Periodic Reporting for period 1 - ChemBioChol (Chemical biology of intracellular cholesterol transport)
Reporting period: 2022-09-01 to 2025-02-28
The overall objective of the project is to develop chemical compounds to study cholesterol transport in cells and its relevance to human health and disease. Cholesterol is a lipid that cannot dissolve in aqueous media found in cells and must be transported between different membranes. This process is mediated by cholesterol transport proteins (CTPs), whose precise functions are not fully understood. Furthermore, very few molecules that can block CTP function have been reported to date and no general strategy to identify them exists. In this project we aim to develop such a strategy, by combining chemical synthesis of sterol-inspired small molecules with biochemical and cell biological method development to determine how well molecules bind to CTPs in cells, and the effects that they have on cholesterol levels and localization. The molecules we design to block CTPs will then also be used to determine the importance of CTPs in diseases including a range of cancers. The specific objectives of the project are:
1. Comprehensive characterisation of CTPs in terms of their affinity and specificity for naturally occurring lipids.
2. Identification and optimisation of CTP binding molecules from sterol-inspired compound collections
3. Development of a general strategy for determining the cellular target binding and selectivity of small molecule CTP modulators.
4. Determining the cellular consequence of selective CTP blockage on cholesterol transport, lipid droplet formation, mTOR signaling and autophagy.
1. Comprehensive characterisation of CTPs in terms of their affinity and specificity for naturally occurring lipids.
2. Identification and optimisation of CTP binding molecules from sterol-inspired compound collections
3. Development of a general strategy for determining the cellular target binding and selectivity of small molecule CTP modulators.
4. Determining the cellular consequence of selective CTP blockage on cholesterol transport, lipid droplet formation, mTOR signaling and autophagy.
We have developed standardised biochemical and biophysical assay systems to determine binding and inhibition of ten different cholesterol transporters, as well as the chemistry to access a large collection of steroid-inspired compounds. The first results from this work have been recently uploaded as preprints (Depta et al. bioRXiv, 2024; and Bro et al. chemRXiv, 2024). By combining the two, we have identified molecules that can selectively block the activity of six out of ten cholesterol transport proteins we have targeted so far. These compounds bind their intended CTP tightly, with minimal off-target activity at other transporters. Importantly, they are able to interact with the CTPs in cells, enabling us to begin studying the roles of CTPs in regulating how lipid pools are maintained in cells.
The most significant achievements so far include the identification of the first small molecule blockers of four sterol transport proteins. We hope that these compounds will be used by us, and others, to study the functions of sterol transporters in detail. More generally, we have understood the requirements for designing blockers with potent activity and excellent selectivity, which can be applied more generally to other lipid binding proteins. We have also recently published a pre-print (Depta et al. bioRXiv 2024) showing how endogenous sterols, which are naturally produced in our bodies, interact with different sterol transporters. This has led to new understanding of their roles, and many testable biological hypotheses.