Abstract 1420 An optimized chemoproteomic approach identifies novel cellular targets of the vitamin E metabolites α-13'-COOH and garcinoic acid

The identification of novel cellular targets of natural products helps to improve the understanding of their biological and pharmacological actions. Most proteomic approaches in use, lead to a large number of false-negative and false-positive hits. We present here an optimised compound-centric chemoproteomic method that leads to the identification of target proteins of the vitamin E derivatives α-13'-carboxychromanol (α-13'-COOH) and garcinoic acid (GA), which are natural metabolites of α-tocopherol and δ-tocotrienol, respectively, in human macrophages. Our approach is particularly suitable for hydrophobic compounds, since the methacrylic resin used is resistant to organic solvents and extreme pH, unlike the more commonly used sepharose resin. With the optimized approach α-13'-COOH and GA were separately coupled to the insoluble methacrylate resin, Toyopearl AF amino 650M, using their carboxyl groups. The coupled resins were incubated with appropriately processed cell lysates. After extensive washing of the resin, the proteins bound to the resin-coupled compounds were eluted and identified using mass spectrometry. 16 proteins were identified and classified according to their functions: lipid metabolism, antioxidant response, glucose metabolism as well as stress and immune response. We validated one of the targets, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), using western blotting. Since HMGCS1 is involved in cholesterol synthesis, this target is in line with our previous findings that α-13'-COOH regulates cellular lipid metabolism. Validation of the remaining proposed targets is ongoing. In conclusion, our compound-centric chemoproteomic approach has enabled us to identify HMGCS1 as a potential molecular target of α-13'-COOH and GA in human macrophages. This project is funded by the Europäischer Fonds für regionale Entwicklung (EFRE; 2019 FGI 0025) and the DAAD.