Lipid Nanoparticles for Membrane Protein Target Drug Discovery

Abstract

Membrane proteins are essential in a plethora of physiological processes and perturbation of their function often leads to disease. As such, membrane proteins are the target for over 50% of pharmaceuticals. Their location within the lipid bilayer has meant that the study of their structure and function still lags behind that of easier to isolate, soluble proteins; this has hindered progress in drug design and development. The application of styrene maleic acid (SMA) polymers to isolate proteins directly from cell membranes, forming SMA lipid particles (SMALPs) where the proteins retain their lipid bilayer environment makes them potentially more suitable for structural and functional studies and could be used for drug development purposes. The aim of this project was to develop polymer-lipid particle approaches for biophysical assays such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) suitable for medium to high throughput screening. To achieve this aim, two model proteins were used, Atm1 and LeuT. Atm1 is an ATP Binding Cassette (ABC) transporter and LeuT is a prokaryotic homologue of neurotransmitter transporters of the neurotransmitter: sodium symporter (NSS) family. Recombinant Atm1 and LeuT were expressed in E. coli cells, extracted, and purified using several different polymers and stability and function investigated. Immobilisation studies for SPR were undertaken, which found that amine-coupling an anti-his antibody to the sensor chip and then binding the his-tagged SMALP-LeuT to that antibody was the most effective method for immobilisation. However, the level of immobilisation was too low to detect small molecule binding. Initial studies with MST showed SMALP-LeuT could be successfully labelled with a fluorophore at the his tag. Detection of leucine binding was highly variable and poorly reproducible. However, binding of the drug desipramine to SMALP-LeuT could be successfully measured and a dose-response curve obtained. Chemical modification of the SMA polymer was tested, to potentially enable immobilisation or labelling of the polymer rather than the protein. Sulfhydryl groups were added to SMA forming SMA-SH, and the modified polymer was able to solubilise and purify proteins. Future work would look at labelling with biotin for immobilisation. Molecular biology approaches to add a SNAP-tag to the Atm1 protein was also started. The SNAP-tag enables site specific labelling with a range of different probes. Finally, two series of novel SMA polymer variants were tested to see if they offered any improvement over SMA2000. The first series were partially esterified SMA polymers, and the second series were benzylamine modified SMA polymers. Whilst all of the novel polymers were able to solubilise and purify membrane proteins, they did not offer an improvement over SMA2000, and were even more sensitive to divalent cations.