Developing biophysical and structural methods for characterisation of small molecule modulators of K2P potassium channels

Thesis

Biophysical techniques are widely used to determine the structure, function and ligand binding properties of a protein. However, their application to membrane proteins has been limited due to the difficulty of obtaining sufficient purified sample.

In this work, I use such methods to examine the thermostability and ligand binding properties of TREKs, two members of the family of tandem pore domain K⁺ channels important for the regulation of cellular excitability. Structures of TREK1 and TREK2 are available and thus when combined with such approaches may help guide the design of better ligands.

I first successfully optimised the DSF method using the CPM dye for the detection of TREK ligands and found this to be a viable approach. In addition, direct binding detection methods such as SPR, ITC and Biolayer Interferometry were also investigated.

A test case of 351 compounds derived from an FDA-approved pharmaceutical agent library were then screened against a range of K2P channels using the CPM assay. Several compounds were identified that appeared to thermostabilize the TREKs. These initial hits were further assessed using label-free DSF and functional studies on channel activity. Two compounds, cilnidipine and rimonabant were identified as a novel inhibitor and activator of TREK2 and TREK1 respectively. The findings are significant for the development of potent and selective binders for the TREKs and other membrane proteins, as they identify a viable workflow for identification of binders using biophysical methods.

Authors: Ang, J

• DOI: 10.5287/ora-avgqpvvyr
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford