target, several fragment hits, and provide an experimental pipeline to validate viral-viral interactions as potential panfiloviral inhibitor targets.
