Paul D Roepe

Georgetown University, Washington, Washington, D.C., United States

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Publications (112)488.31 Total impact

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    Katy S Sherlach, Paul D Roepe
    Frontiers in Physiology 03/2014; 5:108. DOI:10.3389/fphys.2014.00108
  • Paul D Roepe
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    ABSTRACT: Elucidating mechanisms of antimalarial drug resistance accelerates development of improved diagnostics and the design of new, effective malaria therapy. Recently, several studies have emphasized that chloroquine (CQ) resistance (CQR) can be quantified in two very distinct ways, depending on whether sensitivity to the growth inhibitory effects or parasite-kill effects of the drug are being measured. It is now clear that these cytostatic and cytocidal CQR phenotypes are not equivalent, and recent genetic, cell biological, and biophysical evidence suggests how the molecular mechanisms may overlap. These conclusions have important implications for elucidating other drug resistance phenomena and emphasize new concepts that are essential for the development of new drug therapy.
    Trends in Parasitology 02/2014; DOI:10.1016/ · 6.22 Impact Factor
  • Katy S Sherlach, Paul D Roepe
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    ABSTRACT: Determining the antiplasmodial activity of candidate antimalarial drugs in vitro identifies new therapies for drug-resistant malaria. Importantly though, activity can be either growth-inhibitory (cytostatic) or parasite-kill (cytocidal), or both. The simple methods described here can allow for distinction between these activities, as well as definition of drug interactions between two or more compounds. The latter is important in the definition of novel drug combination therapy for malaria. These methods involve live malarial parasite red blood cell culture, routine pharmacology, high-throughput detection of parasite DNA with fluorescent reporters, and routine mathematical analysis of dose-response curves. The techniques and approaches are accessible to most laboratories and require minimal special equipment beyond a fluorescent plate reader and tissue culture facilities. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
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    ABSTRACT: Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L(-1)), dihydroartemisinic acid (70.0±0.3 mg L(-1)), arteannuin B (1.3±0.0 mg L(-1)), isovitexin (105.0±7.2 mg L(-1)) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B's potentiation of artemisinin.
    PLoS ONE 11/2013; 8(11):e80790. DOI:10.1371/journal.pone.0080790 · 3.53 Impact Factor
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    ABSTRACT: Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR(CS)) as quantified by CQ IC50 shift, account for only 10-20% of cytocidal CQR (CQR(CC)) as quantified by CQ LD50 shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR(CS) vs CQR(CC) phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD50. Candidate genes in these loci are consistent with a role for autophagy in CQR(CC), leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR(CC). Taken together, the data show that an unusual autophagy - like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD50 doses (but not IC50 doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR(CC) phenotype.
    PLoS ONE 11/2013; 8(11):e79059. DOI:10.1371/journal.pone.0079059 · 3.53 Impact Factor
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    ABSTRACT: Drug combination therapy is the frontline of malaria treatment. There is an ever-accelerating need for new, efficacious combination therapies active against drug resistant malaria. Proven drugs already in the treatment pipeline, such as the quinolines, are important components of current combination therapy and also present an attractive test bank for rapid development of new concepts. The efficacy of several drug combinations versus chloroquine-sensitive and chloroquine-resistant strains was measured using both cytostatic and cytocidal potency assays. These screens identify quinoline and non-quinoline pairs that exhibit synergy, additivity, or antagonism using the fixed-ratio isobologram method and find tafenoquine -- methylene blue combination to be the most synergistic. Also, interestingly, for selected pairs, additivity, synergy, or antagonism defined by quantifying IC50 (cytostatic potency) does not necessarily predict similar behaviour when potency is defined by LD50 (cytocidal potency). These data further support an evolving new model for quinoline anti-malarials, wherein haem and haemozoin are the principle target for cytostatic activity, but may not be the only target relevant for cytocidal activity.
    Malaria Journal 09/2013; 12(1):332. DOI:10.1186/1475-2875-12-332 · 3.49 Impact Factor
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    ABSTRACT: A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.
