Colloidal Aggregation Confounds Cell-Based Covid-19 Antiviral Screens

Area of Science:

• Biochemistry
• Drug Discovery
• Virology

Background:

• Colloidal aggregation is a major cause of false positives in early drug discovery.
• Cell-based infectivity assays are crucial for identifying antiviral drugs, particularly in drug repurposing for diseases like COVID-19.

Purpose of the Study:

• To investigate the role of colloidal aggregation in SARS-CoV-2 entry inhibitors used in cell-based infectivity assays.
• To evaluate the impact of aggregation on the measured antiviral efficacy of drug candidates.

Main Methods:

• Dynamic light scattering was used to detect colloidal particle formation in 41 drug candidates.
• Drug suspensions were treated with BSA or centrifugation to mitigate aggregation.
• Spike pseudovirus infectivity assays were performed to measure antiviral potency before and after aggregation mitigation.

Main Results:

• 17 out of 41 drug candidates formed colloidal particles and showed detergent-dependent enzyme inhibition.
• Antiviral potencies decreased by at least 10-fold after BSA or centrifugation treatments, indicating an aggregation-based mechanism.
• Fluorescence microscopy revealed that aggregates sequestered labeled spike proteins, consistent with artifactual interactions.

Conclusions:

• Colloidal aggregation is a common artifact in cell-based antiviral drug repurposing assays for COVID-19.
• Presaturation with BSA and centrifugation serve as effective counter-screens to identify and eliminate aggregation-induced artifacts.
• Addressing colloidal aggregation is essential for accurate drug efficacy assessment in antiviral research.