Molecular mechanism of drug transport by the human breast cancer resistance protein (ABCG2)

Lead Research Organisation: University of Cambridge
Department Name: Pharmacology

Abstract

Multidrug transporters are very small molecular machines that are present at the surface of human cells and microorganisms. These ingenious machines bind structurally unrelated drugs and toxins at the inside of the cell, and then move these compounds to the outside of the cell. Hence, multidrug transporters act as drug pumps that are able to lower the drug concentration inside the cell to levels at which these drugs are inactive and useless. As drugs now have lost their therapeutic effect, the cell has become multidrug resistant. Multidrug transporters provide the cell with a defense mechanism against toxic compounds that are harmful. However, the activity of multidrug transporters also interferes with the drug-based control, termed chemotherapy, of human tumours and infectious pathogenic microorganisms, in which drugs are used that should kill the cell. This project focuses on the human breast cancer resistance protein (BCRP), which is one of the major drug pumps in human. Surprisingly, we have been able to functionally express BCRP in Lactococcus lactis, a harmless bacterium which is used in the production of yoghurts and cheeses, and which is very easy to handle. We will now study how BCRP recognizes and binds drugs, and how BCRP is able to move drugs out of the cell. This knowledge may allow us to rationally design new drugs that can poison or circumvent the activity of BCRP and related transporters to improve chemotherapy of human cancers.

Technical Summary

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels, and, hence, cells become multidrug resistant. Multidrug transporters interfere with the chemotherapeutic treatment of cancers and infections by pathogenic microorganisms. This research proposal focuses on the breast cancer resistance protein (BCRP, also termed ABCG2). BCRP is an ATP-binding cassette (ABC) transporter containing a nucleotide-binding domain, followed by a membrane domain with 6 transmembrane spanning segments. The protein is thought to homodimerise to form a functional transporter with the 4 core domains. Surprisingly, our recent observations suggest that the membrane domain of BCRP (BCRP-MD) is functionally analogous to a protonmotive force-driven secondary-active multidrug/proton antiporter. This project will focus in more detail on the role of the protonmotive-force and ATP-binding/hydrolysis in BCRP-mediated drug translocation, and aims to identify residues that are involved in proton translocation and in dimerisation of BCRP. The project entails a multidisciplinary strategy involving molecular biology-based methods as well as biochemical and biophysical techniques. This strategy will add important new information about the molecular mechanism of this unique class of multidrug transporters, and may lead to the development of new modulators that could induce drug uptake by ABC-type multidrug transporters in drug-resistant cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.

Publications

10 25 50
 
Description BBSRC Project Grant
Amount £151,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2011 
End 10/2014
 
Title ABCG2 expressing bacteria 
Description Bacterial (Lactococcus lactis) cells expressing human ABCG2 and mutants derived thereof 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact The bioenergetic properties of membrane transporters are easier to study in bacteria, where the levels of nucleotides and the magnitude and composition of electrochemical transmembrane gradients can be manipulated more easily. As current evidence suggests that certain human multidrug transporters also transport lipids, it is useful to express these transporters in a bacterial lipid environment which is very different from human, and which prevents that the transporters are virtually embedded in their lipid substrates. The latter situation makes a study of drug/lipid transport by ABCG2 more difficult. 
 
Title ABCG2 mutants 
Description Various cell lines were produced that express ABCG2 tagged at N or C-terminus, as well as mutants that are affected in drug and steroid hormone transport 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact Our strains facilitate biochemical studies on human ABCG2 
 
Title Electrophysiological recordings on single purified membrane transporters 
Description With input from my current postdoc Dr Kelvin Agboh and based on work of an earlier postdoc Dr Saroj Velamakanni, we have established methods to measure electrical currents generated by single purified ATP-binding cassette membrane (ABC) transporters in planar bilayers. This sheds new light on the biochemical properties and reaction mechanisms of these proteins. We hope to publish this work in 2016 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The ABC transporters under study are members of a large ABC superfamily. It is therefore likely that this feature of ABC transporters is more widespread 
 
Title Transport assays in proteoliposomes 
Description In this method, we are able to record the transport activity of purified membrane domain of ABCG2 in proteoliposomes in which the protein is functionally reconstituted. It allows a study of the transport mechanism with a degree of detail that cannot be obtained by studies in intact cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This method can be applied on a large variety of multidrug transporters 
 
Title steroid binding assay for ABCG2 
Description equilibrium binding assay for steroid binding to ABCG2 transporter 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2008 
Provided To Others? Yes  
Impact Our method facilitates the measurement of lipid binding to membrane transporters in detergent solution 
 
Description Collaboration with Dr Susan Bates, NIH, WASHINGTON DC, USA 
Organisation National Institutes of Health Clinical Center
Department Medical Oncology
Country United States 
Sector Public 
PI Contribution My research focussed on biochemical properties of mutants of ABCG2
Collaborator Contribution Dr Susan Bates (NIH) studied trafficking and surface expression of the mutants by confocal fluorescence microscopy.
Impact Outcomes were included in our published paper by Velamakanni et al in Molecular Pharmacology
Start Year 2008
 
Description Collaboration with Dr Tai-Ping Fan, Cambridge 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution In thi scollaboration, we studied the effect of ginsenosides on the activity of ABCG2
Collaborator Contribution We provides the ABCG2 test system
Impact We published a paper together
Start Year 2006
 
Description National Science Week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Gave talk for general pubic about role of ABCG2 in multidrug resistance of cancer cells.

Increased awareness about the importance of pharmacological/biochemical research
Year(s) Of Engagement Activity 2007,2008
 
Description Presentation by Postdoc 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talk sparked questions and discussion afterwards

Talk had scientific impact
Year(s) Of Engagement Activity 2006