NS2B-NS3 protease as a target for anti-flaviviral therapeutics: Expression and NMR assignment of proteins from pathogenic strains for drug screening

Flaviviruses are associated with widespread human infections, and their incidence in the northern hemisphere has increased in recent years. Despite their significant clinical impact, there are currently no small molecule therapeutics available for the most representative viruses such as Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) [1]. The viral genome encodes a polyprotein precursor that is subsequently processed by both viral and host proteases into functional units. A key player in this cleavage process is the NS2B-NS3 cofactor-protease complex, making it a promising target for drug discovery [2]. Additionally, the high sequence and structural conservation of this complex across flaviviruses supports the development of broad-spectrum inhibitors targeting these viruses [3]. This study aims to create a platform for screening flaviviral protease inhibitors using NMR. The approach includes: (i) the recombinant expression and purification of NS2B-NS3 constructs from Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) in E. coli; (ii) the assignment of backbone resonance for the proteases; and (iii) the assessment of ligand binding affinity through chemical shift perturbation analysis following protease-inhibitor titrations. NS2B-NS3 constructs from Zika virus (ZIKV) and West Nile virus (WNV) were produced, including both linked (Gly₄-Ser-Gly₄) and unlinked configurations to evaluate their suitability for solution NMR studies. For the Dengue virus (DENV), only the unlinked construct was produced. Backbone resonance assignment was carried out for the ZIKV-linked construct using multidimensional heteronuclear NMR experiments. This process resulted in approximately 62% assignment of the protease backbone, providing a foundation for residue-specific analysis of ligand interactions. The backbone assignment for the DENV and WNV proteases is currently in progress. These results demonstrate the feasibility of producing stable, NMR-compatible flaviviral protease constructs for ligand screening. The assigned constructs will be used in ligand screening and structure-based drug design to develop new pan-flaviviral protease inhibitors.