Amino Acids

, Volume 47, Issue 6, pp 1155–1166 | Cite as

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

  • Imke von Koschitzky
  • Heike Gerhardt
  • Michael Lämmerhofer
  • Michal Kohout
  • Matthias Gehringer
  • Stefan Laufer
  • Mario Pink
  • Simone Schmitz-Spanke
  • Christina Strube
  • Annette Kaiser
Original Article


Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E–Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC–MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum–mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.


Deoxyhypusine hydroxylase Hypusine Plasmodium Malaria Box Iron chelators 



This work was supported by Grants from the DAAD (Project-ID 57068252) and Heinz und Gertie Fischer Stiftung to AK. The authors acknowledge receipt of the antibody IU-88 from Dr. RG Mirmira, Department of Medicine, Indiana University School of Medicine, Indianapolis, USA.

Conflict of interest

The authors declare that they have no competing interest.


  1. Andrus L, Szabo P, Grady RW, Hanauske AR, Huima-Byron T, Slowinska B, Zagulska S, Hanauske-Abel HM (1998) Antiretroviral effects of deoxyhypusyl hydroxylase inhibitors: a hypusine-dependent host cell mechanism for replication of human immunodeficiency virus type 1 (HIV-1). Biochem Pharmacol 55:1807–1818CrossRefPubMedGoogle Scholar
  2. Assaraf YG, Golenser J, Spira DT, Bachrach U (1984) Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malaria parasite, Plasmodium falciparum. Biochem J 222:815–819PubMedCentralCrossRefPubMedGoogle Scholar
  3. Atemnkeng VA, Pink M, Schmitz-Spanke S, Wu XJ, Dong LL, Zhao KH, May C, Laufer S, Langer B, Kaiser A (2013) Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton. PLoS One 8:e58318PubMedCentralCrossRefPubMedGoogle Scholar
  4. Avery VM, Bashyam S, Burrows JN, Duffy S, Papadatos G, Puthukkuti S, Sambandan Y, Singh S, Spangenberg T, Waterson D, Willis P (2014) Screening and hit evaluation of a chemical library against blood-stage Plasmodium falciparum. Malar J 13:190-2875-13-190CrossRefGoogle Scholar
  5. Baird JK, Hoffman SL (2004) Primaquine therapy for malaria. Clin Infect Dis 39:1336–1345CrossRefPubMedGoogle Scholar
  6. Belda-Palazon B, Nohales MA, Rambla JL, Acena JL, Delgado O, Fustero S, Martinez MC, Granell A, Carbonell J, Ferrando A (2014) Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation. Front Plant Sci 5:202PubMedCentralPubMedGoogle Scholar
  7. Bergeron RJ, Xia MX, Phanstiel O IV (1993) Total syntheses of ()-hypusine and its (2S, 9S)-diastereomer. J Org Chem 58:6804–6806CrossRefGoogle Scholar
  8. Birkholtz LM, Williams M, Niemand J, Louw AI, Persson L, Heby O (2011) Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities. Biochem J 438:229–244PubMedCentralCrossRefPubMedGoogle Scholar
  9. Bowman JD, Merino EF, Brooks CF, Striepen B, Carlier PR, Cassera MB (2014) Antiapicoplast and gametocytocidal screening to identify the mechanisms of action of compounds within the malaria box. Antimicrob Agents Chemother 58:811–819PubMedCentralCrossRefPubMedGoogle Scholar
  10. Chawla B, Kumar RR, Tyagi N, Subramanian G, Srinivasan N, Park MH, Madhubala R (2012) A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani. PLoS One 7:e33138PubMedCentralCrossRefPubMedGoogle Scholar
  11. Clement PM, Hanauske-Abel HM, Wolff EC, Kleinman HK, Park MH (2002) The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro. Int J Cancer 100:491–498CrossRefPubMedGoogle Scholar
