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Establishing Target Engagement and Selectivity for CTP Inhibitors in Cells: Synthesis and Application of CHOLesterol Probes for a Comprehensive MAPping of the Cholesterol Interactome

Objective

Cholesterol is a structural component of membranes and plays a key role in human health by regulating the fluidity of membranes and affecting signaling pathways. Intracellular cholesterol transport proteins (CTP) are responsible for maintaining cholesterol homeostasis by regulating local cholesterol concentrations, arising from de novo biosynthesis or dietary sources. Dysregulated cholesterol transport is associated with cardiovascular disease but also Alzheimer's and cancer. Despite the obvious importance of CTPs, a chemical probe to investigate binding, inhibition and selectivity profiling of CTP inhibitors does not exist. CTPs are all structurally related in their cholesterol binding site. Therefore, the development of a selective modulator to study their individual function and possible therapeutic relevance is challenging. Only a few CTP inhibitors have been reported so far and it has not been possible to conclusively evaluate their cellular selectivity. In CholMap, I aim to introduce a universal sterol-based affinity probe with the ability to bind all CTPs and evaluate the selectivity of CTP inhibitors in cells. This sterol-based probe would be bifunctional containing a diazirine, which is transformed into a carbene via UV irradiation for covalent binding to CTPs, and an alkyne for binding to a functional tag via click chemistry. I will synthesize differently constituted probes and test them in vitro for their efficiency. An optimized sterol-based affinity probe would be used in a chemoproteomic workflow to determine the target engagement and the selectivity of different CTP inhibitors. The introduction of a universal sterol-based affinity probe in combination with a chemoproteomic workflow would constitute the first example of CTP selectivity profiling in cells. The results will also enable me to uncover new sterol-binding proteins including new potential drug targets, as well as providing a large dataset accessible to the whole research field.

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Coordinator

DANMARKS TEKNISKE UNIVERSITET
Net EU contribution
€ 247 553,28
Address
ANKER ENGELUNDS VEJ 101
2800 Kongens Lyngby
Denmark

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Region
Danmark Hovedstaden Københavns omegn
Activity type
Higher or Secondary Education Establishments
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Total cost
No data