PhD defence - Hogan Bryce-Rogers

Abstract

In the continued balance of cholesterol levels in human bodies many fundamental mechanisms that are integral to our bodily and cellular functions, have been overlooked. Particularly, the intracellular trafficking of cholesterol within a cell, which is diversely concentrated in different cellular compartments, is governed by a several families of proteins known as sterol transport proteins (STPs). The study of which has culminated in the necessity to selectively and potently inhibit their function using small molecule tool compounds. In this dissertation the discovery of potent and selective tool compounds, the starquins, targeted at steroidogenic-acute regulatory related lipid transfer domain (STARD) containing protein 4 (STARD4) is detailed.

The study utilised a computer aided design to optimise the structure and key interactions between the tool compounds and STARD4. This was followed up by the characterisation of potency and selectivity of the probes against 10 more STPs where the probes showed promise at being tailorable for other 3 other proteins. The core quinoline fused scaffold also observed a tuneable fluorescence (starflin) that resulted in Forster resonance energy transfer (FRET) between a tryptophan on STARD4 and the ligand to develop new assays to examine the most potent ligands’ affinities. The starquin leads were used to gain detailed structural insights into STARD4 ligand binding by the complementary use of the solution phase hydrogen-deuterium exchange mass spectrometry (HDX-MS) and by solving the first holo X-ray crystal structure of STARD4. These techniques detailed the key interactions necessary to potently inhibit STARD4 and further proved that the engagement of the sterol pocket lid is a formidable target for the ligand binding potential. Ultimately, the biological evaluation of the starquins confirmed the inhibition of STARD4 significantly increases free cholesterol levels and can enhance its localisation to the plasma membrane. Thus, the use of these tool compounds will help in the elucidation of STARD4’s roles in diseases such as liver cancer and atherosclerosis.

Principal Supervisor: 
Professor Luca Laraia, DTU Chemistry

Co-supervisor: 
Professor Mads Clausen, DTU Chemistry

Examiners:
Professor Sophie Beeren, DTU Chemistry
Professor Morten Grøtli, University of Gothenburg, Sweden
Professor Thomas Poulsen, University of Aarhus, Denmark

Chairperson:
Associate Professor Kira Astakhova, DTU Chemistry