Sun-Young Han

A Novel Inhibitor of 9-cis-Epoxycarotenoid Dioxygenase in Abscisic Acid Biosynthesis in Higher Plants

Abscisic acid (ABA) is a major regulator in the adaptation of plants to environmental stresses, plant growth, and development. In higher plants, the ABA biosynthesis pathway involves the oxidative cleavage of 9-cis-epoxycarotenoids, which may be the key regulatory step in the pathway catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We developed a new inhibitor of ABA biosynthesis targeting NCED and named it abamine (ABA biosynthesis inhibitor with an amine moiety). Abamine is a competitive inhibitor of NCED, with a Ki of 38.8 μm. In 0.4 m mannitol solution, which mimics the effects of osmotic stress, abamine both inhibited stomatal closure in spinach (Spinacia oleracea) leaves, which was restored by coapplication of ABA, and increased luminescence intensity in transgenic Arabidopsis containing the RD29B promoter-luciferase fusion. The ABA content of plants in 0.4 m mannitol was increased approximately 16-fold as compared with that of controls, whereas 50 to 100 μm abamine inhibited about 50% of this ABA accumulation in both spinach leaves and Arabidopsis. Abamine-treated Arabidopsis was more sensitive to drought stress and showed a significant decrease in drought tolerance than untreated Arabidopsis. These results suggest that abamine is a novel ABA biosynthesis inhibitor that targets the enzyme catalyzing oxidative cleavage of 9-cis-epoxycarotenoids. To test the effect of abamine on plants other than Arabidopsis, it was applied to cress (Lepidium sativum) plants. Abamine enhanced radicle elongation in cress seeds, which could be due to a decrease in the ABA content of abamine-treated plants. Thus, it is possible to think that abamine should enable us to elucidate the functions of ABA in cells or plants and to find new mutants involved in ABA signaling.

5,5′-Substituted Indirubin-3′-oxime Derivatives as Potent Cyclin-Dependent Kinase Inhibitors with Anticancer Activity

To enhance the ability of indirubin derivatives to inhibit CDK2/cyclin E, a target of anticancer agents, we designed and synthesized a new series of indirubin-3-oxime derivatives with combined substitutions at the 5 and 5 positions. A molecular docking study predicted the binding of derivatives with OH or halogen substitutions at the 5 position to the ATP binding site of CDK2, revealing the critical interactions that may explain the improved CDK2 inhibitory activity of these derivatives. Among the synthesized derivatives, the 5-nitro-5-hydroxy analogue 3a and the 5-nitro-5-fluoro analogue 5a displayed potent inhibitory activity against CDK2, with IC(50) values of 1.9 and 1.7 nM, respectively. These derivatives also showed antiproliferative activity against several human cancer cell lines, with IC(50) values of 0.2-3.3 microM. A representative analogue, 3a, showed greater than 500-fold selectivity for CDK relative to selected kinase panel and potent in vivo anticancer activity.

Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to DWF4 protein in the brassinosteroid biosynthesis pathway.

Triadimefon (Bayleton), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14 alpha-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.

Brz220 Interacts with DWF4, a Cytochrome P450 Monooxygenase in Brassinosteroid Biosynthesis, and Exerts Biological Activity

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.

Discovery of aminopyridines substituted with benzoxazole as orally active c-Met kinase inhibitors.

We report the synthesis and biological evaluation of aminopyridines substituted with benzoxazole. The SAR of the aminopyridines was explored to improve the inhibitory activity against c-Met and to decrease hERG affinity. These studies led to the discovery of amide 24 which showed good c-Met inhibitory potency, low affinity to hERG and favorable pharmacokinetic properties in rats.

Indirubin derivatives as potent FLT3 inhibitors with anti-proliferative activity of acute myeloid leukemic cells

Indirubin derivatives were identified as potent FLT3 tyrosine kinase inhibitors with anti-proliferative activity at acute myeloid leukemic cell lines, RS4;11 and MV4;11 which express FLT3-WT and FLT3-ITD mutation, respectively. Among several 5 and 5-substituted indirubin derivatives, 5-fluoro analog, 13 exhibited potent inhibitory activity at FLT3 (IC(50)=15 nM) with more than 100-fold selectivity versus 6 other kinases and potent anti-proliferative effect for MV4;11 cells (IC(50)=72 nM) with 30-fold selectivity versus RS4;11 cells. Cell cycle analysis indicated that compound 13 induced cell cycle arrest at G(0)/G(1) phase in MV4;11 cells.

Brz220 a novel brassinosteroid biosynthesis inhibitor: stereochemical structure–activity relationship

The four stereoisomers of Brz220 (2RS,4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole), a novel brassinosteroid biosynthesis inhibitor, were separated by silica gel column chromatography and chiral high-performance liquid chromatography (HPLC). The absolute configuration of each stereoisomer was determined by a combination of asymmetric synthesis and NMR analysis. The effects of these stereoisomers on cress stem elongation clearly indicate that the (2S)-isomer has the greatest inhibitory activity.

N-Benzylideneaniline and N-Benzylaniline are Potent Inhibitors of Lignostilbene-α,β-dioxygenase, a Key Enzyme in Oxidative Cleavage of the Central Double Bond of Lignostilbene

Lignostilbene-α,β-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and β-carotene 15,15′-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 µM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 µM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.

Design and synthesis of lignostilbene-α, β-dioxygenase inhibitors

Abstract Lignostilbene-α,β-dioxygenase cleaves the olefinic double bond of phenolic stilbenes by a mechanism similar to that of 9- cis -epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis. Several analogues of stilbene were designed and synthesized, and their efficacy as inhibitors of lignostilbene-α,β-dioxygenase was examined. The compound ( Z )-1-(4-hydroxyphenyl)-1-fluoro-2-phenylethene ( 2 ) was found to be a potent inhibitor of this enzyme with an IC 50 of 3 μM.

Characterization of the Indirubin Derivative LDD970 as a Small Molecule Aurora Kinase A Inhibitor in Human Colorectal Cancer Cells

Aurora kinase A plays an essential role in mitosis including chromosome separation and cytokinesis. Aberrant expression and activity of Aurora kinase A is associated with numerous malignancies including colorectal cancer followed by poor prognosis. The aim of this study is to determine the inhibitory effects of LDD970, an indirubin derivative, on Aurora kinase A in HT29 colorectal cancer cells. In vitro kinase assay revealed that, LDD970 inhibited levels of activated Aurora kinase A (IC50=0.37 mM). The inhibitory effects of LDD970 on Aurora kinase A, autophosphorylation and phosphorylation of histone H3 (Ser10), were confirmed by immunoblot analysis. Moreover, LDD970 inhibited migration of HT29 cells and upregulated apoptosis-related protein cleaved PARP. In cell viability assay, LDD970 was observed to suppress HT29 cell growth (GI50=4.22 µM). Although further studies are required, results of the present study suggest that LDD970 provide a valuable insight into small molecule indirubin derivative for therapeutic potential in human colorectal cancer.