In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors

Abstract

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC₅₀ <200μM, with the best compounds having an IC₅₀ of 20μM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC₅₀ <200μM and one compound with an IC₅₀ of 4μM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.

Keywords: Ebola; Inhibitor; Innate immune response; Molecular dynamic modeling; VP35.

Figure 1

Structure of the VP35 IID with positive (blue) and negative (red) charged surfaces. Residues essential for competent dsRNA binding are labeled.

Figure 2

(A) Compounds tested for VP35 IID inhibition via EMSA. P=probe only, C=negative control, F=free probe, B=protein bound probe *=complexation of protein-dsRNA and SMI. Compound concentration is 200 μM. (B) Identity and structures of top performing compounds. (C). Dose response of VP35 IID inhibition by ZINC1630966. Compound was serially diluted two fold from 200 μM. C^A1=200 μM coumermycin A1.

Figure 3

Overlay of 26 crystal structures of VP35 IID. Residues essential for dsRNA binding are shown. Arg305 and Lys339 show large degrees of freedom, whereas Lys319 and Arg322 present low degrees of freedom. Lys309 and Arg312 both show a bimodal appearance.

Figure 4

(A) EMSA of compounds from ensemble-flexible docking. P=probe only, C=negative control, F=free probe, B=protein bound probe. *=complexation of protein-dsRNA and SMI. Compound concentration is 200 μM. (B) Identity, corresponding lane # from (A) and structures of top performing compounds. (C) Dose response of VP35 IID inhibition by ZINC05328460. Compound was serially diluted two fold from 200 μM. C^A1=200 μM coumermycin A1. (D) EMSA of ZINC05328460 and partial fragments of ZINC05328460. Lane 1-ZINC05328460. Lane 2-ZINC19319423. Lane 3-ZINC00394385. Lane 4-Coumermycin A1. All compounds were used at 200 μM.