Chemical proteomic strategies for deciphering neocarzilin´s mode of action in cancer cells

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Natural products address a plethora of different proteins with essential functions. The deconvolution of their targets represents a unique opportunity for unravelling new anti-cancer strategies. We here apply chemical proteomics to identify the targets of the natural product neocarzilin within living cancer cells. Neocarzilin is a potent anti-cancer compound which was identified decades ago but still lacks functional characterization. In preliminary studies we synthetically equipped the natural product with an alkyne tag and treated proteomes to identify its cellular target(s). Interestingly, only one protein, the vesicle amine transport protein (VAT-1) with an essential role in cell migration, has been identified as prominent hit. As neocarzilin also exhibits strong anti-proliferative effects which are independent of VAT-1, other yet unidentified targets must exist. To decipher these targets we aim to improve our initial probe by incorporation of a signature stereocenter present in the natural product but absent in the first generation probe design. Target identification will follow our established chemical proteomic procedures and selected protein hits will be validated for compound binding by overexpression and crystallography. Another strong focus of this work is the already identified hit VAT-1. Our initial validation revealed a putative role in cancer cell migration, an important feature of malignant tumors. Further chemical proteomic studies are required to understand the cellular function of this poorly characterized protein and to exploit its potential for future cancer treatment. The studies proposed here will include co-immunoprecipitation in presence and absence of the natural product to decipher interaction partners, co-crystallization and structure elucidation with the compound to unravel the binding mode and rationalize improved binders. The overall aims of this proposal are to decipher the full spectrum of neocarzilin targets, understand its mode of action, investigate the role of VAT-1 and find improved derivatives with therapeutic potential.