  12. Derbyshire ER, Mazitschek R, Clardy J (2012) Characterization of Plasmodium liver stage inhibition by halofuginone. Chem Med Chem 7:844–849PubMedCentralCrossRefPubMedGoogle Scholar
  13. Frey AG, Nandal A, Park JH, Smith PM, Yabe T, Ryu MS, Ghosh MC, Lee J, Rouault TA, Park MH, Philpott CC (2014) Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci USA 111:8031–8036PubMedCentralCrossRefPubMedGoogle Scholar
  14. Frommholz D, Kusch P, Blavid R, Scheer H, Tu JM, Marcus K, Zhao KH, Atemnkeng V, Marciniak J, Kaiser AE (2009) Completing the hypusine pathway in Plasmodium. FEBS J 276:5881–5891CrossRefPubMedGoogle Scholar
  15. Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, Vanderwall DE, Green DV, Kumar V, Hasan S, Brown JR, Peishoff CE, Cardon LR, Garcia-Bustos JF (2010) Thousands of chemical starting points for antimalarial lead identification. Nature 465:305–310CrossRefPubMedGoogle Scholar
  16. Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, Smithson DC, Connelly M, Clark J, Zhu F, Jimenez-Diaz MB, Martinez MS, Wilson EB, Tripathi AK, Gut J, Sharlow ER, Bathurst I, El Mazouni F, Fowble JW, Forquer I, McGinley PL, Castro S, Angulo-Barturen I, Ferrer S, Rosenthal PJ, Derisi JL, Sullivan DJ, Lazo JS, Roos DS, Riscoe MK, Phillips MA, Rathod PK, Van Voorhis WC, Avery VM, Guy RK (2010) Chemical genetics of Plasmodium falciparum. Nature 465:311–315PubMedCentralCrossRefPubMedGoogle Scholar
  17. Horak J, Gerhardt H, Theiner J, Lindner W (2014) Correlation between amino acid racemization and processing conditions for various wheat products, oil seed press cakes and lignin samples. Food Bioprod Process 92:355–368CrossRefGoogle Scholar
  18. Jain RP, Albrecht BK, DeMong DE, Williams RM (2001) Asymmetric synthesis of ()-hypusine. Org Lett 3:4287–4289CrossRefPubMedGoogle Scholar
  19. Kaiser A (2012) Translational control of eIF5A in various diseases. Amino Acids 42:679–684CrossRefPubMedGoogle Scholar
  20. Kaiser A, Hammels I, Gottwald A, Nassar M, Zaghloul MS, Motaal BA, Hauber J, Hoerauf A (2007) Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: an enzyme with dual enzymatic properties. Bioorg Med Chem 15:6200–6207CrossRefPubMedGoogle Scholar
  21. Kaiser A, Khomutov AR, Simonian A, Agostinelli E (2012) A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases. Amino Acids 42:1651–1659CrossRefPubMedGoogle Scholar
  22. Kang KR, Kim YS, Wolff EC, Park MH (2007) Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A. J Biol Chem 282:8300–8308PubMedCentralCrossRefPubMedGoogle Scholar
  23. Kerscher B, Nzukou E, Kaiser A (2010) Assessment of deoxyhypusine hydroxylase as a putative, novel drug target. Amino Acids 38:471–477CrossRefPubMedGoogle Scholar
  24. Krotoski WA (1989) The hypnozoite and malarial relapse. Prog Clin Parasitol 1:1–19PubMedGoogle Scholar
  25. Lehane AM, Ridgway MC, Baker E, Kirk K (2014) Diverse chemotypes disrupt ion homeostasis in the malaria parasite. Mol Microbiol 94:327–339CrossRefPubMedGoogle Scholar
  26. Milord F, Pepin J, Loko L, Ethier L, Mpia B (1992) Efficacy and toxicity of eflornithine for treatment of Trypanosoma brucei gambiense sleeping sickness. Lancet 340:652–655CrossRefPubMedGoogle Scholar
  27. Nishiki Y, Farb TB, Friedrich J, Bokvist K, Mirmira RG, Maier B (2013) Characterization of a novel polyclonal anti-hypusine antibody. SpringerPlus 2:1–5CrossRefGoogle Scholar
