EXCEED THE SPACE PROVIDED. After over a decade of painstaking work, Wellems and colleagues recently identified the gene that appears to be responsible for conferring chloroquine resistance (CQR) to the malarial parasite Plasmodium falciparum. This gene, pfcrt, encodes what appears to be a polytopic integral membrane protein with 10 putative transmembraneous helices. Pfcrt protein is localized to the digestive vacuolar membrane of the intraerythrocytic parasite. Several distinct patterns of mutations in Pfcrt confer CQR, but the function of mutant proteins is not yet known, nor is the endogenous physiology catalyzed by the wild type protein understood. Deciphering Pfcrt function at the molecular level is central to defining CQR, to anticipating additional drug resistance pathways, and !for devising improved therapy. Analysis of molecular level function in vivo is extraordinarily challenging since the protein is localized to an endo membrane of an intracellular parasite. Thus, over the past several years we have endeavored to engineer high level overexpression of malarial integral membrane proteins in yeast, and have recently reported the high level overexpression of mutant and wild type Pfcrt proteins in both S. cerevisiae and P. pastoris. To our knowledge, this represents the first successful overexpression of malarial polytopic integral membrane proteins in a heterologous system. Based on this success, we now propose a detailed vesicle and proteoliposome based study to decipher the function of Pfcrt. We also propose to extend our success with this approach to analysis of another key membrane protein involved in antimalarial drug resistance, Pf mdr 1. The data to be generated are vital for understanding drug resistance in this and other systems, and will provide a template for design of additional in depth studies of apicomplexan membrane transport proteins. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056312-03
Application #
6838140
Study Section
Special Emphasis Panel (ZRG1-SSS-K (05))
Program Officer
Coyne, Philip Edward
Project Start
2003-06-15
Project End
2006-11-30
Budget Start
2004-12-01
Budget End
2005-11-30
Support Year
3
Fiscal Year
2005
Total Cost
$310,400
Indirect Cost
Name
Georgetown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
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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

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