A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems

In this work, we present and discuss a comprehensive set of both newly and previously synthesized
compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently
inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was
employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA
dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature
pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic
feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information
was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide
compound docking within the protein binding site. The affinity of all compounds towards the enzyme
was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data.
The determination of the interaction spectra of the protein residues involved in inhibitor binding
highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic
cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and
A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical
for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity
of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The
agreement between predicted and experimental data confirmed the proposed common molecular
rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp,
ultimately yielding compound 6b (EC50 ¼ 0.3 mM; IC50 ¼ 0.48 mM) as a new, potent inhibitor of this
Pestivirus.