Plant Molecular Biology

, Volume 92, Issue 1–2, pp 235–248 | Cite as

Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

  • Jaroslav Nisler
  • David Kopečný
  • Radka Končitíková
  • Marek Zatloukal
  • Václav Bazgier
  • Karel Berka
  • David Zalabák
  • Pierre Briozzo
  • Miroslav Strnad
  • Lukáš Spíchal
Article

Abstract

Key message

Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies.

Abstract

Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

Keywords

Cytokinin oxidase/dehydrogenase Crystal structure Molecular docking Organic synthesis Thidiazuron Cytokinin 

Supplementary material

11103_2016_509_MOESM1_ESM.docx (623 kb)
Supplementary material 1 (DOCX 623 KB)

References

  1. Abad A, Agulló C, Cuñat AC, Jiménez R, Vilanova C (2004) Preparation and promotion of fruit growth in kiwifruit of fluorinated N-phenyl-N′-1,2,3-thiadiazol-5-yl ureas. J Agr Food Chem 52:4675–4683CrossRefGoogle Scholar
  2. Aremu AO, Masondo NA, Sunmonu TO, Kulkarni MG, Zatloukal M, Spichal L, Doležal K, Van Staden J (2014) A novel inhibitor of cytokinin degradation (INCYDE) influences the biochemical parameters and photosynthetic apparatus in NaCl-stressed tomato plants. Planta 240:877–889CrossRefPubMedGoogle Scholar
  3. Aremu AO, Stirk WA, Masondo NA, Plačková L, Novák O, Pěnčík A, Zatloukal M, Nisler J, Spíchal L, Doležal K, Finniea JF, Van Staden J (2015) Dissecting the role of two cytokinin analogues (INCYDE and PI-55) on in vitro organogenesis, phytohormone accumulation, phytochemical content and antioxidant activity. Plant Sci 238:81–94CrossRefPubMedGoogle Scholar
  4. Arima Y, Oshima K, Shudo K (1995) Evolution of a novel urea-type cytokinin: horticultural uses of forchlorofenuron. Acta Hortic 394:75–83CrossRefGoogle Scholar
  5. Arndt F, Rusch R, Stillfried HV (1976) SN49537, a new cotton defoliant. Plant Physiol 57:599Google Scholar
  6. Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ, Kramer MD, Morris RO (2001) Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:378–386CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bricogne G, Blanc E, Brandl M, Flensburg C, Keller P, Paciorek W, Roversi P, Sharff A, Smart OS, Vonrhein C, Womack TO (2011) BUSTER version 2.1.0 Cambridge. Global Phasing Ltd, UKGoogle Scholar
  8. Brownlee BG, Hall RH, Whitty CD (1975) 3-Methyl-2-butenal: an enzymatic degradation product of the cytokinin, N-6-(delta-2 isopentenyl) adenine. Can J Biochem 53:37–41CrossRefPubMedGoogle Scholar
  9. Burch LR, Horgan R (1989) The purification of cytokinin oxidase from Zea mays kernels. Phytochemistry 28:1313–1319CrossRefGoogle Scholar
  10. Chatfield JM, Armstrong DJ (1986) Regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern. Plant Physiol 80:493–499CrossRefPubMedPubMedCentralGoogle Scholar
  11. D’Agostino IB, Deruere J, Kieber JJ (2000) Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol 124(:):1706–1717CrossRefPubMedPubMedCentralGoogle Scholar
  12. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132CrossRefPubMedGoogle Scholar
  13. Eswar N, Webb B, Marti-Renom M, Madhusudhan M, Eramian D, Shen M, Pieper U, Sali A (2006) Comparative protein structure modeling using Modeller. Curr Protoc Bioinform, Chap 5: Unit 5Google Scholar
  14. Frébort I, Šebela M, Galuszka P, Werner T, Schmülling T, Peč P (2002) Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications. Anal Biochem 306:1–7CrossRefPubMedGoogle Scholar
  15. Frébortová J, Galuszka P, Werner T, Schmülling T, Frébort I (2007) Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae. Biol Plantarum 51:673–682CrossRefGoogle Scholar
  16. Galuszka P, Popelková H, Werner T, Frébortová J, Pospíšilová H, Mik V, Köllmer I, Schmülling T, Frébort I (2007) Biochemical characterization and histochemical localization of cytokinin oxidases/dehydrogenases from Arabidopsis thaliana expressed in Nicotiana tabaccum L. J Plant Growth Regul 26:255–267CrossRefGoogle Scholar
