Interference of small compounds and Mg with dsRNA-binding fluorophores compromises the identification of SARS-CoV-2 RdRp inhibitors.

The COVID-19 pandemic highlighted the need for the rapid development of antiviral therapies. Viral RNA-dependent RNA polymerases (RdRp) are promising targets, and numerous virtual screenings for potential inhibitors were conducted without validation of the identified hits. Here we have tested a set of presumed RdRp inhibitors in biochemical assays based on fluorometric detection of RdRp activity or on the electrophoretic separation or RdRp products. We find that fluorometric detection of RdRp activity is unreliable as a screening method because many small compounds interfere with fluorophore binding to dsRNA, and this effect is enhanced by the Mg metal ions used by nucleic acid polymerases. The fact that fluorimetric detection of RdRp activity leads to false-positive hits underscores the requirement for independent validation methods. We also show that suramin, one of the proposed RdRp inhibitors that could be validated biochemically, is a multi-polymerase inhibitor. While this does not hinder its potential as an antiviral agent, it cannot be considered an specific inhibitor of SARS-CoV-2 RdRp.