Channelling a path for substrates through a multidrug transporter

Understanding nature's "bouncers": the mechanism of multidrug pumps

All cells are surrounded by a protective barrier known as the cell membrane. Cells control what they transport across this membrane, in order to be able to take up (import) or remove (export) specific chemicals. Examples of important transport include taking up nutrients from the diet into the cells lining the gut, export of toxins and waste chemicals into urine and bile, and secretion of vitamins and nutrients into milk in mammary tissue. These transport processes are carried out by "pump proteins" in the cell membrane.

Pump proteins usually transport a specific molecule in a set direction (i.e. either into the cell or out of the cell). Some pump proteins are unusual in that they export from cells not just one type of chemical, but hundreds of different and unrelated chemicals. These proteins are called "multidrug (MDR) pumps" and they are the cell's equivalent of a nightclub bouncer, with actions that can have a big impact. For example, antibiotic resistance in bacteria - a huge healthcare challenge - can occur because multidrug pumps remove antibiotics from their target cells. In humans, MDR pumps protect cancer cells from chemotherapy because the pumps are expressed more in tumour cells. They kick the anticancer drugs designed to kill tumours back out of cells and cause chemotherapy to fail. These MDR pumps can also affect how we all respond to medication for a wide range of conditions including statins for heart disease and anticonvulsants for epilepsy. MDR pumps in the gut, liver and kidney control how these medicines are absorbed and removed from the body. MDR pumps are not always "bad news" - the chemicals industry wants to use bacteria to make chemicals (such as fuels) from simple sugars, rather than using dwindling fossil fuels. MDR pumps are important in this "green chemistry", because they can be used to export the useful chemical products from bacteria for harvesting.

A deep understanding of how MDR pumps work, and how we can hijack or block this process, is therefore really important. In other pump proteins that transport only one chemical, the route this chemical takes through the protein as it is 'pumped' can be well defined. However, one of the main challenges in understanding MDR pumps is that these can deal with so many different drugs and chemicals so mapping their route through the pump protein is very difficult. We are going to tackle this big question for one MDR pump, ABCG2, in our proposal. What makes our research unique is that we have developed a new way to "see" how ABCG2 recognises chemical substrates at a microscopic level. We will use molecular "cookie cutters" to make tiny rings of cell membrane, each containing just one pump protein. We then watch a fluorescent drug (that glows when we shine laser light on it) bind our membrane "cookies" containing ABCG2. This tells us how well our substrates recognise the pump as it works, and how other drugs might stop this process. We can test our substrate route map by changing parts of the pump protein involved to see what happens to its function, and using computer modelling to simulate the interactions. We will also look for novel drug types that bind ABCG2 and regulate function, expecting that these will be useful experimental tools and starting points for future medicines.

The techniques will be adapted to look at any pump protein interacting with its substrate, which will excite people interested in the fundamental roles of pump proteins in all aspects of physiology. We are already discussing with pharmaceutical and chemical production companies how a better understanding of pump:substrate interactions benefits the drug design process and chemicals manufacturing. This research project will therefore advance understanding of the basic biochemistry of an important human MDR pump, and cast a broader light on the large family of membrane pump proteins.

ABCG2 is one of three human multidrug transporters whose ability to recognise multiple chemically distinct compounds is important for chemoresistance and drug pharmacokinetics. Our molecular understanding of how ABCG2 pumps substrates, and its pharmacological inhibition, remains poor despite recent cryo-electron microscopy structural data for ABCG2. We have made significant methodological advances to study ABCG2 transport at a molecular level. We developed a unique quantitative measurement of drug-transporter interaction, using fluorescence correlation spectroscopy (FCS) applied to ABCG2 purified in native lipid membrane nanodiscs (obtained by styrene maleic acid copolymer extraction; SMALP). In this proposal we will combine this method with complementary approaches that will deliver insight into mechanisms of transport, and structure activity relationships (SAR) for novel substrates and inhibitors. We will also demonstrate how our approach can be a more general paradigm to assess drug target affinity at other biomedically important membrane transporters and receptors.

We will define the molecular transport pathway for substrates through ABCG2 using structural modelling, to guide residues for mutation, and flow cytometry, to determine effects on multidrug transport. Dissection of ABCG2 pharmacology through the catalytic cycle will be possible through purification of wild type and mutant ABCG2 isoforms into SMALP nanodiscs and quantitative solution based FCS. Integration of this data with structural modelling will improve our existing mechanistic understanding of ABCG2. Solution based FCS in microplate format will enable screening of chemical compound libraries to provide SAR data for ABCG2, and will permit the identification of lead compounds underpinning longer-term opportunities for development and optimisation of selective ABCG2 inhibitors.

Our research will uniquely allow for pharmacological analysis of a purified membrane protein in a native lipid environment using highly sensitive advanced imaging techniques. This aligns with the priority area "technology development for the biosciences" and will have impacts in pharmaceutical research, green chemistry, skills provision and outreach. In this study we will reveal molecular details of the transport mechanism of the human multidrug pump ABCG2. This has immediate impact on the pharmaceutical industry. All new drugs are required by the Federal Drug Administration and European Medicines Agency to be examined as possible substrates for 3 human multidrug pumps, namely ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance protein 1) and ABCG2. Our work here will establish novel experimental paradigms for enabling characterisation of ABCG2:substrate interactions in the lipid membrane, and could be extended to include ABCC1 and ABCB1. This will complement the existing pipelines for screening new drugs (including those which determine transport rates in living cells). An advantage of our approach is that our FCS technology lends itself to the use of stored protein in membranes, as SMALP solubilised proteins have a good thermostability and resistance to freeze-thaw cycles.

In this research we will extend the use of a new reagent in membrane protein isolation and analysis, namely styrene maleic acid (SMA). Techniques that enable the study of membrane proteins are in ever greater demand, particularly as structural biology now has the power of higher resolution electron microscopy at its command. This has intensified the search for agents which enable purification of membrane proteins in their native environment. Detergents remain beset with the difficulty that they remove the annular lipids which in many cases may have a functional effect on the protein. SMA and other co-polymers have the potential to extract membrane proteins and retain the annular lipid interactions, and this research proposal will provide further evidence for the use of such agents in a range of biophysical settings.

We will develop new experimental tools to generate SAR data for membrane transporters and identify novel lead compounds as transporter substrates and inhibitors. This will have a significant long-term benefit to the green chemicals industry. The challenge for this industry is the synthesis of primary chemicals from renewable feedstocks in engineered strains of yeast or bacteria. A frequent hindrance to the economic viability of these processes is product toxicity, i.e. where the chemical being made (e.g. butanol, toluene, styrene) accumulates to a concentration that causes cell death in the producing strain. In such instances, biotechnologists are looking to identify transporters to remove the product from the cell so enabling product synthesis to continue and also facilitating product recovery from the media. The impact of this current research proposal for bio-based chemicals manufacturing is the potential to quantify substrate:transporter interactions through our FCS technique so enabling screening of candidate transporters for a range of chemicals of interest, or the screening of wild type and systematically mutated versions of a candidate transporter to identify gain of function mutations for transport of the chemical of interest.

The proposal would have a significant economic impact by training a PDRA in an outstanding environment in a cross-disciplinary demand area, identified as vulnerable in RCUK skills analysis. The PDRA would be able to make a significant long-term contribution to the economically valuable biotechnology industry in the UK.

Transporters in medicine and bioengineering are an excellent topic for public engagement work and all the applicants and the PDRA would capitalise on this through events such as Nottingham's community engagement event ("Wonder") and other outreach activities.