Development of a Proximity Labeling Method to Identify the Protein Targets of Bioactive Small Molecules

Identifying the direct protein targets of a bioactive small molecule gives insight into the compound’s mechanism of action, its efficacy, and possible toxicity. Target identification is becoming an increasingly important part of the drugdevelopment process. However, given the transient and heterogeneous nature of interactions between small molecules and proteins, this step is often difficult, greatly slowing the development of new therapeutics. For this reason, new methods to rapidly identify the direct protein targets of bioactive small molecules are of great importance. Enrichment strategies coupled with quantitative mass spectrometry have shown great promise in target identification. Here we will present our progress toward developing an engineered enzymatic tagging method that enables specific labeling and enrichment of protein targets from complex lysates. This method couples the binding of a small molecule to a proximity-based labeling event. Labeled target proteins are enriched and subsequently identified using quantitative LC-MS/MS. We will discuss several variations of this method, and highlight our progress towards applying proximity labeling to small-molecule target identification and validation. B.2 Modelling Atherosclerosis: Molecular Changes in the Ascending Aorta of Cholesterol-fed Rabbits Jingshu Xu, Mia Jüllig, Martin J. Middleditch, Garth J.S. Cooper School of Biological Sciences, University of Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand; Department of Pharmacology, Medical Sciences Division, University of Oxford, Oxford, UK; Centre for Advanced Discovery and Experimental Therapeutics, NIHR Manchester Biomedical Research Centre, the University of Manchester, Manchester, UK