    Journal of Biological Chemistry 06/2013; DOI:10.1074/jbc.M113.453159 · 4.60 Impact Factor
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    ABSTRACT: The function of P. falciparum chloroquine resistance transporter (PfCRT) can be quantified using a S. cerevisiae model system (Baro, N. K., Pooput C and Roepe P.D. Biochemistry. 50, 6701 - 6710). We further optimize this system to distinguish PfCRT isoforms found in P. falciparum strains and isolates from across the globe. We create and express 13 naturally occurring pfcrt alleles associated with a range of chloroquine resistant (CQR) phenotypes. Using galactose induction of PfCRT we quantify PfCRT and chloroquine (CQ) dependent yeast growth inhibition, and [3H]-CQ transport specifically due to a given PfCRT isoform. Surprisingly, we find poor correlation between these parameters vs CQ IC50 observed in strains of malaria harboring the same isoforms. This suggests that increased CQ transport due to PfCRT mutation is necessary, but not sufficient, for the range of CQ IC50 observed in globally distributed CQR P. falciparum isolates.
    Biochemistry 05/2013; 52(24). DOI:10.1021/bi400557x · 3.19 Impact Factor
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    ABSTRACT: Historically, the most successful molecular target for antimalarial drugs has been heme biomineralization within the malarial parasite digestive vacuole. Heme released from catabolized host red blood cell hemoglobin is toxic, so malarial parasites crystallize heme to nontoxic hemozoin. For years it has been accepted that a number of effective quinoline antimalarial drugs (e.g. chloroquine, quinine, amodiaquine) function by preventing hemozoin crystallization. However, recent studies over the past decade have revealed a surprising molecular diversity in quinoline-heme molecular interactions. This diversity shows that even closely related quinoline drugs may have quite different molecular pharmacology. This paper reviews the molecular diversity and highlights important implications for understanding quinoline antimalarial drug resistance and for future drug design.
    Journal of Medicinal Chemistry 04/2013; 56(13). DOI:10.1021/jm400282d · 5.48 Impact Factor
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    ABSTRACT: Ezrin is a key regulator of osteosarcoma metastasis. Our earlier work has established that NSC305787 can directly bind to ezrin and inhibit its activity, which results in reduced metastasis. The objective of this study was to evaluate common antimalarial drugs including quinine, quinidine, mefloquine, chloroquine and amodiaquine that share significant structural features with lead compound NSC305787 for their potential to inhibit ezrin function. Compounds were evaluated on inhibiting chemotaxis of osteosarcoma cells and inhibiting cancer cell invasion of HUVEC monolayer, both of which were measured by electric impedance based methods. Although tested compounds share structural similarities with NSC305787, they were ineffective in binding to ezrin and inhibiting its biochemical and biological functions. The current results will guide us in designing and synthesis of novel improved molecules that will specifically target and inhibit ezrin function.
    xCELLigence User Symposium, Wahington DC; 04/2013
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    ABSTRACT: Bioassay-guided fractionation of an ethanol extract of the leaves and inflorescence of Mallotus oppositifolius collected in Madagascar led to the isolation of the two new bioactive dimeric phloroglucinols mallotojaponins B (1) and C (2), together with the known mallotophenone (3). The structures of the new compounds were determined on the basis of spectroscopic evidence, including their 1D- and 2D-NMR spectra, mass spectrometry, and an X-ray crystal structure. Compounds 1 and 2 showed potent antimalarial activity against chloroquine-resistant Plasmodium falciparum, with IC(50) values of 0.75 ± 0.30 and 0.14 ± 0.04 μM, while 3 was inactive in this assay. Compounds 1-3 also displayed strong antiproliferative activity against the A2780 human ovarian cancer cell line (IC(50) 1.10 ± 0.05, 1.3 ± 0.1 and 6.3 ± 0.4 μM, respectively).
