Researchers at the Wroclaw University of Science and Technology and their colleagues have recently published exciting work improving our understanding of the two proteases encoded by the SARS-CoV-2 genome, the main protease (Mpro) and the papain-like protease (PLpro). These proteases are required for viral gene expression and for viral replication, so they are attractive targets for antiviral drug development. In addition, PLpro can inhibit the host innate immune response via deubiquitination activity, so inhibition of this protease may provide another mechanism of antiviral activity.
In each publication, the scientists used hybrid combinatorial substrate libraries to understand the rules governing substrate specificity of these proteases. Then, these rules guided the design of activity-based probes and irreversible inhibitors for each protease.
To characterize binding of their compounds to the proteases and the deubiquitinase activity of PLpro, the authors took full advantage of the fluorescence detection capabilities of the Sapphire Biomolecular Imager.
In Science Advances, the group examined SARS-CoV-2 PLpro, designing and characterizing two inhibitors. Using a biotinylated ubiquitinated probe, they found no evidence for cross-reactivity between their inhibitors and human deubiquitinases, an important requirement for potential antiviral drugs targeted to this protease. The biotinylated probe, bound to proteins in cell lysates, was detected on blots using an Alexa Fluor 647¬–labeled streptavidin as a probe and imaging the blots with the Sapphire Imager. To study the deubiquitinase activity of SARS-CoV-2 PLpro, binding and cleavage of ubiquitin-containing substrates was detected in SYPRO Ruby-stained gels, also imaged using the Sapphire Imager.
In Nature Chemical Biology, the group focused on SARS-CoV-2 Mpro, designing an inhibitor that demonstrated significant antiviral activity against SARS-CoV-2 in a cell culture assay, and an activity-based fluorescent probe that could be used to visualize active Mpro in cells taken from patients with COVID-19 infection. To study the binding of their compounds to SARS-CoV-2 Mpro, compounds labeled with Cy5 or BODIPY were detected directly on blots. Biotinylated versions were detected using Alexa Fluor 647¬–labeled streptavidin as a probe.
This research spotlights numerous avenues for development of potential anti-coronavirus drugs and tools for disease characterization and diagnostics.
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