Discovery of Novel Hepatitis C Virus NS5B Polymerase Inhibitors by Combining Random Forest, Multiple e-Pharmacophore Modeling and Docking

<div><p>The NS5B polymerase is one of the most attractive targets for developing new drugs to block Hepatitis C virus (HCV) infection. We describe the discovery of novel potent HCV NS5B polymerase inhibitors by employing a virtual screening (VS) approach, which is based on random forest (RB-VS), e-pharmacophore (PB-VS), and docking (DB-VS) methods. In the RB-VS stage, after feature selection, a model with 16 descriptors was used. In the PB-VS stage, six energy-based pharmacophore (e-pharmacophore) models from different crystal structures of the NS5B polymerase with ligands binding at the palm I, thumb I and thumb II regions were used. In the DB-VS stage, the Glide SP and XP docking protocols with default parameters were employed. In the virtual screening approach, the RB-VS, PB-VS and DB-VS methods were applied in increasing order of complexity to screen the InterBioScreen database. From the final hits, we selected 5 compounds for further anti-HCV activity and cellular cytotoxicity assay. All 5 compounds were found to inhibit NS5B polymerase with IC₅₀ values of 2.01–23.84 μM and displayed anti-HCV activities with EC₅₀ values ranging from 1.61 to 21.88 μM, and all compounds displayed no cellular cytotoxicity (CC₅₀ > 100 μM) except compound N2, which displayed weak cytotoxicity with a CC₅₀ value of 51.3 μM. The hit compound N2 had the best antiviral activity against HCV, with a selective index of 32.1. The 5 hit compounds with new scaffolds could potentially serve as NS5B polymerase inhibitors through further optimization and development.</p></div

E-pharmacophore features and the scores for each feature in e-pharmacophore hypotheses generated from the six crystal structures.

<p>E-pharmacophore features and the scores for each feature in e-pharmacophore hypotheses generated from the six crystal structures.</p

Antiviral activity and cytotoxicity of the 5 hits.

<p>Antiviral activity and cytotoxicity of the 5 hits.</p

Validation of the six e-pharmacophore hypotheses.

<p>Validation of the six e-pharmacophore hypotheses.</p

Redocked binding modes of the co-crystalized inhibitors in the active site of NS5B polymerase.

<p>(A-B) the palm I region, (C-D) the thumb I region, (E-F) the thumb II region.</p

Structures of the 5 hit compounds.

<p>Structures of the 5 hit compounds.</p

Deafness genes detection array.

<p>The <i>mtDNA 12S rRNA</i> 1555A>G homoplasmic mutation. The box in the scanned image of the microarray chip represents the <i>mtDNA 12S rRNA</i> 1555A>G square areas. The upper dark dots indicate the wild-type is absent and the green dots below indicate the 1555A>G homoplasmic mutation (A). Sequence chromatograms showing the homoplasmic 1555A>G mutation as indicated by the arrow (B).</p

The binding poses of compound N1 –N5 in the respective binding pocket of NS5B polymerase.

<p>(A) N1; (B) N2; (C) N3; (D) N4; (E) N5. Potential hydrogen bonding interaction are shown as dashed lines.</p

Mutations analysis and conservations of the identified variants in the <i>MYH14</i> and <i>WFS1</i> genes.

<p>Chromatogram of exon 3 of the <i>MYH14</i> gene showing heterozygous mutation c.541G>A in affected individuals (left panel; arrow) and heterozygous mutation c.449C>T in exon 4 of the <i>WFS1</i> gene (right panel; arrow) (A). Protein sequence alignment showing conservation of residues MYH14 A181 (left panel; arrow) and WFS1 A150 (right panel; arrow) across six species. Sequence alignment of the non-muscle class II myosin showing conservation of MYH14 A181 (left panel; red underlined) (B). Diagram of the human MYH14 consisting of a N-terminal myosin domain, a myosin head region, a motor domain, two IQ motifs, a coiled-coil region and a tail domain (C).</p

E-pharmacophore hypotheses with energetically favorable sites from the six crystal structures.

<p>Pink sphere represents hydrogen-bond acceptor (A); orange ring represents aromatic ring (R); blue sphere represents hydrogen-bond donor (D); red sphere represents negatively ionizable (N); green spheres represent hydrophobic (H).</p