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    • Albert Einstein College of Medicine: Doctoral Dissertations
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    • Albert Einstein College of Medicine: Doctoral Dissertations
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    Characterization of Purine Transport in Plasmodium as a Therapeutic Target: a Structural Perspective

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    Date
    2017
    Author
    Arora, Avish
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    Abstract
    Malaria infects over 200 million people annually and is caused by infection with Plasmodium parasites. Like other parasitic protozoa, Plasmodium species lack de novo purine synthesis. Instead, they must import purines from their host to fulfill metabolic requirements. The Plasmodium falciparum Equilibrative Nucleoside Transporter (PfENT1) is the primary purine import pathway into the parasites. We have recently discovered small molecule inhibitors of PfENT1 and validated it as an antimalarial drug target.;Firstly, we sought to characterize the Plasmodium berghei ENT1 (PbENT1), which has 60% amino acid sequence identity with PfENT1. We heterologously expressed PbENT1 in purine-auxotrophic Saccharomyces cerevisiae yeast cells. We determined that PbENT1 expressing yeast can take up [ 3H]adenosine and [3H]uridine. In addition, we showed that uptake of these radiolabeled substrates is inhibited by various purines and pyrimidines. All nine compounds that were identified as inhibitors of PfENT1 using our yeast high-throughput screen showed similar activity against PbENT1.;Second, we utilized the substituted cysteine accessibility method (SCAM) to identify water-accessible residues along transmembrane segment 2 (TM2) of PfENT1. PfENT1 Cysmutant expressing yeast were then treated with methanethiosulfonate (MTS) reagents, which preferentially react with water-accessible cysteine residues. MTS reagents reacted with Cys substituted for Phe-63, Tyr-65, Asn-66, Gln-69, Ile-70, and Gly-72 resulting in inhibition of transport. Also, MTS reagents reacted with Cys substituted for Lys-64, Thr-67, Phe-68, and Thr71, resulting in marked potentiation. Potentiation appears to be only partially due to increased substrate affinity, but primarily due to increased transport rate.;Thirdly, we characterized the transport pathway of Adenosine Monophosphate (AMP) into red blood cell (RBC) -free parasites. We found that RBC-free P. falciparum parasites could take up [3H]AMP, although at a rate ~100 fold less than [3H]adenosine. The purines adenosine, inosine, and hypoxanthine also inhibited [3H]AMP transport at almost the identical IC50 values of ~1 muM. The IC50 values were unchanged even when PfENT1 was completely inhibited. From this work, it appears that there is a low-affinity AMP transporter expressed in Plasmodium falciparum. Along with our structual characterization of PfENT1, we hope that this body of knowledge will help to develop novel anti-malarial therapies targeting purine uptake.
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    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:10586300
    https://hdl.handle.net/20.500.12202/406
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    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

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