  17. Gemrotová M, Kulkarni MG, Stirk WA, Strnad M, Van Staden J, Spíchal L (2013) Seedlings of medicinal plants treated with either a cytokinin antagonist (PI-55) or an inhibitor of cytokinin degradation (INCYDE) are protected against the negative effects of cadmium. Plant Growth Regul 71:137–145CrossRefGoogle Scholar
  18. Goldschmidt H, Bardach B (1892) Zur Kenntniss der Diazoamidokörper. Chem Ber 25:1347–1378CrossRefGoogle Scholar
  19. Hare PD, Van Staden J (1994) Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol 35:1121–1125Google Scholar
  20. Holub J, Hanuš J, Hanke DE, Strnad M (1998) Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul 26:109–115CrossRefGoogle Scholar
  21. Houba-Hérin N, Pethe C, d’Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626CrossRefPubMedGoogle Scholar
  22. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66:125–132CrossRefPubMedPubMedCentralGoogle Scholar
  23. Karplus PA, Diederichs K (2012) Linking crystallographic model and data quality. Science 336:1030–1033CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kopečný D, Pethe C, Šebela M, Houba-Hérin N, Madzak C, Majira A, Laloue M (2005) High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica. Biochimie 87:1011–1022CrossRefPubMedGoogle Scholar
  25. Kopečný D, Šebela M, Briozzo P, Spíchal L, Houba-Hérin N, Mašek V, Joly N, Madzak C, Anzenbacher P, Laloue M (2008) Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor. J Mol Biol 380:886–899CrossRefPubMedGoogle Scholar
  26. Kopečný D, Briozzo P, Popelková H, Šebela M, Končitíková R, Spíchal L, Nisler J, Madzak C, Frébort I, Laloue M, Houba-Hérin N (2010) Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie 92:1052–1062CrossRefPubMedGoogle Scholar
  27. Kopečný D, Končitíková R, Popelka H, Briozzo P, Vigouroux A, Kopečná M, Zalabák D, Šebela M, Skopalová J, Frébort I, Moréra S (2015) Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. FEBS J 283:361–377CrossRefPubMedGoogle Scholar
  28. Kurita K, Matsumura T, Iwakura Y (1976) Trichloromethyl chloroformate. Reaction with amines, amino acids, and amino alcohols. J Org Chem 41:2070–2071CrossRefGoogle Scholar
  29. Laloue M, Fox JE (1989) Cytokinin oxidase from wheat: partial purification and general properties. Plant Physiol 90:899–906CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lomin SN, Krivosheev DM, Steklov MY, Arkhipov DV, Osolodkin DI, Schmülling T, Romanov GA (2015) Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands. J Exp Bot 66:1851–1863CrossRefPubMedPubMedCentralGoogle Scholar
  31. Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, Mercy M, Kopečný D, Majira A, Rogowsky P, Laloue M (2004) Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557CrossRefPubMedGoogle Scholar
  32. Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118CrossRefPubMedGoogle Scholar
  33. Mok MC, Mok, DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T (1982) Cytokinin activity of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron). Phytochem 21:1509–1511CrossRefGoogle Scholar
  34. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333CrossRefPubMedGoogle Scholar
  35. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 16:2785–2791CrossRefGoogle Scholar
  36. Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C (2004) Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16:1365–1377CrossRefPubMedPubMedCentralGoogle Scholar
  37. Nisler J, Zatloukal M, Popa I, Doležal K, Strnad M, Spíchal L (2010) Cytokinin receptor antagonists derived from 6-benzylaminopurine. Phytochem 71:823–830CrossRefGoogle Scholar
  38. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefPubMedGoogle Scholar
  39. Romanov GA, Kieber JJ, Schmülling T (2002) A rapid cytokinin response assay in Arabidopsis indicates a role for phospholipase D in cytokinin signalling. FEBS Lett 515(:):39–43CrossRefPubMedGoogle Scholar
  40. Romanov GA, Spíchal L, Lomin SN, Strnad M, Schmülling T (2005) A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein. Anal Biochem 347:129–134CrossRefPubMedGoogle Scholar
