Zika virus (ZIKV) is a mosquito-borne flavivirus that poses a significant global health threat, similar to related viruses such as dengue, West Nile, and yellow fever viruses. Transmission primarily occurs through the bite of infected Aedes mosquitoes, and cases of sexual and vertical transmission have also been reported (Petersen et al., 2016). ZIKV infection has been associated with severe clinical outcomes, including congenital microcephaly and other neurological abnormalities in infants, as well as Guillain-Barré syndrome in adults (Parra et al., 2016). The ZIKV outbreak in 2015–2016 highlighted its epidemic potential (Yadav et al., 2016). Despite its clinical and societal impact, no approved antiviral therapies or vaccines are currently available, underscoring the urgent need to develop effective antivirals targeting ZIKV.

The ZIKV genome encodes a single large polyprotein that must be cleaved into functional viral proteins, comprising both structural and non-structural components (Shi and Gao, 2017). These proteins are essential for viral particle formation, replication, assembly, and evasion of host immune responses. The viral protease is a two-component complex consisting of a cofactor region of approximately 40 residues from NS2B and the N-terminal domain of NS3. This NS2B-NS3 protease plays a critical role in processing the viral polyprotein by cleaving it at multiple sites, making it a key target for antiviral drug development. Inhibition of its activity can effectively block viral maturation and replication (Brecher et al., 2013, Kang et al., 2017).

Protease inhibitor development can be pursued through various strategies. Cell-based assays such as phenotypic screening allow direct assessment of inhibitor effects in a physiologically relevant environment (Abrams et al., 2020, Anindita et al., 2024, Brecher et al., 2017). To elucidate mechanisms of action, it is essential to confirm direct interactions between developed inhibitors and the viral protease, which requires purified protein. Structure-based drug design also represents a powerful approach, relying on high-resolution structures of the protease and its inhibitor-bound complexes (Braun et al., 2020, Li and Kang, 2020, Shin et al., 2021). Since NS2B is a membrane-associated protein whose proper folding depends on a membrane-mimicking environment, a variety of engineered constructs have been designed to facilitate structural, biochemical characterization and exploring protease-inhibitor interaction (Fig. 1).

Several classes of ZIKV protease inhibitors have been reported (Feng, 2024), including covalent, irreversible, and allosteric inhibitors (Li et al., 2017b, Li et al., 2018b, Ontoria et al., 2026). However, none have advanced to clinical trials due to limitations in potency and efficacy. Among these, allosteric inhibitors have great potential for antiviral development, as they can achieve favourable physicochemical properties while circumventing challenges posed by the highly hydrophilic active site of the protease (Yao et al., 2019). The NS2B cofactor region plays an indispensable role in stabilizing NS3 folding and maintaining protease activity, and the enzyme undergoes conformational changes depending on the presence or absence of substrates or inhibitors (Mahawaththa et al., 2017, Phoo et al., 2016, Su et al., 2009, Zhang et al., 2016). To facilitate the discovery and evaluation of allosteric inhibitors, a novel construct g18ZiPro was designed. This construct contains only the N-terminal segment of the NS2B cofactor linked to the N-terminal domain of NS3 via a glycine-rich linker (Fig. 1). Expressed and purified from E. coli, g18ZiPro lacks protease activity. Its crystal structure was successfully determined, and the 1H-15N-HSQC spectrum was obtained and assigned. Fragment-based screening using this construct identified two hit compounds, providing a valuable foundation for rational allosteric inhibitor design. Thus, g18ZiPro represents a useful tool for characterizing and advancing allosteric inhibitors targeting ZIKV protease.