Attached below is the article, “Purine import into malaria parasites as a target for
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Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: Malaria: Advances in Pathophysiology, Biology, and Drug Development
Purine import into malaria parasites as a target for
antimalarial drug development
I. J. Frame,1,â?? Roman Deniskin,1,â?? Avish Arora,1 and Myles H. Akabas1,2,3
Department of Physiology and Biophysics, 2 Department of Neuroscience, 3 Department of Medicine, Albert Einstein College
of Medicine, Bronx, New York
Address for correspondence: Myles H. Akabas, M.D., Ph.D., Department of Physiology and Biophysics, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. firstname.lastname@example.org
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life
cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are
imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine
salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes
four putative ENTs (PfENT1â??4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary
purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed
in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human
blood (<10 â®M). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target. Keywords: purines; nucleoside transporter; malaria; drug development Introduction Plasmodium species parasites, like many other protozoan parasites, are purine auxotrophs, unable to perform de novo purine biosynthesis. They rely on the host to provide purines that they modify through enzymes of the purine salvage pathway to generate the purine nucleotides necessary for cellular metabolic processes, including RNA and DNA synthesis, cellular energetics (ATP), and the synthesis of purine-containing molecules, such as NADH, NADPH, coenzyme A, and S-adenosylmethionine, among others. Purine metabolic pathways in Plasmodium parasites have been extensively reviewed and will not be discussed further in this work.1â??8 Purine monomers exist in three major forms: nucleobases, nucleosides, and nucleotides. Two â?? These authors contributed equally to this work. families of membrane transporters have been identified that transport purine nucleobases and nucleosides, the equilibrative nucleoside transporters (ENT, SLC29 family)9,10 and the concentrative nucleoside transporters (CNT, SLC28).11 The ENTs and CNTs are distinct gene families with no apparent sequence or structural homology. Although the gene family names suggest that the ENT family are facilitated transporters and the CNTs ion-coupled transporters, that distinction does not always hold, because some ENTs may be protonâ?? purine symporters.12,13 The Plasmodium falciparum genome contains four ENT homologues, PfENT1â?? 4, and no CNT homologues.14â??17 Thus, as discussed in detail later, ENTs are likely to be the major purine-import pathway into Plasmodium parasites. In the subsequent sections, we will review previous studies on the structure, function, and pharmacology of non-Plasmodium ENTs and then we will review the Plasmodium ENTs. We will then discuss other aspects of purine uptake and metabolism doi: 10.1111/nyas.12568 C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1â??10 1 Purine import as an antimalarial drug target Frame et al. of relevance to ENTs as potential drug targets for novel antimalarial compounds. Purine transport and metabolism by red blood cells Equilibrative nucleoside transporters: cloning, structure, and pharmacology Red blood cells (RBCs) provide the host environment for asexual-stage Plasmodium blood-stage parasites. Like Plasmodium parasites, RBCs are unable to synthesize purines by de novo biosynthesis. RBCs import purines and modify them via a subset of purine salvage pathway enzymes (Fig. 1). Thus, purines in the plasma are the source for both the RBCs and the parasites. Four ENT homologues have been identified in the human genome. In humans, hENT1 and hENT2 are the major plasma membrane purine transporters.18,19 They are 40% sequence identical. hENT3 is present in intracellular membranes and mutations in hENT3 cause a variety of human disorders.20â??23 The fourth human ENT homologue was initially characterized as a plasma membrane monoamine transporter (PMAT), but at acidic pH it transports purines.12,24,25 Both hENT1 and hENT2 transport both purines and pyrimidines. Both have a strong preference for nucleosides as substrates as compared to nucleobases.9,26,27 The pharmacology of hENT1 and hENT2 is quite distinct. hENT1 is inhibited by low nanomolar concentrations of nitrobenzylthioinosine (NBMPR), dipyridamole, and dilazep.18 In contrast, these compounds only inhibit hENT2 in the 10 â®M concentration range.19 Residues responsible for these differences have been identified through experiments involving chimeric constructs and site-directed