  28. Njuguna JT, von Koschitzky I, Gerhardt H, Lammerhofer M, Choucry A, Pink M, Schmitz-Spahnke S, Bakheit MA, Strube C, Kaiser A (2014) Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium. Bioorg Med Chem 22:4338–4346CrossRefPubMedGoogle Scholar
  29. Park JH, Aravind L, Wolff EC, Kaevel J, Kim YS, Park MH (2006) Molecular cloning, expression, and structural prediction of deoxyhypusine hydroxylase: a HEAT-repeat-containing metalloenzyme. Proc Natl Acad Sci USA 103:51–56PubMedCentralCrossRefPubMedGoogle Scholar
  30. Pearson RG (1963) Hard and soft acids and bases. J Am Chem Soc 85:3533–3539CrossRefGoogle Scholar
  31. Saeftel M, Sarite RS, Njuguna T, Holzgrabe U, Ulmer D, Hoerauf A, Kaiser A (2006) Piperidones with activity against Plasmodium falciparum. Parasitol Res 99:281–286CrossRefPubMedGoogle Scholar
  32. Spangenberg T, Burrows JN, Kowalczyk P, McDonald S, Wells TN, Willis P (2013) The open access malaria box: a drug discovery catalyst for neglected diseases. PLoS One 8:e62906PubMedCentralCrossRefPubMedGoogle Scholar
  33. Teng R, Junankar PR, Bubb WA, Rae C, Mercier P, Kirk K (2009) Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by (1)H NMR spectroscopy. NMR Biomed 22:292–302CrossRefPubMedGoogle Scholar
  34. Thompson GM, Cano VS, Valentini SR (2003) Mapping eIF5A binding sites for Dys1 and Lia1: in vivo evidence for regulation of eIF5A hypusination. FEBS Lett 555:464–468CrossRefPubMedGoogle Scholar
  35. Tice CM, Ganem B (1983) Chemistry of naturally occurring polyamines. 8. Total synthesis of (+)-hypusine. J Org Chem 48:5048–5050CrossRefGoogle Scholar
  36. Umland TC, Wolff EC, Park MH, Davies DR (2004) A new crystal structure of deoxyhypusine synthase reveals the configuration of the active enzyme and of an enzyme.NAD.inhibitor ternary complex. J Biol Chem 279:28697–28705CrossRefPubMedGoogle Scholar
  37. von Koschitzky I, Kaiser A (2013) Chemical profiling of deoxyhypusine hydroxylase inhibitors for antimalarial therapy. Amino Acids 45:1047–1053CrossRefGoogle Scholar
  38. Vu VV, Emerson JP, Martinho M, Kim YS, Munck E, Park MH, Que L Jr (2009) Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center. Proc Natl Acad Sci USA 106:14814–14819PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Imke von Koschitzky
    • 1
  • Heike Gerhardt
    • 2
  • Michael Lämmerhofer
    • 2
  • Michal Kohout
    • 3
  • Matthias Gehringer
    • 4
  • Stefan Laufer
    • 4
  • Mario Pink
    • 5
  • Simone Schmitz-Spanke
    • 5
  • Christina Strube
    • 6
  • Annette Kaiser
    • 1
    • 7
  1. 1.Institute for Pharmacogenetics, Medical Research CentreUniversity Duisburg-EssenEssenGermany
  2. 2.Institute of Pharmaceutical Sciences, Pharmaceutical (Bio) AnalysisEberhard-Karls-University TübingenTübingenGermany
  3. 3.Department of Organic ChemistryInstitute of Chemical Technology PraguePragueCzech Republic
  4. 4.Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical SciencesEberhard-Karls-University TübingenTübingenGermany
  5. 5.Institute and Outpatient Clinic of Occupational, Social and Environmental MedicineUniversity of Erlangen-NürnbergErlangenGermany
  6. 6.Institute for ParasitologyUniversity of Veterinary Medicine HannoverHannoverGermany
  7. 7.Fraunhofer Institut IMEAachenGermany

Personalised recommendations