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Description
The two-tag assay is a cell-based assay that was initially developed in the Wolkowicz lab to monitor host mediated proteolytic events within the natural milieu of the cell. The assay was first designed to observe proteolytic events critical to viral maturation within the classical secretory pathway and perform chemical library screenings for drug discovery purposes. The cleavage boundary of HIV-1 envelope was used as proof-of-principle which demonstrated the robustness of the assay. Once this tool proved to be useful, the premature membrane of Dengue virus was adapted to the assay to show it could be exploited to target another family of viruses, Flaviviridae. A close relative to Dengue virus is Zika virus, which also requires host proteases for premature membrane cleavage to release infectious viral particles. Zika virus is the most recent Flavivirus to be adapted to the two-tag assay where it is compared to Dengue virus to see if what was previously learned about Dengue in the context of the assay can also be used in understanding mechanisms for drugs discovery against Zika virus. Though the two-tag assay can help elucidate events that occur within the classical secretory pathway (CSP), it is not limited to this compartment of the cell. A protease that also requires cleavage by resident proteases of the CSP to become catalytically active, Matrix Mettaloproteinase-14 (MMP-14), executes it proteolytic activity at the cell surface. Using the assay as an advantageous tool for phenotypic reporters of cleavage and non-cleavage events, proteolytic activity at the cell surface can also be monitored using MMP-14 as proof-of- principle. MMP-14 proves to be a great target for drug discovery in this assay considering it is highly expressed in cancer cells and a main contributor to metastasis. Here, it is demonstrated that the cell surface activity of MMP-14 can be monitored and the effects of known MMP pan inhibitors such a doxycycline and small molecule inhibitor, Batimastat, can be described with the assay. The results of MMP-14 provide confidence in the use of the two-tag assay to monitor cell surface cleavage events and its continued discovery for new drugs against disease.
