Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity

Zhou, Saisai; Mu, Suyu; Yu, Shuqi; Tian, Yang; Lu, Sijia; Li, Zhen; Wu, Hao; Zhao, Jiaying; Chen, Huanchun; Sun, Shiqi; Song, Yunfeng

doi:10.3390/v17060785

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Open AccessArticle

Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity

by

Saisai Zhou

^1,2

,

Suyu Mu

³,

Shuqi Yu

^1,2,

Yang Tian

^1,2,

Sijia Lu

^1,2,

Zhen Li

^1,2,

Hao Wu

^1,2,

Jiaying Zhao

^1,2,

Huanchun Chen

^1,2,

Shiqi Sun

³

and

Yunfeng Song

^1,2,*

¹

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

²

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

³

Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

^*

Author to whom correspondence should be addressed.

Viruses 2025, 17(6), 785; https://doi.org/10.3390/v17060785

Submission received: 14 April 2025 / Revised: 23 May 2025 / Accepted: 27 May 2025 / Published: 29 May 2025

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

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Abstract

Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at >620 μM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and π-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS⁻ and 2-PyOH as competitive, and DCMQ/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a ΔTm ≥ 1.5 °C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 μM, exhibiting two orders of magnitude higher potency than 2-MPO (400 μM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses.

Keywords: FMDV; 2C protein; helicase; antiviral inhibitor

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MDPI and ACS Style

Zhou, S.; Mu, S.; Yu, S.; Tian, Y.; Lu, S.; Li, Z.; Wu, H.; Zhao, J.; Chen, H.; Sun, S.; et al. Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity. Viruses 2025, 17, 785. https://doi.org/10.3390/v17060785

AMA Style

Zhou S, Mu S, Yu S, Tian Y, Lu S, Li Z, Wu H, Zhao J, Chen H, Sun S, et al. Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity. Viruses. 2025; 17(6):785. https://doi.org/10.3390/v17060785

Chicago/Turabian Style

Zhou, Saisai, Suyu Mu, Shuqi Yu, Yang Tian, Sijia Lu, Zhen Li, Hao Wu, Jiaying Zhao, Huanchun Chen, Shiqi Sun, and et al. 2025. "Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity" Viruses 17, no. 6: 785. https://doi.org/10.3390/v17060785

APA Style

Zhou, S., Mu, S., Yu, S., Tian, Y., Lu, S., Li, Z., Wu, H., Zhao, J., Chen, H., Sun, S., & Song, Y. (2025). Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity. Viruses, 17(6), 785. https://doi.org/10.3390/v17060785

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Discovery of Small Molecules Against Foot-And-Mouth Disease Virus Replication by Targeting 2C Helicase Activity

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