Award details

Ion transport by the human Breast Cancer Resistance Protein (ABCG2)

Reference	BB/I002383/1
Principal Investigator / Supervisor	Professor Hendrik Willem van Veen
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Pharmacology
Funding type	Research
Value (£)	331,418
Status	Completed
Type	Research Grant
Start date	03/10/2011
End date	02/10/2014
Duration	36 months

Abstract

The human Breast Cancer Resistance Protein (ABCG2) is an ATP-binding cassette transporter that affects the pharmacokinetic properties of drugs in our body due to its wide distribution at important pharmacological barriers. In addition, ABCG2 expression confers resistance on cancer cells by reducing the intracellular concentration of cytotoxic drugs to subtoxic levels. Although the clinical need for inhibitors that could modulate ABCG2 activity is emphasized in the literature, the rational development of such compounds will require further insights into the possible routes for modulation, and will require a greater understanding of the molecular mechanisms that underlie ABCG2 activity. ABCG2 has been characterized in the literature as an ATP-dependent transporter which couples ATP-binding and hydrolysis to the transmembrane efflux of drugs. Surprisingly, analogous to previous observations on the bacterial ABC transporter LmrA, we found that the membrane domain of human ABCG2 in the absence of the nucleotide-binding domain (termed ABCG2-MD) is active as a secondary-active multidrug efflux transporter that, in ABCG2-MD, couples drug efflux to the uptake of protons by an electrogenic drug-proton antiport mechanism. The hybrid use of primary and secondary forms of metabolic energy by membrane transport systems is an intriguing and important concept that has been described for a number of other ATP-dependent transporters. In this project we will explore this novel fundamental property of ABCG2-MD in more detail and extend our observations to full-length ABCG2. We aim to characterize proton transport and transport of other ions by ABCG2 in electrophysiological experiments and conventional transport assays using radioligands and fluorescence spectroscopy, and identify amino acid residues in ABCG2 important for ion transport. We will assess how ion transport is integrated in the transport cycle of ABCG2, and how ion transport is linked to multidrug transport.

Summary

Drug transporters, such as the Breast Cancer Resistance Protein (ABCG2), are recognized as key players in the distribution of drugs in human. The localization of this efflux pump in organs responsible for drug biotransformation and excretion gives ABCG2 an important gatekeeper function in controlling drug access to metabolizing enzymes and excretory pathways. ABCG2 is also found in cancer cells, where it mediates the extrusion of drug to the cell's exterior. In doing so, it prevents entry of anticancer drugs into cells and, hence, impairs the chemotherapeutic treatment of this life-threatening disease. In this proposal, we will study fundamental aspects of the transport mechanism of human ABCG2. In particular, we will study the previously unknown ability of ABCG2 to transport ions such as protons. We are interested to learn which ions are transported (in addition to protons, for example sodium, potassium, chloride ions), how ABCG2 transports these ions, and why? What is the relationship between ion transport and drug transport? ABCG2 is thought to be active as a dimer, which we will test by mass spectrometry, and is likely to have drug binding sites that are alternately exposed to the inside surface of the membrane (where drugs bind) and outside surface of the membrane (where drugs are released). Based on the available crystal structures of multidrug binding proteins (transporters and transcriptional regulators), we hypothesize that (i) protons and other ions might displace drugs from binding sites in ABCG2 during drug release and/or (ii) their binding might support structural changes in the two ABCG2 units and their interface, that are associated with the reorientation of the drug binding sites. Fundamental knowledge about ABCG2 activity will allow the rational development of inhibitors (also termed modulators) of this efflux pump that could be used to target drugs to specific parts of the human body, and to improve chemotherapy of cancers.

Impact Summary

ABCG2-mediated drug efflux significantly reduces the achievable levels of drugs in body compartments such as the brain, and affects our ability to treat cancers by chemotherapy. Our aim is to study the fundamental property of ABCG2 to transport small ions and to analyze the potential role of these ions in drug binding and release, and in specific steps of the transport cycle of ABCG2. Our research has clear social and economic impact as increased knowledge of the biochemical mechanism of ABCG2 will generate new avenues for modulation of its activity in a clinical setting, which will offer considerable potential for modulator development programs in pharmaceutical industries and commercial institutes. These modulators could rejuvenate anticancer drugs when tumor cells are resistant, and improve pharmacokinetic properties of drugs in our body. Dissemination to society in general will be effected via school outreach activities (the PI is active in this area through his work as a teaching fellow at Clare College, Cambridge), and via community events such as Cafe Scientifique talks, the Cambridge Science Festival, and via a publicly accessible website with a lay summary of our findings. The PI and postdoctoral worker will engage in academic dissemination through peer reviewed publications and talks at conferences and workshops, whilst wider public dissemination is more likely to be undertaken by the PI. Our project web site will be mounted by IT staff at Cambridge University and thereafter populated by material provided by the staff on the project. We will engage the print and broadcast media via the Office of Communications of Cambridge University and BBSRC Media Office with articles written by them and ourselves following a briefing session, and external release following approval by the PI. For radio and TV interviews, both Cambridge University and the BBSRC offer media appearance training. The PI has attended these to ensure maximum impact for any such events.The PI and Prof. Robinson (University of Oxford) already have a record of collaboration on the application of Mass Spectrometry on ABC transporters. Our management structure will involve frequent (fourmonthly) progress meetings to consider progress, make use of any emergent information from our own efforts or from other laboratories, and identify any action needed (e.g. contact PR company or other commercial entity), and the person to carry out the action to maximize project impact. The PI and Dr Fertig (Nanion, Munich) will collaborate on the application of electrophysiological techniques and the Port-o-patch patch clamp system in studies on ion transport by ABCG2. As the Port-o-patch equipment is mostly applied on channel proteins, the optimization of methods that involve the existing equipment for measurements on membrane transporters might broaden the market for this equipment, which could be beneficial for Nanion and perhaps for related companies in this field. The researchers at Nanion have a strong academic interest in this project, and will give staff on this project full support and access to their experience built up over the past years. Meetings will take place twice per year, whereas communication by email and telephone will occur on a weekly/monthly basis. After publication of primary research papers, methods on the use of the Port-o-patch in ABC transporter research will be made available to a broader public via 'Application notes', which can be downloaded from Nanion's website.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Pharmaceuticals
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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