title: Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase.
abstract: Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. 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.
authors.affiliation: 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@gmail.com.|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@gmail.com.|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@gmail.com.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Nisler
authors.affiliation: 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.kopecny@upol.cz.
authors.forename: David
authors.initials: D
authors.lastname: Kopečný
authors.affiliation: 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.
authors.forename: Radka
authors.initials: R
authors.lastname: Končitíková
authors.affiliation: 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.|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.
authors.forename: Marek
authors.initials: M
authors.lastname: Zatloukal
authors.affiliation: 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.|Department of Physical Chemistry, Faculty of Science, Palacký University, 17. listopadu 1192/12, 771 46, Olomouc, Czech Republic.
authors.forename: Václav
authors.initials: V
authors.lastname: Bazgier
authors.affiliation: Department of Physical Chemistry, Faculty of Science, Palacký University, 17. listopadu 1192/12, 771 46, Olomouc, Czech Republic.|Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, 17. listopadu 1192/12, 771 46, Olomouc, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Berka
authors.affiliation: 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.
authors.forename: David
authors.initials: D
authors.lastname: Zalabák
authors.affiliation: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, 78026, Versailles, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Briozzo
authors.affiliation: 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.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.affiliation: 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.|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.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Spíchal
mesh_terms: D003583:Cytokinins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010671:Phenylurea Compounds / Q000737:chemistry*; D013830:Thiadiazoles / Q000737:chemistry*
chemical_list: D003583:Cytokinins; D004791:Enzyme Inhibitors; D010671:Phenylurea Compounds; D013830:Thiadiazoles; C016785:thidiazuron; D010088:Oxidoreductases; C019984:cytokinin oxidase
keywords: Crystal structure; Cytokinin; Cytokinin oxidase/dehydrogenase; Molecular docking; Organic synthesis; Thidiazuron