Synthesis, identification, and biological characterisation of sterol transport protein inhibitors

Sterol transport proteins (STPs) are an important class of proteins performing an integral role in a range of cellular processes including cholesterol homeostasis, vesicular and non-vesicular trafficking, and regulating organelle contact sites. Despite their importance, the identification of selective modulators of STPs has proved challenging, owing to the high degree of structural similarity in their sterol binding domains. To overcome this challenge, an approach was developed to synthesise a screening collection of sterol-inspired compounds enriched in hits against STPs. By fusing a representative steroidal scaffold to a series of heterocyclic scaffolds, a collection of 65 sterol-inspired compounds were prepared for use in screening.The relevance of the sterol-inspired compound collection in affording selective hits against STPs was demonstrated by screening against the Aster family of STPs (Asters-A, -B, and –C) using a combination of differential scanning fluorimetry (DSF), fluorescence polarisation (FP), and fluorescence resonance energy transfer (FRET)-based assays. Hit compounds were identified and validated against Aster-A and Aster-C which after further optimisation resulted in the discovery of a new chemotype of Aster-A inhibitor, Asterpyrin-1, and the most potent and selective inhibitor of Aster-C known to date, Astercin-2. Finally, the inhibitors were used to investigate Aster-related biology in a cellular setting, revealing differential effects on the levels and localisation of cholesterol and cholesterol esters, and demonstrating the utility of these sterol-inspired compounds as tool compounds