Experience from an HTS campaign: Investigation of susceptibility and rescue of SARS-CoV-2 nsp3 protease assay from metal contamination

Different biochemical assays yield different rates of false positives than others either due to the nature of the enzyme, the technology associated with the bioassay, or properties of the compounds being screened. Ensuring that the right counter-screens are in place to identify false positives without wasting time and resources on them is of great importance. Herein we describe the results of a high throughput screen (HTS) against non-structural protein 3 (nsp3) protease PL^Pro, which resulted exclusively in false-positive hits. By triaging hit compounds through purification of metal chelating resin, we identified contamination by either copper or palladium as the most likely source of false positives from the library screening campaign. We then performed a systematic assessment of the vulnerability of nsp3 protease screening to metal contamination and evaluated common additives to combat the inhibitory effects of different metal salts. We further conducted a thorough survey of the literature reports of nsp3 HTS campaigns with a focus on the presence of additives and what metal susceptibility was likely, given the results of our work. We conclude that the majority of reported nsp3 screens are susceptible to copper contamination with a smaller proportion also potentially susceptible to palladium contamination.