Drug resistant malaria kills 600,000 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 rates of infection. Current and future treatment of the many different strains of drug resistant Plasmodium falciparum malaria that now exist requires a more complete understanding of multiple genotypes and phenotypes. 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 drugs fail. We must stay ahead of the resistance curve and define molecular mechanisms that guide ongoing drug and vaccine research. Our laboratory has helped to lead the field in the molecular level analysis of the PfCRT and PfMDR1 proteins, two transporters that are crucial for several forms of antimalarial drug and multidrug resistance. Recently, dozens of new isoforms of these proteins have been discovered, but only a handful have yet been studied. Elucidating function and resistance - conferring ability of these isoforms is crucial for defining local drug resistance and selecting region- and genotype-specific drugs whose efficacy is not compromised by local isoforms of these drug resistance transporters. In this grant period we will:
Aim 1. Elucidate the molecular basis of PfCRT functional diversity and define K76T phenotypes. (1.1) Quantitatively compare all known PfCRT isoforms (1.2) Perform pfcrt gene editing experiments in P. falciparum to test in vitro conclusions, and Aim 2. Utilize a similar approach in order to elucidate PfMDR1 functional diversity, including: (2.1) Drug binding and drug transport in vitro, and (2.2) Pfmdr1 gene editing experiments in P. falciparum to test in vitr conclusions Aim 3. Elucidate the three-dimensional structures of PfCRT and PfMDR1 proteins. (3.1) Use a shot-gun crystallization strategy with multiple CRT orthologs, (3.2) Leverage tools within the TransportPDB resource, and (3.3) Generate tight-binding Nanobodies (Nbs) to chaperone crystal lattice formation.
Mutations in the pfcrt and pfmdr1 genes have been linked to resistance to antimalarial drugs, and at least 6 PfMDR1 and 53 PfCRT protein isoforms have been defined across the globe. This project quantitatively compares the function of all PfMDR1 and PfCRT isoforms using yeast and malarial parasite model systems, and also endeavors to solve the three dimensional structure of these proteins via x-ray crystallography.
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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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