Targeting of the human multidrug resistance protein ABCB1 at the blood-brain barrier as an approach for improved brain pharmacotherapy and cancer chemotherapy

P-glycoprotein, or ABCB1 is a multi-drug transporter found in many epithelial cells and is very important for pharmacokinetics at the blood-brain barrier where it has a protective function. The protein is also found overexpressed in cancer cells, where its function is undesirable as it leads to resistance of the cancer to chemotherapy. Murine ABCB1 expressed in the yeast P. pastoris has been purified in detergent and has been crystallised and its structure determined by X-ray crystallography. The human ortholog has also been expressed and purified in this system, but not crystallised. In this project the aim is to extend these studies by reconstituting the murine and human ABCB1 orthologs into lipid bilayers which will allow:

(i) Functional studies of the protein under conditions mimicking those encountered at the blood-brain barrier and (ii) Structural studies of the protein in a system that is closer to the native environment (ie in a lipid bilayer at a high protein:lipid ratio). The functional studies will examine the ATPase activity of the protein in the presence of various potential substrates and inhibitors. If a suitable assay can be set up, high throughput screening of potential ABCB1 inhibitor compounds will be carried out. The functional studies will also look at uptake of ABCB1 substrates into the lipid vesicles and how the lipid composition of the membrane affects the transport of the substrates. The structural studies will initially be set up to search for conditions producing 2D crystals of the protein in the membrane. If achieved, the crystals will be examined by high resolution cryo-electron microscopy using the Polara TEM facility in Manchester. We will look at the structure of the protein in various stages of the transport cycle and in a more native-like lipid environment. So far X-ray crystallography has only been possible for one of the stages in the cycle (the nucleotide-free state). Importantly, we wish to search for the subtle structural changes induced by substrate (drug) binding to the protein and how this triggers ATP hydrolysis at the cytoplasmic domains which power the movement of drugs back across the blood brain barrier or the plasma membrane of cancer cells. These structural data will allow the development of improved models for the functioning of the protein and will hopefully identify crucial residues that can be targeted to regulate the activity of the protein in a controlled and reversible fashion. Such a goal would allow the gatekeeper role of the protein at the blood-brain barrier to up- or down-regulated in order to optimise chemotherapy. Moreover the treatment of cancers where multidrug resistance is frequently encountered would be significantly enhanced if the protein can be down regulated during chemotherapy.