    Journal of Natural Products 01/2013; 76(3). DOI:10.1021/np300750q · 3.95 Impact Factor
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    ABSTRACT: The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum [Karle JM, Karle IL, Gerena L, Milhous, WK. 1992. Antimicrob. Agents Chemother. 36: 1538-1544]. We synthesized 9-epi-QN (eQN) and 9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to QN and QD we observe a large decrease in cytostatic activity (higher IC(50)) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observe relatively small changes in cytocidal activity (LD(50)). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency, and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-VIS titrations show that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicates binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with 2:1 stoichiometry (drug:FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic vs. cytocidal dosages of quinoline antimalarial drugs, and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes.
    Antimicrobial Agents and Chemotherapy 10/2012; 57(1). DOI:10.1128/AAC.01234-12 · 4.45 Impact Factor
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    ABSTRACT: We report an improved, non-hazardous, high throughput assay for in vitro quantification of antimalarial drug inhibition of β-hematin (hemozoin) crystallization performed under conditions that are more physiologic relative to previous assays. The assay uses differential detergent solubility of crystalline and non-crystalline forms of heme and is optimized via the use of lipid catalyst. Using this assay, we quantify the effect of pH on the crystal growth inhibitory activities of current quinoline antimalarials, evaluate the catalytic efficiencies of different lipids, and test for possible correlation between hemozoin inhibition by drugs vs. their antiplasmodial activity. Consistent with several previous reports, we find good correlation between hemozoin inhibition potency vs. cytostatic antiplasmodial potency (IC(50)) for a series of chloroquine (CQ) analogues. However, we find no correlation between hemozoin inhibition potency and cytocidal antiplasmodial potency (LD(50)) for the same drugs, suggesting that cellular targets for these two layers of 4-aminoquinoline drug activity differ. This important concept is also explored further for QN and its stereoisomers in the accompanying manuscript (Gorka, A.P., Sherlach, K.S., de Dios, A.C. and Roepe, P.D. following paper, this issue).
    Antimicrobial Agents and Chemotherapy 10/2012; 57(1). DOI:10.1128/AAC.01709-12 · 4.45 Impact Factor
  • Anthony P Sinai, Paul D Roepe
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    ABSTRACT: Programmed cell death (PCD) pathways remain understudied in parasitic protozoa in spite of the fact that they provide potential targets for the development of new therapy. The best understood PCD pathway in higher eukaryotes is apoptosis although emerging evidence also points to autophagy as a mediator of death in certain physiological contexts. Bioinformatic analyses coupled with biochemical and cell biological studies suggest that parasitic protozoa possess the capacity for PCD including a primordial form of apoptosis. Recent work in Toxoplasma and emerging data from Plasmodium suggest that autophagy-related processes may serve as an additional death promoting pathway in Apicomplexa. Detailed mechanistic studies into the molecular basis for PCD in parasitic protozoa represent a fertile area for investigation and drug development.
    Trends in Parasitology 07/2012; 28(9):358-64. DOI:10.1016/ · 6.22 Impact Factor
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    ABSTRACT: Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.
    Bioorganic & medicinal chemistry 03/2012; 20(10):3292-7. DOI:10.1016/j.bmc.2012.03.042 · 2.95 Impact Factor
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    ABSTRACT: Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.
    Cellular Microbiology 12/2011; 14(4):589-607. DOI:10.1111/j.1462-5822.2011.01745.x · 4.82 Impact Factor
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    ABSTRACT: We have used a novel gas chromatography/mass spectrometry (GC/MS)-based approach to quantify perchloroethylene (PCE) residues in dry-cleaned fabrics. Residual PCE was extracted from fabric samples with methanol and concentration was calculated by the gas chromatographic peak area, standardized using PCE calibration data. Extracts examined were from samples of 100% wool, polyester, cotton, or silk, which were dry cleaned from one to six times in seven different Northern Virginia dry-cleaning establishments. Additional experiments were conducted to investigate the kinetics of PCE release in the extraction solvent and to the open air. We found that polyester, cotton, and wool retained ≥ µM levels of PCE, that these levels increased in successive dry-cleaning cycles, and that PCE is slowly volatilized from these fabrics under ambient room air conditions. We found that silk does not retain appreciable PCE. Measured differences across dry-cleaning establishments and fabric type suggest more vigorous monitoring of PCE residues may be warranted. Environ. Toxicol. Chem. 2011;30:2481-2487. © 2011 SETAC.