  41. Romanov GA, Lomin SN, Schmülling T (2006) Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay. J Exp Bot 57:4051–4058CrossRefPubMedGoogle Scholar
  42. Schrödinger Release 2014-3 (2014) Maestro, version 9.9. Schrödinger, LLC, New YorkGoogle Scholar
  43. Šmehilová M, Galuszka P, Bilyeu KD, Jaworek P, Kowalska M, Šebela M, Sedlářová M, English JT, Frébort I (2009) Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize. J Exp Bot 60:2701–2712CrossRefPubMedGoogle Scholar
  44. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  45. Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, Schmülling T (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45:1299–1305CrossRefPubMedGoogle Scholar
  46. Stolz A, Riefler M, Lomin SN, Achazi K, Romanov GA, Schmülling T (2011) The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors. Plant J 67:157–168CrossRefPubMedGoogle Scholar
  47. Storoni LC, McCoy AJ, Read RJ (2004) Likelihood-enhanced fast rotation functions. Acta Crystallogr D Biol Crystallogr 60:432–438CrossRefPubMedGoogle Scholar
  48. Suttle JC, Mornet R (2005) Mechanism-based irreversible inhibitors of cytokinin dehydrogenase. J Plant Physiol 162:1189–1196CrossRefGoogle Scholar
  49. Suzuki T, Miwa K, Ishikawa K, Yamada H, Aiba H, Mizuno T (2001) The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant Cell Physiol 42:107–113CrossRefPubMedGoogle Scholar
  50. The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger. LLC, New YorkGoogle Scholar
  51. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem 31:455–461PubMedPubMedCentralGoogle Scholar
  52. Vyroubalová Š, Václavíková K, Turečková V, Novák O, Šmehilová M, Hluska, T, Ohnoutková L, Frébort I, Galuszka P (2009) Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels. Plant Physiol 151:433–447CrossRefPubMedPubMedCentralGoogle Scholar
  53. Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550CrossRefPubMedPubMedCentralGoogle Scholar
  54. Whitty CD, Hall RH (1974) A cytokinin oxidase in Zea mays. Can. J Biochem 52:789–799Google Scholar
  55. Wuts PGM, Greene TW (1991) Greene’s protective groups in organic synthesis, 4th edition, Wiley, New YorkGoogle Scholar
  56. Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T (2001) The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol 42:1017–1023CrossRefPubMedGoogle Scholar
  57. Zalabák D, Galuszka P, Mrízová K, Podlešáková K, Gu R, Frébortová J (2014) Biochemical characterization of the maize cytokinin dehydrogenase family and cytokinin profiling in developing maize plantlets in relation to the expression of cytokinin dehydrogenase genes. Plant Physiol Biochem 74:283–293CrossRefPubMedGoogle Scholar
  58. Zatloukal M, Gemrotová M, Doležal K, Havlíček L, Spíchal L, Strnad M (2008) Novel potent inhibitors of A. thaliana cytokinin oxidase/dehydrogenase. Bioorg Med Chem 16:9268–9275CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Jaroslav Nisler
    • 1
    • 2
    • 3
  • David Kopečný
    • 4
  • Radka Končitíková
    • 4
  • Marek Zatloukal
    • 1
    • 2
  • Václav Bazgier
    • 1
    • 5
  • Karel Berka
    • 5
    • 6
  • David Zalabák
    • 7
  • Pierre Briozzo
    • 8
  • Miroslav Strnad
    • 1
  • Lukáš Spíchal
    • 1
    • 2
  1. 1.Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research (CRH), Institute of Experimental Botany AS CRPalacký UniversityOlomoucCzech Republic
  2. 2.Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  3. 3.Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical TechnologyUniversity of Chemistry and Technology in PraguePragueCzech Republic
  4. 4.Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  5. 5.Department of Physical Chemistry, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  6. 6.Regional Centre of Advanced Technologies and Materials, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  7. 7.Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  8. 8.Institut Jean-Pierre Bourgin, INRA, AgroParisTechUniversité Paris-SaclayVersaillesFrance

Personalised recommendations