[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-594] Accelerating Antiviral Drug Screening for Dengue Viruses and Enteroviruses Using Cell–Based Protease Assay Platforms

○tzu ching lo¹, Ping-Chung Kuo², Chih-Hsuan Tsai¹ (1. Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University (Taiwan), 2. School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC (Taiwan))

Keywords:

Dengue virus,Enterovirus D68,viral protease,fluorescence-based protease assay,antiviral screening platform,baculovirus

Dengue viruses (DENV) and enteroviruses (EV) are major human pathogens responsible for significant global morbidity, and effective antiviral therapies remain unavailable. Viral proteases, including NS3 of DENV and 3C of EV, are essential for viral polyprotein processing and represent highly attractive drug targets due to their conserved structures and critical roles in viral replication. To establish a rapid and scalable antiviral screening strategy, we developed in vitro protease-based antiviral screening platforms integrating both Escherichia coli expression and baculovirus surface display systems. Recombinant protease construct of NS3 of DENV type 2 was generated by fusing NS3 with its cofactor NS2B (i.e., DENV NS2B–NS3), whereas recombinant 3C of EV-D68 (i.e., EV-D68 3C) was expressed solely. Both constructs were expressed via E. coli and baculovirus expression vectors, with the latter expressing the proteases in Sf21 insect cells through virus infection. Protease activity was assessed directly from crude lysates of E. coli and Sf21 insect cells using fluorescence-based substrates in a 96-well plate format. Key assay parameters, including enzyme volume, substrate concentration, reaction time, and buffer composition, were systematically examined to optimize assay performance. For inhibitor screening, compounds were pre-incubated with proteases prior to substrate addition, and dose–response relationships were analyzed under optimized conditions. Known inhibitors of both proteases specifically inhibited the activity of corresponding proteases, demonstrating that the platforms have antiviral screening capabilities. In summary, we developed robust and flexible protease-based antiviral screening platforms for DENV and EV. These platforms eliminate the need for extensive protein purification and enable rapid evaluation of enzyme activity and inhibition, providing a scalable framework for antiviral drug discovery.