Characteristics of chloroquine translocation by pfCRT

Lead Research Organisation: University of Brighton
Department Name: Sch of Pharmacy & Biomolecular Sciences

Abstract

Malaria continues its reign as one of the largest causes of death in the developing world. Currently, the main therapeutic strategy is drug administration, or chemotherapy. The drug chloroquine is one of the most widely used anti-malarial agents. Chloroquine enters the malarial parasite whilst it resides in red blood cells (erythrocytes). During this period the parasite grows by virtue of digesting the protein rich erythrocyte environment. This digestion occurs within a specific compartment of the parasite, known as the food vacuole. Chloroquine also enters the food vacuole and inhibits a specific pathway involved in digestion of erythrocyte proteins. In turn, this causes a build up of toxic by-products and ultimately leads to death of the parasite. Unfortunately, in many regions worldwide, the malaria parasites have built up a resistance to chloroquine, and many other anti-malarial drugs. There are numerous pathways contributing to resistance in malaria, but the main one associated with chloroquine resistance is caused by mutations in the PfCRT gene. The PfCRT gene produces a protein that resides on the surface of the food vacuole and is thought to confer resistance by altering the amount of chloroquine accumulating in this compartment. It is unclear what this protein does normally in the parasite and what consequences the mutations have on its activity.
Our primary aim is to determine how the PfCRT protein contributes to resistance against chloroquine and whether its actions can be overcome.
To enable us to reach this objective, we have developed a novel experimental system to directly examine PfCRT activity in isolation. The system will enable us to examine the following key issues:
(i) Providing information on which anti-malarial drugs (other than chloroquine) are targeted by PfCRT and therefore succumb to resistance.
(ii) Catalogue compounds capable of inhibiting PfCRT, which could potentially restore chloroquine accumulation and overcome resistance. Positive compounds could be used in future chemical programs to develop more potent agents.
(iii) Determine whether PfCRT pumps drugs in an energy dependent manner or by simply acting as a pore through which chloroquine can exit the food vacuole.
Providing a greater understanding of how PfCRT causes resistance to chloroquine in malaria will significantly enhance future strategies to overcome its unwanted activity and thereby circumvent the resistance to chemotherapy.

Technical Summary

Chemotherapy has been, and remains, a mainstay of therapy in the world-wide battle against malaria. Unfortunately, the emergence of resistance has negated the efficacy of a large number of chemotherapeutics including chloroquine (CQ). A large number of genetic studies have revealed that the gene encoding PfCRT plays a major role in conferring resistance to CQ. The PfCRT protein is a member of the drug/metabolite exchange family of transporters and is located in the digestive vacuole of sensitive and resistant parasites. The precise ?physiological? role of PfCRT remains unknown and the form expressed in resistant parasites contains a number of mutations. Investigations with resistant parasites suggest that PfCRT contributes to resistance by reducing the accumulation of CQ at its site of action; namely the digestive vacuole.
It is assumed, but not demonstrated, that PfCRT confers resistance by directly modulating CQ transport across the digestive vacuole membrane. There is a great deal of controversy surrounding the mechanism of action of PfCRT and debate rages as to whether it modulates CQ translocation by acting as a transporter or a channel.
This lack of fundamental understanding exists largely due to the absence of a simple experimental system to monitor CQ transport. We have recently generated a heterologous expression system to enable the extraction, purification and reconstitution of PfCRT into synthetic vesicles. Reconstituted PfCRT can be utilised to directly examine CQ transport. Our aim is to characterise the biochemical pharmacology of PfCRT to increase our understanding of fundamental issues surrounding the ability of this protein to confer resistance. In particular we propose to: (i) reveal the specificity of PfCRT to anti-malarial drugs, (ii) ascertain the potential to pharmacologically inhibit its activity, (iii) characterise the bioenergetics of CQ transport by PfCRT and (iv) reveal the molecular mechanism of drug translocation by the protein. A variety of biochemical and biophysical approaches will be utilised to achieve these main research objectives.
A molecular understanding of how PfCRT confers resistance will greatly enhance efforts to overcome its unwanted ability to confer resistance against CQ based chemotherapy.

Publications


10 25 50
 
Description Post Doctorial Bridging fund
Amount £10,232 (GBP)
Organisation University of Brighton 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2013 
End 12/2013
 
Title Developed an expression system for pfCRT in Hi5 insect cells 
Description Codon optimised cDNA for a resistant (Dd2) & a sensitive (Hb3) isoform, obtained from Prof. Fiddock, was used to generate a recombinant baculovirus expression system. Both isoforms were successfully expressed in Trichplusia ni (High-5) insect cells. 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact This expression system has allowed us to purify and reconstitute both isoforms of pfCRT into proteoliposomes for biochemical and electrophysiological experiments. 
 
