Interaction of 8-anilinonaphthalene-1-sulfonate with SARS-CoV-2 main protease and its application as a fluorescent probe for inhibitor identification

The 3C-like main protease of SARS-CoV-2 (3CL^Pro) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CL^Pro is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CL^Pro inhibitors rely on peptide-based colorimetric or fluorogenic substrates. However, the COVID-19 pandemic has limit or delay access to these substrates, especially for researchers in developing countries attempting to screen natural product libraries. We explored the use of the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) as an alternative assay for inhibitor identification. Fluorescence enhancement upon binding of ANS to 3CL^Pro was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CL^Pro inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CL^Pro was explored with molecular modeling. Our results suggested that ANS could be employed in a competitive binding assay to facilitate the identification of novel SARS-CoV-2 antiviral compounds.