    Environmental Toxicology and Chemistry 11/2011; 30(11):2481-7. DOI:10.1002/etc.665 · 2.83 Impact Factor
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    ABSTRACT: Investigation of extracts from the plant Athroisma proteiforme (Humbert) Mattf. (Asteraceae) for antimalarial activity led to the isolation of the five new sesquiterpene lactones 1–5 together with centaureidin (6). The structures of the new compounds were deduced from analyses of physical and spectroscopic data, and the absolute configuration of compound 1 was confirmed by an X-ray crystallographic study. Athrolides C (3) and D (4) both showed antiplasmodial activities with IC50 values of 6.6 (3) and 7.2 μM (4) against the HB3 strain and 5.5 (3) and 4.2 μM (4) against the Dd2 strain of the malarial parasite Plasmodium falciparum. The isolates 1–6 also showed antiproliferative activity against A2780 human ovarian cancer cells, with IC50 values ranging from 0.4 to 2.5 μM.
    Journal of Natural Products 10/2011; 74(10). DOI:10.1021/np200499d · 3.95 Impact Factor
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    Nicholas K Baro, Chaya Pooput, Paul D Roepe
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    ABSTRACT: Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.
    Biochemistry 08/2011; 50(31):6701-10. DOI:10.1021/bi200922g · 3.19 Impact Factor
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    ABSTRACT: With one exception (Gligorijevic et al., Mol Biochem Parasitol 2008;159:7-23.) all previous quantification of chloroquine (CQ) potency vs. P. falciparum has been by growth inhibition assays, meaning potency is defined as cytostatic potential and quantified by IC(50) values. In this study we investigate the cytocidal potency of CQ and other common quinoline antimalarial drugs (quantified as LD(50)). Similar to results from assays for cytostatic potency, we are able to readily distinguish drug resistant from drug sensitive P. falciparum parasites as well as different degrees of resistance. However, we find that fold-resistance to CQ and other quinoline drugs quantified via LD(50) ratios differs quite dramatically from fold resistance calculated via IC(50) ratios. Also, importantly, we find that verapamil chemoreversal of CQ resistance differs when quantified via cytocidal vs. cytostatic assays, as do patterns of "multidrug" resistance in well-known laboratory strains of P. falciparum. The results have important implications for development of new antimalarial drugs and for fully defining the genetic loci that confer clinically relevant antimalarial drug resistance phenomena.
    Molecular and Biochemical Parasitology 04/2011; 178(1-2):1-6. DOI:10.1016/j.molbiopara.2011.03.003 · 2.24 Impact Factor

Publication Stats

4k Citations
488.31 Total Impact Points


  • 1998–2014
    • Georgetown University
      • • Department of Chemistry
      • • Department of Biochemistry and Molecular and Cellular Biology
      • • Department of Physics
      • • Lombardi Cancer Center
      Washington, Washington, D.C., United States
  • 2012
    • University of North Carolina at Wilmington
      Wilmington, North Carolina, United States
  • 1987–2002
    • Brandeis University
      • Department of Chemistry
      Волтам, Massachusetts, United States
    • Boston University
      • Department of Physics
      Boston, Massachusetts, United States
  • 1997
    • Cornell University
      Итак, New York, United States
  • 1993–1997
    • Memorial Sloan-Kettering Cancer Center
      • • Department of Pathology
      • • Division of Molecular Pharmacology & Chemistry
      New York, New York, United States
  • 1990–1992
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
    • University of California, Los Angeles
      Los Ángeles, California, United States
  • 1989–1990
    • Roche Institute of Molecular Biology
      Nutley, New Jersey, United States
  • 1987–1988
    • University of Massachusetts Boston
      • Department of Physics
      Boston, MA, United States