Fragment screening targeting Ebola virus nucleoprotein C-terminal domain identifies lead candidates

Abstract

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 ¹H 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 ¹H,¹⁵N NMR spectra of ¹⁵N-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.

Keywords: Drug development; Ebola virus; Fragment screening; Nucleoprotein C-terminal domain.

Fig. 1.. Identification of fragments binding to eNP-CTD.

(A) The C-terminal domain of eNP (residues 641-739) was screened with 439 fragment compounds from the Maybridge Ro3 Core Library, formulated as mixtures of 4-5 compounds such that 15 μM protein was exposed to 75 μM of each compound. (B) Example of overlaid one-dimensional proton spectra of a compound peak with 15 μM protein (blue) and without (red). Significant protein-binding effects, defined as ≥ 10% loss of peak intensity or ≥ 0.002 ppm (1.2 Hz) change in chemical shift or both, for at least one compound peak, were found for 48 compounds.

Fig 2.. Affinity determination by ¹H,¹⁵N HSQC NMR and mapping of perturbations onto protein structure.

(A) Forty-seven compounds were tested by ¹H,¹⁵N protein-observe HSQC experiments (shown here, Maybridge CC69846) at single concentrations or in titration series and significantly perturbed peaks noted. The Q739 backbone amide and an unassigned W (Trp) sidechain amide are shown as examples of commonly perturbed peaks. (B) The nine residues perturbed by CC69846 could be grouped into Sites 1, 3, and 4; Site 2 was perturbed by other compounds. Structure models were prepared using Pymol. (C) Titration data for all perturbed residues within a site for three replicate CC69846 titrations were combined and fitted to give a single binding curve and K_d for each site.

Fig. 3.. Six compounds binding to discrete sites with K_d < 7.0 mM.

Binding sites and K_d values for each compound are shown.

Figure 4.. Correspondence of small-molecule binding hotspots with perturbed sites.

FTMap placed up to 16 compounds onto various sites on the crystal structure of eNP-CTD (4QB0) (Dziubanska et al., 2014) to identify small-molecule binding hotspots. For comparison, commonly perturbed residues at the four experimentally-determined binding sites are indicated by colored patches.

Figure 5.. Groove between Sites 3 and 4 and Docking of CC68513.

(A) The shallow groove between Site 3 (cyan, H669 and M670) and Site 4 (dark blue, H654) with moderately perturbed residues D663 and Y667 (medium blue) is shown with the distance between M670 and H654 side chains. (B) Two poses of compound CC68513 (magenta) placed within the groove by docking program HADDOCK are shown in the same orientation as above, with hydrogen bonds to D663 and Q659 (the latter not perturbed or highlighted) as predicted by Pymol. Compound CC68513 itself is 9.5 Å long. Three other poses suggested by HADDOCK have been omitted for clarity.