FRET Probes for Detection of Both Active and Inactive Zika Virus Protease

• Published in: Biochemistry (Easton) 2024-11, Vol.63 (24), p.3300-3309
• Main Authors: Cruz, Kristalle G., Alexander, Kevin, Makhaik, Sparsh, Hardy, Jeanne A.
• Format: Article
• Language: English
• Subjects: Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Humans; Protease Inhibitors - chemistry; Protease Inhibitors - pharmacology; Viral Proteases - chemistry; Viral Proteases - metabolism; Zika Virus - enzymology
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.4c00415

Proteases are a privileged class of enzymes due to their catalysis of an irreversible post translational modification, namely cleavage of substrate proteins. Protease activity is essential for human pathways including inflammation, blood clotting, and apoptosis. Proteases are also essential for the propagation of many viruses due to their role in cleavage of the viral polyprotein. For these reasons, proteases are an attractive and highly exploited class of drug targets. To fully harness the power of proteases as drug targets, it is essential that their presence and function are detectable throughout the course of the protease lifetime, from inactive zymogen to the fully cleaved (mature) protease. A number of methods for detection of proteases have been developed, however, many rely on catalytic activity, so are not useful throughout the proteolytic life cycle. Here, we build on our observation that the MH1 family of benzofuran-aminothiazolopyridine inhibitors of Zika virus protease (ZVP) undergo a unique FRET interaction with tryptophan residues in the protease. The full FRET signal is only observed in higher potency binding interactions. Moreover, this approach can distinguish two inactive variants of ZVP based on their folded or unfolded state. These studies also probe the physicochemical basis of the FRET signal. Exploiting these types of FRET interactions may offer an orthogonal approach for detection of this protease, which takes advantage of the relationship between the novel ligand and the core of the protein and is therefore useful throughout the protease maturation cycle. Depending on chemical properties, this approach may be applicable in other proteases and other protein classes.