Chalcones from Angelica keiskei (ashitaba) inhibit key Zika virus replication proteins

Abstract

Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC₅₀s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC₅₀ of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC₅₀ values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.

Keywords: Angelica keiskei; Ashitaba; Chalcones; Polymerase; Protease; Zika virus.

Figure 1.

Structures of 4-hydroxyderricin (4-HD), xanthoangelol (XA) and xanthoangelol-E (XA-E) isolated from Angelica keiskei Koidzumi.

Figure 2.. 3D intermolecular interactions of ZIKV NS2B-NS3 protease catalytic site in docking poses:

A) 4HD (colored in orange), B) XA (colored in pink) and C) XA-E (colored in mauve). Hydrophobic interactions are presented as white transparent surface and hydrogen bonds as green dotted lines. Hydrogen, nitrogen and oxygen atoms are colored in white, blue and red, respectively.

Figure 3.. 3D intermolecular interactions of ZIKV NS2B-NS3 protease allosteric pocket in docking poses:

A) 4HD (colored in orange), B) XA (colored in pink) and C) XA-E (colored in mauve). Hydrophobic interactions are presented as white transparent surface and hydrogen bonds as green dotted lines. Hydrogen, nitrogen and oxygen atoms are colored in white, blue and red, respectively.

Figure 4.. ZIKV enzymatic protease assays.

A) Activity assays at 200 μM for 4-HD, XA, and XA-E. Concentration-response curves adjusted with Hill to determine IC₅₀ ± Δ IC₅₀ values for B) 4-HD, C) XA and D) XA-E. The positive control aprotinin had an IC₅₀ of 0.13 ± 0.02 μM.

Figure 5.. 3D intermolecular interactions of ZIKV NS5 RdRp (N-pocket) docking poses:

A) 4HD (colored in orange); B) XA (colored in pink) and C) XA-E (colored in mauve). Hydrophobic interactions are presented as white transparent surface; hydrogen bonds, as green dotted lines and π-cation interaction, as red dotted lines. Hydrogen, nitrogen and oxygen atoms are colored in white, blue and red, respectively.

Figure 6.. ZIKV polymerase assays.

A) Activity assays at 20 μM for 4-HD, XA, and XA-E. B) Concentration-response curves adjusted with Hill to determine IC₅₀ ± Δ IC₅₀ values for XA.

Figure 7.. Ensemble docking of XA against ZIKV NS5 RdRp.

A) Vina score versus RFscore of the XA - ZIKV NS5 RdRp clustered docking poses. The best docking pose is represented by cluster 1 (colored in blue) and pose 1 (circle) that had Vina score of −9.0 Kcal·mol⁻¹ and RFscore of −7.3 Kcal·mol⁻¹; B) XA-RdRp best docking pose. Hydrophobic interactions are presented as white transparent surface and hydrogen bonds, as green dotted lines. Hydrogen, nitrogen and oxygen atoms are colored in white, blue and red, respectively; C) Superposition of XA docking pose (colored in cyano) and DENV RdRp allosteric co-crystallized inhibitor (colored yellow) and D) 2D chemical structural comparison of XA and DENV RdRp allosteric inhibitor: common ligand moieties are highlighted: hydroxyls (colored in orange), delocalized π electrons (colored in green) and aromatic rings with hydroxyls/methoxyls (colored in blue).

Figure 8.. The inhibitory and cytotoxic effects of the chalcones on ZIKV.

Inhibition of ZIKV growth by 4HD (A), XA (B) and XA-E (C) and cytotoxicity of these compounds (D). Two-step assays were used to quantify ZIKV. First, Vero cells were infected with ZIKV 1h, compounds added, and supernatants harvested after 48 hrs. Virus titers in supernatants were then determined by plaque assay. Cytotoxicity was determined by measuring the release of lactase dehydrogenase from Vero cells. Statistical analysis was performed using a one-way ANOVA with Tukey’s post-test, *p < 0.05.