Rational design of 2 H-chromene-based antiphytovirals that inhibit virion assembly by outcompeting virus capsid-RNA interactions

Abstract

Although the determination of the structural basis of potato virus Y (PVY) coat protein (CP) provides the possibility for CP-based antiviral drug design, the role of many specific residues on CP in regulating virion pathogenicity is largely unknown, and fewer small-molecular drugs have been discovered to act on these potential sites. In this study, a series of derivatives of 2,2-dimethyl-2H-chromene are rationally designed by employing a molecular hybridization strategy. We screen a case of phytovirucide C50 that could form a stable H-bond with Ser¹²⁵ of PVY CP to exert antiviral properties. Ser¹²⁵ is further identified to be crucial for CP-viral RNA (vRNA) interaction, enabling PVY virion assembly. This interaction can be significantly inhibited through competitive binding with compound C50. The study enhances our understanding of anti-PVY drug mechanisms and provides a basis for developing new CP-targeting virus particle assembly inhibitors.

Keywords: Biochemistry; Chemistry; Virology.

Graphical abstract

Figure 1

Rational design of 2H-chromene-based antiphytovirals (A) Active ingredients in common commercial antiviral agents for plants. (B) Some reported phytoviral CP inhibitors. (D) 2.2-dimethyl-2H-chromene-based lead optimization incorporating sulfonamides and Schif bases. (C) Molecular basis of our previous anti-PVY agent A.

Figure 2

Production of the intended compounds (A) Manufacturing pathways for the target molecules. Reagent and conditions: (i) Et₃N, DCM, 0°C 30 min, then at rt 3 h, 50%–80% yield; (ii) Et₃N, CaCl₂, EtOH, reflux, 3 h, 50%–60% yield; (iii) cat. AcOH, EtOH, reflux, 5 h, 40%–75% yield. (B) X-ray crystal structure of compound C6 (CCDC: 2297246).

Figure 3

Effect of C50 against PVY infection (A) Effect of C50 against PVY infection in Chenopodium amaranticolor. Ningnanmycin (NNM) was used as a control. The concentration was marked below the inoculated leaves. (B) Green fluorescence expression map of Nicotiana benthamiana leaf blades treated with PVY-GFP infectious C50 and NNM under UV illumination. (C and D) Quantitative assessment of PVY CPand GFP accumulation in systemically infected leaves of N. benthamiana plants at 7 days post-agroinfiltration (dpai), utilizing RT-qPCR method. After injection of Agrobacterium with pCamPVY-GFP, a solution of 1% Tween 80 containing C50 (500 μg/mL), NNM (500 μg/mL), and DMSO (as a control) was sprayed on the leaves of N. benthamiana. RT-qPCR normalization was achieved using EF1α as an internal control. Data are presented as mean ± SD from three biological replicates per treatment, with statistical significance indicated by different letters (p < 0.05, one-way ANOVA).

Figure 4

3D-QSAR analysis (A) CoMFA and (B) CoMSIA models for comparing experimental versus predicted pEC₅₀. (C and D) CoMFA 3D isopotential maps illustrating (C) steric and (D) electrostatic contributions. (E–H) CoMSIA 3D isopotential maps depicting (E) steric, (F) electrostatic, (G) hydrophobic, and (H) hydrogen bond acceptor fields. (I) Relationship between structure and anti-PVY activity.

Figure 5

Ser¹²⁵ is a key target for binding compound C50 to PVY CP (A) Computational binding analysis of C50 to PVY CP using molecular docking techniques. (B–F) Comprehensive results of molecular dynamics simulations of C50 and PVY CP. (B) RMSD analysis, (C) RMSF analysis, (D and E) interaction analysis, (F) ligand torsion diagram. (G) Analysis comparing the binding of PVY CP^wt and PVY CP^S125A mutant proteins to compounds.

Figure 6

Effect of mutation on Ser¹²⁵ in CP on PVY infection (A) Illustrative representation of the pCamPVY-GFP genome structure, highlighting the Ser¹²⁵ residue (marked by red arrows) within the core domain of PVY CP. The blue-bordered box encapsulates the site-specific mutants, wild-type plasmids, viruses, and their respective sequences. (B) Comparative visualization of disease symptoms (top) and green fluorescent expression (bottom) under UV illumination in N. benthamiana leaves inoculated with wild-type and mutated PVY strains. (C and D) Quantitative assessment of PVY CP accumulation in systemically infected leaves of N. benthamiana plants at 7 dpai, utilizing RT-qPCR (C) and western blot (D) methods. RT-qPCR normalization was achieved using EF1α as an internal control, while Ponceau S-stained RuBisCO served as a loading control. Data are presented as mean ± SD from three biological replicates per treatment, with statistical significance indicated by different letters (p < 0.05, one-way ANOVA). (E) Examination of cell-to-cell movement dynamics in N. benthamiana plants infected with wild-type and mutated PVY strains at 3 dpai. (F) Enumeration of PVY particles within 70 μm² microscopic fields, presented as mean ± SD derived from five fields per treatment. Statistical significance is denoted by different letters (p < 0.05, one-way ANOVA). (G) Particles of PVY-GFP and PVY^S125A-GFP under transmission electron microscope.

Figure 7

Mechanism of anti-PVY action of compound C50