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.

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

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

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

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

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

Institut Jean Pierre Bourgin INRA AgroParisTech Université Paris Saclay 78026 Versailles France

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

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

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