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Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

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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 IC 50 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.

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Acknowledgments

The authors gratefully acknowledge support through the projects LO1204 and LO1305 from the Ministry of Education, Youth and Sports of the Czech Republic and the grant 15-22322S and 15-19266S from the Czech Science Foundation. The work was also supported by student projects IGA_PrF_2016_028 and IGA_PrF_2016_018 of Palacký University, Olomouc.

Author contributions

J. Nisler, L. Spíchal and M. Strnad designed the research, V. Bazgier and K. Berka performed molecular modeling and docking, J. Nisler and M. Zatloukal synthesized the compounds, J. Nisler, R. Končitíková, D. Zalabák contributed to enzyme kinetics, R. Končitíková, D. Kopečný and P. Briozzo preformed X-ray crystallographic study, J. Nisler and D. Kopečný wrote the paper.

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Authors and Affiliations

  1. Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research (CRH), Institute of Experimental Botany AS CR, Palacký University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic

    Jaroslav Nisler, Marek Zatloukal, Václav Bazgier, Miroslav Strnad & Lukáš Spíchal

  2. Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic

    Jaroslav Nisler, Marek Zatloukal & Lukáš Spíchal

  3. Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, University of Chemistry and Technology in Prague, Technická 5, 166 28, Prague, Czech Republic

    Jaroslav Nisler

  4. Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic

    David Kopečný & Radka Končitíková

  5. Department of Physical Chemistry, Faculty of Science, Palacký University, 17. listopadu 1192/12, 771 46, Olomouc, Czech Republic

    Václav Bazgier & Karel Berka

  6. Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, 17. listopadu 1192/12, 771 46, Olomouc, Czech Republic

    Karel Berka

  7. Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71, Olomouc, Czech Republic

    David Zalabák

  8. Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, 78026, Versailles, France

    Pierre Briozzo

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  1. Jaroslav Nisler
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  2. David Kopečný
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  3. Radka Končitíková
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  4. Marek Zatloukal
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  5. Václav Bazgier
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  6. Karel Berka
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  7. David Zalabák
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  8. Pierre Briozzo
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  9. Miroslav Strnad
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  10. Lukáš Spíchal
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Correspondence to Jaroslav Nisler or David Kopečný.

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Nisler, J., Kopečný, D., Končitíková, R. et al. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase. Plant Mol Biol 92, 235–248 (2016). https://doi.org/10.1007/s11103-016-0509-0

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