mutagenesis.28â??38 ENTs are polytopic membrane proteins. When hENT1 was initially cloned, hydrophobicity analysis predicted it to have 11 transmembrane segments.18,19 Experimental data show that the Nterminus is cytoplasmic and the C-terminus is extracellular, suggesting an odd number of membranespanning segments. Glycosylation site insertion analysis is consistent with 11 membrane-spanning segments.39â??41 No X-ray crystal structures of ENTs have been solved to date, but using the Rosetta molecular modeling software, an ab initio model of the Leishmania donovani LdNT1.1 transporter, an ENT family member, has been constructed.42 Experimental studies using disulfide cross-linking between engineered cysteine residues have verified some predictions of the model.43,44 The watersurface accessibility of residues in transmembrane segments of several ENT family members have been analyzed by the substituted cysteine accessibility method (SCAM).45,46 SCAM experiments have identified residues that may line the ENT substrate permeation pathway in TM4, 5, 6, and 9â??11.47â??52 2 Figure 1. Simplified illustration of the purine transport and metabolism pathways in a P. falciparumâ??infected RBC. More detailed metabolic pathways are available in other reviews.7,8 Colored circles represent transporters and channels for the given substrates. The number of circles for a given transporter does not indicate relative abundance or transport capacity; they simply indicate the pathway a given substrate may take to cross a particular membrane. The light blue circle with the question mark represents the AMP-transport pathway that has been shown to exist functionally but whose molecular identity is unknown.99 In the interest of simplifying the figure, the parasitophorus vacuole membrane that surrounds the parasite in not shown because it is nonselectively permeable to small molecules such as purines.17 Purine transport pathways into various subcellular organelles that contain DNA that must be replicated during the parasite life cycle, such as the mitochondrion and apicoplast, are not shown.118,119 PfENT2, whose substrate specificity is unknown but is localized in the parasite endoplasmic reticulum, is also not shown.103 PfENT3, whose cellular localization and substrate specificity is unknown, is also not shown. PfENT4 is shown in the parasite plasma membrane, but localization experiments have not been performed. hENT1, human ENT1; hFNT1, human facilitated nucleobase transporter 1; NPP, new permeability pathway; hAK, human adenosine kinase; hADA, human adenosine deaminase; hPNP, human purine nucleoside phosphorylase; hHGPRT, human hypoxanthine guanine phosphoribosyl transferase; IMP, inosine monophosphate; PfADA, P. falciparum adenosine deaminase; PfPNP, P. falciparum purine nucleoside phosphorylase; PfHGXPRT, P. falciparum hypoxanthine guanine xanthine phosphoribosyl transferase; XMP, xanthine monophosphate. C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1â??10 Frame et al. Human plasma contains micromolar concentrations of purines. Early determinations of the plasma purine concentrations, particularly adenine/adenosine/ATP, were likely overestimates, owing to hemolysis and release from RBCs during sample acquisition and storage. With better techniques, more accurate measurements have been obtained that more likely reflect the composition of human plasma in situ.53,54 The major purines found in human blood are hypoxanthine (1â??5 â®M) and inosine ( 1 â®M).53,55â??58 Adenosine is the other purine found in plasma. Adenosine acts as a hormoneregulating local vasodilation and platelet aggregation. The plasma concentration of adenosine is regulated by local circumstances and ranges from nanomolar values up to 1â??5 â®M.53,54,57,59 Thus, in humans, the total plasma purine concentration is below 10 â®M.57 The human RBC plasma membrane has two major purine transport pathways. Nucleosides enter via hENT1 and nucleobases largely enter via an NBMPR-insensitive nucleobase facilitated transporter (hFNT1) whose molecular identity is uncertain.60â??66 In P. falciparumâ??infected RBCs, these two purine-import pathways seem to provide the primary pathway for purine entry into the RBC cytoplasm, but there is a component of adenosine uptake into infected RBCs that is insensitive to NBMPR and may be mediated by the new permeability pathway.67â??71 The RBC cytoplasm contains a subset of purine salvage pathway enzymes. They have two important roles: to remove adenosine from the blood and to provide the purine molecules necessary for RBC function. Adenosine is an important extracellular signaling molecule through its interactions with adenosine receptors, members of the G proteinâ??coupled receptor superfamily.72â??74 The main pathway for removal of adenosine from plasma is uptake into RBCs. In the RBC, adenosine is either