Methods and strategies for tackling assay interference associated with small molecules

Biochemical and cell-based assays are essential to discovering and optimising efficacious and safe drugs, agrochemicals and cosmetics. However, false assay readouts stemming from colloidal aggregation, chemical reactivity, chelation, light signal attenuation and emission, membrane disruption and other interference mechanisms remain significant challenges in screening synthetic compounds and natural products. To address assay interference, a range of powerful experimental approaches and strategies are available. Moreover, in silico methods for identifying suspicious assay readouts and predicting likely interference compounds, including rule-based, similarity-based, statistical and machine-learning approaches, are now gaining traction and recognition. This Review begins with a balanced and timely overview of the scope and limitations of experimental and theoretical approaches for tackling assay interference. It then focuses on computational methods, discusses strategies to integrate them with experimental approaches, and provides recommendations for best practices. The Review closes with a summary of the critical facts and learnings and an outlook on possible future developments.