Colloidal Aggregation Confounds Cell-Based Covid-19 Antiviral Screens

Colloidal aggregation is one of the largest contributors to false positives in early drug discovery. Here, we consider aggregation's role in cell-based infectivity assays in Covid-19 drug repurposing. We investigated the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate the impact of aggregation on antiviral efficacy in cells, we presaturated the colloidal drug suspensions with BSA or spun them down by centrifugation and measured the effects on spike pseudovirus infectivity. Antiviral potencies diminished by at least 10-fold following both BSA and centrifugation treatments, supporting a colloid-based mechanism. Aggregates induced puncta of the labeled spike protein in fluorescence microscopy, consistent with sequestration of the protein on the colloidal particles. These observations suggest that colloidal aggregation is common among cell-based antiviral drug repurposing and offers rapid counter-screens to detect and eliminate these artifacts.