Target profiling of electrophilic compounds by using chemical proteomics strategies

Abstract

Target identification elucidates the mode of action of bioactive molecules and therefore is critical in modern drug discovery. Various methods for target identification have been developed in recent years including chemical proteomic, genomic methods, and bioinformatic prediction. However, most methods investigate compounds individually. It remains challenging to efficiently examine multiple compounds in parallel, particularly for structurally distinct compounds. Celastrol (Cel) has intriguing bioactivities and has been recognised as one of five promising natural products for drug discovery. In this study we have achieved two objectives (i) to employ a classic compound-centric chemical proteomics strategy to identify the molecular target of Cel; (ii) to extend target identification to other compounds that are similar to Cel, by developing a novel chemical proteomics-genomics strategy for high-throughput target profiling of electrophilic compounds in parallel. First, we identified catechol-O-methyltransferase (COMT) as a binding target of Cel by using compound-centric chemical proteomics. After comprehensive characterization of Cel-COMT interactions, Cel was demonstrated to inhibit the enzymatic activity of COMT and thus increased dopamine level in neuroendocrine chromaffin cells significantly, which explained the known dopaminergic and neuroprotective effects of Cel. Our study not only identified a novel binding target of Cel, but also provided a new scaffold and a cysteine hot spot for developing a new generation of COMT inhibitors to treat neurological disorders. Next, we established a chemical proteomics-genomics strategy for multiplexed target profiling of bioactive small molecules. We selected Cel as a representative electrophilic bioactive compound, and other three structurally distinct electrophilic compounds that are of high, medium, and low similarity to Cel, according to their perturbations of global transcription. An reactive cysteine profiling method was developed by using two complementary chemical probes to scrutinize reactive cysteines as potential binding sites of tested electrophiles. Then, target profiling of the four electrophilic compounds was performed by using the chemical proteomics approach, leading to identification of known and novel binding targets. Our results demonstrated that the cysteine reactivity of electrophilic compounds is associated with their influence on gene transcription. Therefore, the integrated approach combining genomics and chemical proteomics enables multiplexing of target identification of structurally distinct compounds.