Structure-based drug design for envelope protein E2 uncovers a new class of bovine viral diarrhea inhibitors that block virus entry

Abstract

Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV.

Cell-to-Cell Transmission Is the Main Mechanism Supporting Bovine Viral Diarrhea Virus Spread in Cell Culture

“…Monoclonal antibodies (MAbs) BVD/CA 17 and 26 against CD46 (56) were a kind gift from Till Rümenapf, University of Veterinary Medicine Austria. The effective concentrations for the inhibition of BVDV infection with recombinant E2 and CD46 MAbs were estimated in a cytopathic effect reduction assay (57). The inhibitory effect on virus entry was tested on MDBK cell monolayers incubated with recombinant E2 (1 g/ml) and CD46 MAbs (3 g/ml) for 1 h before infection with BVDV.…”

“…Drugs used in entry assays were first tested for cytotoxicity in MDBK cells. Cells in 96-well plates were incubated for 3 h with increasing amounts of drugs and rinsed twice with PBS, and after 72 h at 37°C, viability was measured using crystal violet staining as previously described (57).…”

Discovery of Novel Bovine Viral Diarrhea Inhibitors Using Structure-Based Virtual Screening on the Envelope Protein E2

“…E2 mediates receptor recognition on the cell surface and is required for fusion of virus and cell membranes after the endocytic uptake of the virus during entry (Ronecker et al, 2008 ; Wang et al, 2009 ). In this work, we expand on a structure-based approach to seek hit small-molecules that dock into the druggable pocket at the interface between domains I and II of the envelope protein E2 of BVDV (Pascual et al, 2018 ). Around a million compounds from different chemical libraries were screened in a high-throughput docking (HTD) fashion.…”

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells

“…3 The molecular system was described in the dihedral space using the ECEPP/3 force field 67,68 within the ICM program 36,37 and prepared in a similar fashion as in earlier works. [69][70][71] Docking was performed within the orthosteric binding site after deleting all water molecules and co-factors, using a flexible-ligand-rigid-receptor approach as implemented in ICM. In the docking algorithm the torsional degrees of freedom (DOF) of the smallmolecules and their six rigid coordinates were considered flexible within the receptor energy field, and subjected to a Monte Carlo global energy minimization.…”