Over the last decade, small-molecule microarrays (SMMs) have proven to be a general, robust and scalable screening platform for discovering protein-small molecule interactions that lead to modulators of protein function. SMMs are manufactured by robotically arraying stock solutions of compounds onto functionalized substrates such as glass microscope slides. The arrays are then incubated with proteins of interest and putative protein-small molecule interactions are typically detected using fluorescently labeled antibodies and a standard fluorescence slide scanner. The high-throughput and miniaturized nature of the microarray-based binding assay allows for screening of large panels of proteins against tens of thousands of compounds in a relatively short time frame. Our group has developed multiple approaches to manufacturing SMMs including fluorous-based homogeneous display and isocyanate-based covalent attachment for heterogeneous display. The isocyanate approach allows us to make SMMs that contain compounds not intentionally synthesized or modified for immobilization onto a solid substrate allowing us to make arrays containing bioactive small molecules, including FDA-approved drugs, synthetic drug-like compounds, natural products, and products of diversity-oriented synthesis on a single slide. Based on an in silico evaluation of more than 400,000 compounds coming from a variety of these sources, we estimate that 77% of the compounds are compatible with immobilization using the isocyanate strategy and we routinely prepare SMMs containing compounds from Broad Institute screening collection. Our group and other groups have used SMMs to identify small molecules that target proteases, kinases, histone deacetylases, and transcription factors. Notably, our group recently used this approach to identify a direct binder to the ‘undruggable’ extracellular growth factor Sonic hedgehog (Shh) that inhibits the Shh signaling pathway in human cell lines and primary keratinocytes. Projects are underway to expand the utility of the SMM platform to other applications including target identification and diagnostics.
courtesy of HHMI