Evaluation of Compound Optical Interference in High-Content Screening

Compound optical interference remains an inherent problem in chemical screening and has been well documented for biochemical assays and less so for automated microscopy-based assays. It has also been the assumption that the latter should not suffer from such interference because of the washing steps involved in the process, thus eliminating the residual nonspecific compound effects. Instead, these compounds may have no relevance to the actual target, and as such, compound optical interference contributes to a number of false-positives, resulting in a high attrition rate during subsequent follow-up studies. In this report, we analyze the outcome of a high-content screen using enhanced green fluorescent protein as a reporter in a gain-of-function cell-based assay in search of modulators of the micro RNA (miRNA) biogenesis pathway. Using a previously validated image-based biosensor, we screened a diverse library collection of ~315,000 compounds covering natural and synthetic derivatives in which 1130 positives were identified to enhance green fluorescence expression. Lateral confirmation and dose-response studies revealed that all of these compounds were the result of optical interference and not specific inhibition of miRNA biogenesis. Here, we highlight the chemical classes that are susceptible to compound optical interference and discuss their implications in automated microscopy-based assays.