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

TY - JOUR

T1 - Experience from an HTS campaign

T2 - Investigation of susceptibility and rescue of SARS-CoV-2 nsp3 protease assay from metal contamination

AU - Georgiou, Irene

AU - O'Neill, Sandra

AU - Robinson, Colin

AU - Cunningham, Fraser

AU - O'Byrne, Sean N.

AU - Gray, David

AU - Gilbert, Ian H.

AU - Scott, Duncan E.

N1 - Copyright: © 2026 The Authors. Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening.

PY - 2026/5

Y1 - 2026/5

N2 - 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 PLPro, 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.

AB - 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 PLPro, 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.

KW - False-positives

KW - HTS

KW - Metal contamination

KW - nsp3

KW - PLpro protease

KW - SARS-CoV-2

UR - https://www.scopus.com/pages/publications/105032871537

U2 - 10.1016/j.slast.2026.100409

DO - 10.1016/j.slast.2026.100409

M3 - Article

C2 - 41833692

AN - SCOPUS:105032871537

SN - 2472-6303

VL - 38

JO - SLAS Technology

JF - SLAS Technology

M1 - 100409

ER -