Toward High-throughput, Quantitative Platforms to Identify the Targets of Small Molecules

Abstract

Target identification is a major challenge in probe and drug discovery. Current binding assays are unable to detect interactions between unoptimized probes and difficult targets, such as transcription factors. Here, we developed generalizable, high-throughput platforms that can rapidly identify the mechanism(s) of action of small molecules emerging from high-throughput screening (HTS) campaigns. Specifically, this project established a solid phase method to rapidly modify small molecules with moieties of interest using isocyanate-based chemistries. This chemical method can be used to quickly generate photoaffinity labeling analogs of small molecules that can be used in a covalent ELISA and mass spectrometry workflow to determine whether small molecules bind to a target of interest and identify off-target binders. Additionally, we created a synergistic critical path for assessing the mechanism of action and on-target activity of small molecules through the generation of an on-target transcriptional profile and application of the L1000 gene-expression platform. Together, these workflows and chemical tools will enable high-throughput studies of small molecule-protein interactions with a wide range of affinities and abundances and facilitate prioritization of small molecules that bind and modulate the function of difficult targets.