Flavonoids from Pterogyne nitens as Zika virus NS2B-NS3 protease inhibitors

Abstract

Although the widespread epidemic of Zika virus (ZIKV) and its neurological complications are well-known there are still no approved drugs available to treat this arboviral disease or vaccine to prevent the infection. Flavonoids from Pterogyne nitens have already demonstrated anti-flavivirus activity, although their target is unknown. In this study, we virtually screened an in-house database of 150 natural and semi-synthetic compounds against ZIKV NS2B-NS3 protease (NS2B-NS3p) using docking-based virtual screening, as part of the OpenZika project. As a result, we prioritized three flavonoids from P. nitens, quercetin, rutin and pedalitin, for experimental evaluation. We also used machine learning models, built with Assay Central® software, for predicting the activity and toxicity of these flavonoids. Biophysical and enzymatic assays generally agreed with the in silico predictions, confirming that the flavonoids inhibited ZIKV protease. The most promising hit, pedalitin, inhibited ZIKV NS2B-NS3p with an IC₅₀ of 5 μM. In cell-based assays, pedalitin displayed significant activity at 250 and 500 µM, with slight toxicity in Vero cells. The results presented here demonstrate the potential of pedalitin as a candidate for hit-to-lead (H2L) optimization studies towards the discovery of antiviral drug candidates to treat ZIKV infections.

Keywords: Antiviral; Drug discovery; Emerging arboviruses; Enzyme inhibitors; Flavonoid; NS3 protein; Protease; Pterogyne nitens; Virtual screening; Zika virus.

Figure 1.. 3D docking poses and 2D interaction diagrams of flavonoids from Pterogyne nitens and the ZIKV NS2B-NS3p cleavage site.

The catalytic site (His51, Asp75, and Ser135) is represented in sticks and the surrounding pockets: S1’ (Ser135 residue of NS3), S1 (Asp129 residue of NS3), S2 (Ser81 residue of NS2B) and S3 (Tyr161 residue of NS3), represented in sticks and colored surface. Docking poses of (A-B) Rutin (orange sticks), (C-D) Quercetin (blue sticks), and (E-F) Pedalitin (magenta sticks). In the 2D interaction diagrams, the interactions among flavonoids and protein residues are represented: as magenta arrows (hydrogen bonds) and as green lines (π-π stacking interactions). The VMD software [45] was used for the visual inspection of docking poses and to render the 3D molecular images.

Figure 2.. NS2B-NS3p characterization in the presence of rutin, quercetin and pedalitin.

(A) Thermogram with Boltzmann adjustment and the respective Midpoint Temperature. (B) Percentage of NS2B-NS3p activity inhibition by rutin, quercetin and pedalitin. (C) Docking pose of pedalitin at ZIKV NS2B-NS3pro allosteric pocket (docking score −6.9 kcal·mol), highlighting the Hbonds with Asn152 and Trp83 of NS3.

Figure 3.. Antiviral activity of flavonoids against ZIKV in cell culture.

Vero cells non-infected (NI) or infected with ZIKV strain MR766 at a multiplicity of infection (MOI) of 0.01 were treated or not with the flavonoids: quercetin, rutin or pedalitin at different concentrations. (A) Vero cells were treated with flavonoids or with cell culture media + DMSO (vehicle) and evaluated for cell viability using an MTT assay after 72 h of incubation. (B) ZIKV-infected Vero cells were treated with flavonoids or with the vehicle and assessed for cell viability. Cell culture supernatant from ZIKV-infected culture was recovered and assessed for viral load using plaque assay, at 48 h (C) and 72 h (D) post-infection (p.i.). Cell viability data were expressed in percentage, normalized to NI controls treated with vehicle. Viral load data were expressed as plaque-forming units per mL of cell culture supernatant.

Figure 4.. Antiviral activity of rutin, pedalitin, and quercetin by focus reduction assay.

Vero cells were infected with 100 FFU of ZIKV (strain MR766) and were treated with the flavonoids rutin, pedalitin, and quercetin at different concentrations. (A) Representative image of one experiment showing ZIKV infected-cell foci after the addition of peroxidase substrate (TrueBlue detection reagent – KPL). (B) Inhibition of formation of focus-forming units of ZIKV after treatment with different concentrations of rutin (green), pedalitin (carmine) and quercetin (blue), respectively. Dashed line represents the mean of untreated control. Bars represent mean ± SD. Statistical differences by Kruskal-Wallis followed by Dunn’s test are indicated by asterisks (*p<0.05).