title: Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.
abstract: The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Jie
authors.initials: J
authors.lastname: Song
authors.affiliation: Division of Biological Chemistry & Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Baker
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Monja
authors.initials: M
authors.lastname: Rothert
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Henke
authors.affiliation: Division of Biological Chemistry & Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Jeacock
authors.affiliation: Division of Biological Chemistry & Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Horn
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Eric
authors.initials: E
authors.lastname: Beitz
mesh_terms: D000818:Animals; D051397:Aquaglyceroporins / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D006801:Humans; D010419:Pentamidine / Q000494:pharmacology*; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects*; D014353:Trypanosomiasis, African / Q000188:drug therapy
chemical_list: D051397:Aquaglyceroporins; D014344:Trypanocidal Agents; D010419:Pentamidine
references.citation: Nature. 1998 Jan 29;391(6666):499-502
references.pmid: 9461219
references.citation: Proc Natl Acad Sci U S A. 2012 Aug 14;109(33):13254-9
references.pmid: 22847446
references.citation: Biochim Biophys Acta. 2012 May;1818(5):1218-24
references.pmid: 22326891
references.citation: Antimicrob Agents Chemother. 1994 May;38(5):1123-8
references.pmid: 8067749
references.citation: J Biol Chem. 2004 Oct 8;279(41):42669-76
references.pmid: 15294911
references.citation: FEBS Lett. 2003 Dec 18;555(3):500-4
references.pmid: 14675763
references.citation: Mol Aspects Med. 2012 Oct-Dec;33(5-6):553-61
references.pmid: 22342689
references.citation: J Antimicrob Chemother. 2014 Mar;69(3):651-63
references.pmid: 24235095
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6053-8
references.pmid: 11972053
references.citation: J Biol Chem. 2010 Dec 17;285(51):40081-7
references.pmid: 20947511
references.citation: Adv Exp Med Biol. 2010;679:57-69
references.pmid: 20666224
references.citation: J Biol Chem. 2004 Jul 23;279(30):31010-7
references.pmid: 15138256
references.citation: Mol Microbiol. 2006 Sep;61(6):1598-608
references.pmid: 16889642
references.citation: Biochim Biophys Acta. 2014 May;1840(5):1566-73
references.pmid: 24140393
references.citation: Protein Sci. 1998 Jun;7(6):1458-68
references.pmid: 9655351
references.citation: Biochem Pharmacol. 1976 Feb 1;25(3):271-6
references.pmid: 1267824
references.citation: FEBS J. 2011 Mar;278(5):740-8
references.pmid: 21205205
references.citation: Mol Cell Biol. 1997 Aug;17(8):4282-93
references.pmid: 9234685
references.citation: PLoS Pathog. 2015 Jun;11(6):e1004942
references.pmid: 26110623
references.citation: Nucleic Acids Res. 1994 Dec 25;22(25):5767-8
references.pmid: 7838736
references.citation: Acta Trop. 1997 Nov;68(2):139-47
references.pmid: 9386789
references.citation: Front Pharmacol. 2014 Sep 01;5:199
references.pmid: 25225485
references.citation: Pharmacol Ther. 2015 Nov;155:22-35
references.pmid: 26277280
references.citation: Front Pharmacol. 2014 Sep 12;5:207
references.pmid: 25309439
references.citation: Parasite. 2002 Dec;9(4):375-9
references.pmid: 12514955
references.citation: J Protozool. 1976 May;23(2):349-56
references.pmid: 6797
references.citation: J Biol Chem. 2002 Apr 19;277(16):13548-55
references.pmid: 11821433
references.citation: Nat Commun. 2015;6:6284
references.pmid: 25669138
references.citation: PLoS One. 2012;7(5):e37435
references.pmid: 22624030
references.citation: Mol Biochem Parasitol. 2008 Sep;161(1):76-9
references.pmid: 18588918
references.citation: Science. 2000 Oct 20;290(5491):481-6
references.pmid: 11039922
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D362-9
references.pmid: 22080560
references.citation: Cell Physiol Biochem. 2013;32(4):880-8
references.pmid: 24217645
references.citation: Mol Biochem Parasitol. 2011 Mar;176(1):55-7
references.pmid: 21093499
references.citation: Nat Rev Microbiol. 2009 Nov;7(11):775-86
references.pmid: 19806154
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10996-1001
references.pmid: 22711816
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):269-74
references.pmid: 16407156
references.citation: Cell Mol Life Sci. 2007 Sep;64(18):2413-21
references.pmid: 17571212
references.citation: Nature. 2013 Sep 19;501(7467):430-4
references.pmid: 23965626
references.citation: Science. 2013 Jun 14;340(6138):1346-9
references.pmid: 23766328
references.citation: EMBO J. 2009 Aug 5;28(15):2188-94
references.pmid: 19574955
references.citation: Trends Parasitol. 2013 May;29(5):207-12
references.pmid: 23601931
references.citation: J Biol Chem. 1998 Sep 18;273(38):24737-43
references.pmid: 9733774