Antiviral compounds are crucial to controlling the SARS-CoV-2 pandemic. Approved drugs have been tested for their efficacy against COVID-19, and new pharmaceuticals are being developed as a complementary tool to vaccines However, there are not any effective treatment against this disease yet. In this work, a cheap and fast purification method of natural tyrosinase from Agaricus bisporus fresh mushrooms was developed in order to evaluate the potential of this enzyme as a therapeutic protein by the inhibition of SARS-CoV-2 3CLpro protease activity in vitro. Tyrosinase showed a mild inhibition of 3CLpro of around 15%. Thus, different variants of this protein were synthesized through chemical modifications, covalently binding different tailormade glycans and peptides to the amino terminal groups of the protein. These new tyrosinase conjugates were purified and characterized by circular dichroism and fluorescence spectroscopy analyses, and their stability under different conditions. Then all these tyrosinase conjugates were tested in 3CLpro protease inhibition. From them, the conjugate between tyrosinase and dextranaspartic acid (6kDa) polymer showed the highest inhibition, with an IC50 of 2.5 µg/ml and IC90 of 5 µg/ml, results that highlight the potential use of modified tyrosinase as a therapeutic protein and opens the possibility of developing this and other enzymes as pharmaceutical drugs against diseases.

This work was supported by the Spanish National Research Council (CSIC) (project CSIC PTI-Global Health SGL2103036 (J.M.P). This work was supported by Fundación hna (grant to A.V.-C. and O.A.).

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