phosphorylated by adenosine kinase to convert it into AMP or deaminated by adenosine deaminase to inosine, which is converted to hypoxanthine by purine nucleoside phosphorylase.8,64 Hypoxanthine can then be phosphoribosylated by hypoxanthine guanine phosphoribosyl transferase to form IMP, which can be further metabolized to guanylates.4,75 Alternatively, hypoxanthine can be released into the plasma and either oxidized to xanthine or uric acid or cleared by metabolic Purine import as an antimalarial drug target processes in other cells. The composition of the purine pool inside RBCs maintained in supraphysiological (367 â®M) hypoxanthine-containing media was recently reported.76 The relationship to the purine composition in cells maintained in physiological purine-containing media (<10 â®M) is uncertain because previous studies have shown changes in RBC purine composition and content as a function of media purine supplementation.77 Inside the RBC, the main constituent of the purine pool is ATP, which is present at a concentration of 2 mM. Given the volume of an RBC, this would not even provide a sufficient amount of purine to allow eight-fold replication of the Plasmodium genome, the minimal amount of DNA replication that occurs during the 48-h intraerythrocytic life cycle. This implies that purines must be imported into the RBC in order to supply sufficient amounts of purines to the developing intracellular parasite. Plasmodium equilibrative nucleoside transporters Sequence analysis of the P. falciparum genome identified four putative ENT homologues (PfENT1â??4).14,16 Extensive information about the four genes and their expression patterns in parasite life cycle stages is available on the PlasmoDB website (http://plasmodb.org/plasmo/). The gene identification numbers are PfENT1, PF3D7_1347200; PfENT2, PF3D7_0824400; PfENT3, PF3D7_1469400; and PfENT4, PF3D7_0103200. Curiously, PfENT3 is only found in Plasmodium species that infect primates and humans.78 Homologues of PfENT1, 2, and 4 are found in all Plasmodium species sequenced to date. The four PfENT homologues have diverged significantly both from hENT1 and from each other. PfENT1 is 17% identical to hENT1 in amino acid sequence. Individually, PfENT1 is 15â??22% identical to PfENT2â??4. Only 2% of residues are identical between the four homologues. PfENT1 The primary import pathway for purines to supply the parasite purine salvage pathway is via PfENT1. PfENT1 mRNA is found in all parasite life-cycle stages. The mRNA level has a small peak in the early trophozoite stage and then decreases in late schizonts.79,80 PfENT1 peptides have been identified in all asexual and sexual blood stages C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1â??10 3 Purine import as an antimalarial drug target Frame et al. and in sporozoites using mass spectrometry.81â??88 PfENT1 was cloned and heterologously expressed in Xenopus laevis oocytes.89,90 Interestingly, the native pfent1 nucleotide sequence can be functionally expressed in oocytes and did not require codon optimization for expression. There were significant differences in the substrate and inhibitor profiles and affinities reported in the initial pair of papers.89,90 Some of these differences arose from technical issues related to the effects of substrate metabolism on the radioactive substrate uptake experiments in oocytes that formed the basis of the papers.91,92 PfENT1 transports both purine and pyrimidine nucleobases and nucleosides with affinities in the hundreds of micromolar to millimolar range.90â??93 The characteristics of purine nucleobase uptake by RBC-free parasites is similar to the characteristics determined for heterologously expressed PfENT1.93 Uptake experiments in isolated parasites and in Xenopus oocytes must be done with care so as to distinguish the kinetic properties of the transporter from those of metabolic enzymes that may metabolize the transported substrates.92,94 Difficulties separating transport and metabolism have adversely affected some studies in isolated parasites.94,95 Human hENT1 inhibitors including NBMPR, dipyridamole, and dilazep do not inhibit PfENT1 expressed in oocytes at concentrations up to 10 â®M.50,90,91 These characteristics are similar to those reported for intact, RBC-free parasites.91,93 Immunoelectron microscopy with anti-PfENT1 antibodies show that it is mainly localized in the parasite plasma membrane.96 Knockout of pfent1 is conditionally lethal.97,98 At purine concentrations found in human blood (<10 â®M), pfent1 parasites are not viable. Growth can be rescued by addition of purines, hypoxanthine, adenosine, or inosine to the growth media at concentrations above 50 â®M. Maximal growth is not observed until a purine concentration of 500 â®M.97 The growth rescue at high purine concentrations suggests that the pfent1 parasites have a