Characteristics of chloroquine translocation by pfCRT

Lead Research Organisation: University of Oxford
Department Name: RDM Clinical Laboratory 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.

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