Drug resistant malaria kills millions annually. Reversing horrific trends in incidence and mortality requires a balanced approach in vaccine and drug research, as well as field based efforts to control vector populations and infection rates. Current and future treatment of the many different strains of drug resistant malaria that now exist requires a more complete understanding of multiple genotypes and phenotypes. We must not be lulled into a false sense of (temporary) security provided by current artemisinin (ART) based therapies;black market ART is already circulating and generating ART resistance. The struggle against drug resistant malaria is ongoing and must be met continuously;else we have learned nothing from the past 50 years while watching CQ and other drugs fail. We must "stay ahead of the resistance curve" and define molecular mechanisms that guides ongoing drug and vaccine research. Our laboratory has helped to lead the field in molecular level analysis of PfCRT and PfMDR1 proteins using heterologous expression systems. In this competitive renewal period we will:
Aim 1) Continue to define binding functions of PfCRT isoforms via heterlogous expression in yeast and analysis of purified membrane, ISOV, and PL preparations harboring these proteins. We will use recently developed techniques and chemical probes for drug, amino acid, and ion binding and transport. We will also synthesize additional probes (e.g.,AzB-MQ, AzBCQ side chain length variants, AzB-QN) for PfCRT function. These probes will also be used in Aim 3.
Aim 2) Continue to define drug transport functions of PfCRT isoforms using ISOV and PLs and radio labeled and fluorescent (e.g. NBD CQ) probes. We will also synthesize additional probes (e.g., NBD-MQ, NBD-QN) using previously synthesized intermediates and similar chemistry relative to successful synthesis of NBD-CQ.
Aim 3) Test hypotheses for function of PfMDR1 following a similar approach, and also using high throughput plate based ATPase assays we have developed and published [93, 93B]. We will investigate the unusual (relative to other ABCB transporters) drug - influenced "communication" between the two symmetrical halves of PfMDR1 [93]. We will analyze binding, transport and ATPase properties of ISOV and PLs harboring known ratios of various PfCRT and PfMDR1 proteins to test for interactions between the two transporters.

Public Health Relevance

Drug resistant malaria continues to both evolve and spread, and globally causes over 1 million deaths annually. This project aims to define, at a molecular level, how mutated proteins cause that drug resistance. Such information is central to development of new drugs and other therapies to combat drug resistant malaria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056312-09
Application #
8274341
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
2003-06-15
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2012
Total Cost
$338,503
Indirect Cost
$117,980
Name
Georgetown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R et al. (2016) Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology. Malar J 15:186
Callaghan, Paul S; Hassett, Matthew R; Roepe, Paul D (2015) Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT). Biochemistry 54:5083-94
Sherlach, Katy S; Roepe, Paul D (2014) Determination of the cytostatic and cytocidal activities of antimalarial compounds and their combination interactions. Curr Protoc Chem Biol 6:237-48
Roepe, Paul D (2014) To kill or not to kill, that is the question: cytocidal antimalarial drug resistance. Trends Parasitol 30:130-5
Baro, Nicholas K; Callaghan, Paul S; Roepe, Paul D (2013) Function of resistance conferring Plasmodium falciparum chloroquine resistance transporter isoforms. Biochemistry 52:4242-9
Brunner, Ralf; Ng, Caroline L; Aissaoui, Hamed et al. (2013) UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells. J Biol Chem 288:22576-83
Gorka, Alexander P; Sherlach, Katy S; de Dios, Angel C et al. (2013) Relative to quinine and quinidine, their 9-epimers exhibit decreased cytostatic activity and altered heme binding but similar cytocidal activity versus Plasmodium falciparum. Antimicrob Agents Chemother 57:365-74
Dinio, Theresa; Gorka, Alexander P; McGinniss, Andrew et al. (2012) Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum. Bioorg Med Chem 20:3292-7
Sinai, Anthony P; Roepe, Paul D (2012) Autophagy in Apicomplexa: a life sustaining death mechanism? Trends Parasitol 28:358-64
Roepe, Paul D (2011) PfCRT-mediated drug transport in malarial parasites. Biochemistry 50:163-71

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