secondary, low-affinity, low-capacity purine transport pathway besides PfENT1. The results of purine uptake experiments into pfent1 parasites released from the host RBCs differ between two papers from the same group.97,98 The initial paper reported that uptake rates of adenosine and inosine were about 50% of uptake amounts in wild-type parasites, but no hypoxanthine uptake 4 was observed.97 The subsequent paper reported no uptake of adenosine, inosine, or hypoxanthine by pfent1 parasites, but restoration of uptake in pfent1 parasites expressing PfENT1 through complementation from a plasmid.98 Parasites lacking pfent1 did not import xanthine, guanine, or guanosine, implying that PfENT1 is the only import pathway for these purines.98 Although there are discrepancies in the results as to whether the pfent1 parasites can transport adenosine and inosine, the results in the second paper by the same authors presumably represent their view of the transport properties of the pfent1 parasites. This emphasizes the importance of PfENT1 in the uptake of purine nucleobases and nucleosides. Even though the pfent1 parasites cannot transport physiologically relevant nucleosides and nucleobases, they can grow on hypoxanthine-, adenosine-, inosine-, or adenine-supplemented media because the RBC purine salvage pathway likely converts these purines into a form that can be transported and used by the parasites, possibly as nucleotides.99 The identity of the specific chemical forms of the purines imported by pfent1 parasites remains unknown. The lethal impact of pfent1 knockout for parasites grown in purine concentrations found in human blood suggests that it might be a target for the development of novel antimalarial drugs.89,90,92,97,100 However, no inhibitors of PfENT1 have been identified to date. Experiments are in progress in our lab using a high-throughput screen that we have developed to identify PfENT1 inhibitors and to characterize their effects on the proliferation of malaria parasites in culture. Rodent malaria parasites lacking the PfENT1 homologue Knockout of the Plasmodium yoelli homologue of pfent1, pynt1, resulted in parasites that survived within the mouse. The pynt1 parasites displayed a growth defect compared to wild type. Peak parasitemia was only 2% for mice infected with 5000 pynt1 parasites but reached 30% with the same number of wild-type parasites. Infection with 50 pynt1 parasites did not yield observable parasitemia, but conferred immunity to challenge with wild-type P. yoelli and Plasmodium berghei strains. The authors noted that pynt1 parasites were unable to complete ookinete development in the mosquito.101 C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1â??10 Frame et al. A P. berghei pbnt1 knockout in the ANKA strain used as a model for cerebral malaria was also reported.102 Mice infected with the pbnt1 parasites did not develop cerebral malaria symptoms or pathology.102 The observations from both rodent malaria parasite models is clear. Removing the ENT1 protein is clearly detrimental to parasite growth and virulence. The latter report supports the idea that blocking ENT1 with a small molecule might prevent the development of cerebral malaria. PfENT2 Like PfENT1, PfENT2 is expressed in all parasite blood stages but was not detected in sporozoites.81â??88 PfENT2 mRNA levels peak significantly in the early-to-late trophozoite period.79,80 Attempts to heterologously express either native or codon-optimized PfENT2 in either Xenopus oocytes or in Saccharomyces cerevisiae (yeast) did not provide evidence for functional purine transport.91,103 PfENT2-expressing yeast were somewhat more sensitive to the pyrimidine analog 5-fluorouridine, but there was no other evidence of pyrimidine transport.103 Immunoelectron microscopy showed localization in internal parasite membranes, likely to be predominantly the endoplasmic reticulum.103 PfENT3 PfENT3 is the least studied of the Plasmodium ENTs. Curiously, pfent3 homologues are not found in the genomes of murine or avian Plasmodium species that have been sequenced to date.78 However, homologues are found in all species that infect primates or humans. PfENT3 mRNA expression is fairly constant through the 48-h intraerythrocytic life cycle.79,80 PfENT3 transcript levels appear to increase in response to antimycin A exposure.104 No evidence for PfENT3 expression has been reported in proteomic studies.81â??88 This may be due to low levels of protein expression or to specific expression in only a limited set of life-cycle stages. Attempts to express either native or yeast codon-optimized PfENT3 in either Xenopus oocytes or in S. cerevisiae did not result in evidence of functional purine or pyrimidine transport, although protein expression w ... Purchase answer to see full attachment
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