Development of NS2B-NS3 protease inhibitor that impairs Zika virus replication

Wen-Wei Lin; Yi-Jung Huang; Yen-Tseng Wang; Yun-Syuan Lin; Nonsikelelo Mazibuko; Chien-Shu Chen; Tian-Lu Cheng; Chih-Shiang Chang; Yu-Ling Leu; Chiao-Yun Chen; Chih-Hung Chuang

doi:10.1016/j.virusres.2023.199092

Development of NS2B-NS3 protease inhibitor that impairs Zika virus replication

Virus Res. 2023 May:329:199092. doi: 10.1016/j.virusres.2023.199092. Epub 2023 Apr 5.

Authors

Affiliations

¹ Department of Laboratory Medicine, School of Post Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
² Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biochemistry, School of Post Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
³ Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biochemistry, School of Post Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁴ Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan.
⁵ Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁶ Department of Laboratory Medicine, School of Post Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
⁷ School of Pharmacy, China Medical University, Taichung 40402, Taiwan; Drug Development Center, China Medical University, Taichung 40402, Taiwan.
⁸ Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan.
⁹ Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan; School of Post-Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Electronic address: ccy77015@gmail.com.
¹⁰ Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Electronic address: a4132600@gmail.com.

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes severe neurological disorders, such as microcephaly in fetuses. Most recently, an outbreak of ZIKV started in Brazil in 2015. To date, no therapeutic agents have been approved to treat ZIKV infection in the clinic. Here, we screened a small molecule inhibitor that can inhibit the function of ZIKV non-structural protein 2B (NS2B)-NS3 protease (ZIKV NS2B-NS3 protease), thereby interfering with viral replication and spread. First, we identified the half maximal inhibitory concentration (IC₅₀) of compound 3 (14.01 μM), 8 (6.85 μM), and 9 (14.2 μM) and confirmed that they are all non-competitive inhibitors. In addition, we have used the blind molecular docking method to simulate the inhibition area of three non-competitive inhibitors (compound 3, 8, and 9) with the ZIKV NS2B-NS3 protease. The results indicated that the four allosteric binding residues (Gln139, Trp148, Leu150, and Val220) could form hydrogen bonds or non-bonding interactions most frequently with the three compounds. The interaction might induce the reaction center conformation change of NS2B-NS3 protease to reduce catalyzed efficiency. The concentration of compounds required to reduce cell viability by 50% (CC₅₀), and the concentration of compounds required to inhibit virus-induced cytopathic effect by 50% (EC₅₀) of three potential compounds are >200 μM, 2.15 μM (compound 3), > 200 μM, 0.52 μM (compound 8) and 61.48 μM, 3.52 μM (compound 9), and Temoporfin are 61.05 μM, 2 μM, respectively. To select candidate compounds for further animal experiments, we analyzed the selectivity index (SI) of compound 3 (93.02), 8 (384.61), 9 (17.46), and Temoporfin (30.53, FDA-approved drug against cancer). Compound 8 has the highest SI value. Therefore, compound 8 was selected for verification in animal models. In vivo, compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in a ZIKV-infected suckling mouse model. We conclude that compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

Keywords: Non-competitive; Small molecule inhibitor; ZIKV NS2B-NS3 protease; Zika virus (ZIKV).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antiviral Agents / therapeutic use
Enzyme Inhibitors / metabolism
Mice
Molecular Docking Simulation
Peptide Hydrolases / metabolism
Protease Inhibitors / pharmacology
Serine Endopeptidases / metabolism
Viral Nonstructural Proteins / chemistry
Virus Replication
Zika Virus Infection*
Zika Virus* / physiology

Substances

Protease Inhibitors
Viral Nonstructural Proteins
Antiviral Agents
Enzyme Inhibitors
Serine Endopeptidases
Peptide Hydrolases