Title Electrochemical detection of GSH flux using microelectrodes 
Description Multi-walled Carbon nanotubes (MWCNT) modified with Prussian blue (PB) electrodes provided good sensitivity for the detection of glutathione and exceptional stability; whilst MWCNT just coated with PB electrodes provided excellent sensitivity but poor stability. Such findings provide important implications on the fabrication, suitability and use of chemically modified electrodes for electrochemical detection of glutathione transporters such as pfCRT. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This analytical technique may potentially allow the quantitative measurement of glutathione flux in real time. 
 
Title Purification and reconstitution of mutant and wild type pfCRT 
Description A method of purifying pfCRT has been developed along with reconstitution of the purified protein into proteoliposomes. His tagged protein is solubilised with detergent (DDM) and purified using metal affinity chromatography. The pure protein is eluted from the column with imidazole and reconstituted into liposomes compose of E.coli lipids or PC/PE with bio-beads. The proteoliposomes can be used for tritiated chloroquine uptake experiment. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This data was presented at the Gordon RC conference in Switzerland 
 
Description Collaboration with Dr D Baker LSHTM 
Organisation London School of Hygiene and Tropical Medicine (LSHTM)
Department Malaria Centre
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Collaborating in the area of calcium signalling in plamodium falciparium. At present investigating plasmodium ion channels in planar lipid bilayer.
Collaborator Contribution Prepared and provided live parasite for electrophysiological investiigations
Impact We are actively investigating malarial ryanodine receptors in planar lipid bilayers.
Start Year 2013
 
Description Structural studies on pfCRT with Dr Simone Weyand 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Our expertise and baculovirus has been donated to Dr Simone Weyand laboratory for pfCRT expression purification and structural studies. We have transferred our expression and purification expertise to this laboratory as Dr Weyand has expertise in crystallography and Cryo EM.
Collaborator Contribution Our laboratory developed all the pfCRT expression and purification protocols.
Impact None as yet as the collaboration is at a very early stage.
Start Year 2015
 
Description Structural studies on the BK channel with Dr Simone Weyand and Dr Alice Rothnie 
Organisation Aston University
Department School of Engineering and Applied Science
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution My laboratory expresses the his tagged BK channel in HEK293 cells. Membranes are harvested and function determined in black lipid bilayers. The protein is purified in Dr Alice Rothnies laboratory at Aston University, We also check the function of purified BK channels prior to CryoEM studies at Simon Weyands laboratory
Collaborator Contribution Dr Simon Weyand's research group are carrying out the CryoEM studies on the purified channel
Impact This data was presented in 2016 at the informal SMALP meeting in Birmingham
Start Year 2016
 
Description Structural studies on the BK channel with Dr Simone Weyand and Dr Alice Rothnie 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution My laboratory expresses the his tagged BK channel in HEK293 cells. Membranes are harvested and function determined in black lipid bilayers. The protein is purified in Dr Alice Rothnies laboratory at Aston University, We also check the function of purified BK channels prior to CryoEM studies at Simon Weyands laboratory
Collaborator Contribution Dr Simon Weyand's research group are carrying out the CryoEM studies on the purified channel
Impact This data was presented in 2016 at the informal SMALP meeting in Birmingham
Start Year 2016
 
Description Visit to Rowena Martin, Richard Callaghan and Stefan Broer to investigate transporter currents in oocytes expressing pfCRT 
Organisation College of Medicine, Biology & Environment (CMBE)
Department Research School of Biology
Country Australia, Commonwealth of 
Sector Academic/University 
PI Contribution Personally undertook voltage clamp experiments, in oocytes in order to study currents associated with pfCRT.
Collaborator Contribution The Australia partners I collaborated with have an expression for pfCRT in Oocytes. The purpose of this visit was to gain experience in measuring transporter currents in their systems in order to develop a similar approach to pfCRT at the university of Brighton.
Impact Dr Callaghan and I are undertaking fresh experiments with nanodiscs and giant unilaminar vesicles (GUV's), with the view to carry out fresh uptake experiments, binding experiments and patch clamp experiments.
Start Year 2013
 
Description Presentation LSHTM Malaria Centre Retreat 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Malaria Centre Retreat 2013, Thursday 21st March and Friday 22nd March 2013

Purification, reconstitution and uptake/efflux assays of wild type and mutant pfCRT
Rebecca J. Wright1, Eva K. Schmitt2, Marcus C. Allen1, Patrick G. Bray3, Ian D. Kerr4, Richard Callaghan2

200 hundred delegates from a range of countries present their research work on management control treatment and eradication of Malaria.


Active collaboration with Dr D Baker at LSHTM in the area of Calcium signalling in Plasmodium falciparium.
Year(s) Of Engagement Activity 2013
 
Description Presentation at Oxford University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Presentation on the importance of biomedical research and its involvement in the University. This is a yearly event and is directed to A-level students from disadvantaged areas/regions in the UK.

Considerable debate on research topics and stimulated students to seek Nuffield Bursaries
Year(s) Of Engagement Activity 2010
 
Description School visit - Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact The presentations were to a 6th form group comprising approximately 40-50 students at the Cherwell High School.

Two students later came to the laboratory to undertake work experience.
Year(s) Of Engagement Activity 2010