A Chemical Genetics Strategy that Identifies Small Molecules which Induce the Triple Response in Arabidopsis.

To explore small molecules with ethylene-like biological activity, we conducted a triple response-based assay system for chemical library screening. Among 9600 compounds, we found N-[(1,3,5-trimethyl-1H-pyrazol-4-yl)methyl]-N-methyl-2-naphthalenesulfonamide (EH-1) displayed promising biological activity on inducing a triple response in Arabidopsis seedlings. Chemical synthesis and structure-activity relationship (SAR) analysis of EH-1 analogues with different substitution patterns on the phenyl ring structure of the sulfonamide group indicated that 3,4-dichloro-N-methyl-N-(1,3,5-trimethyl-1H-pyrazol-4-yl-methyl) benzenesulfonamide (8) exhibits the most potent biological activity. To determine the mechanism of action, we conducted RNA sequencing (RNA-Seq) analysis of the effect of EH-1 and 1-aminocyclopropane-1-carboxylate (ACC), the precursor of ethylene biosynthesis, following the quantitative real-time polymerase chain reaction (RT-PCR) confirmation. Data obtained from RNA-Seq analysis indicated that EH-1 and ACC significantly induced the expression of 39 and 48 genes, respectively (above 20 fold of control), among which five genes are up-regulated by EH-1 as well as by ACC. We also found 67 and 32 genes that are significantly down-regulated, respectively, among which seven genes are in common. For quantitative RT-PCR analysis. 12 up-regulated genes were selected from the data obtained from RNA-Seq analysis. We found a good correlation of quantitative RT-PCR analysis and RNA-Seq analysis. Based on these results, we conclude that the action mechanism of EH-1 on inducing triple response in Arabidopsis is different from that of ACC.

Fenarimol, a Pyrimidine-Type Fungicide, Inhibits Brassinosteroid Biosynthesis.

The plant steroid hormone brassinosteroids (BRs) are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz), the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM), a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 µM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL) but not by gibberellin (GA). Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 µM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.

Asymmetric synthesis and effect of absolute stereochemistry of YCZ-2013, a brassinosteroid biosynthesis inhibitor.

The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2-propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding optically pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisation of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indicated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity with IC50 values of approximately 24 ± 3 and 24 ± 2 nM, respectively. The IC50 values of the trans isomers of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 ± 50 and 3900 ± 332 nM, respectively. Co-application of brassinolide (10nM), the most potent BR, and GA3 (1 µM) to Arabidopsis seedlings grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the induced dwarfism of Arabidopsis seedlings, whereas GA3 showed no effect.

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis.

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure-activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G(2)) is a highly selective inhibitor of BR biosynthesis, with an IC(50) value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.

Synthesis of novel brassinosteroid biosynthesis inhibitors based on the ketoconazole scaffold.

Brassinosteroids (BRs) are steroidal plant hormones that control several important agronomic traits such as plant architecture, seed yield, and stress tolerance. Inhibitors that target BR biosynthesis are candidate plant growth regulators. We synthesized novel triazole derivatives, based on the ketoconazole scaffold, that function as inhibitors of BR biosynthesis. The biological activity of the test compounds was evaluated by determining their ability to induce dwarfism in Arabidopsis seedlings grown in the dark. The chemically induced dwarfism of Arabidopsis seedlings was further evaluated by a rescue experiment using the co-application of brassinolide and/or gibberellins (GA). The structure-activity relationship studies revealed a potent BR biosynthesis inhibitor, 2RS, 4RS-1-{2-(4-chlorophenyl)-4-[2-(2-ethoxyphenyl)-ethyl]-1,3-dioxolan-2-ylmethyl}-1H-1,2,4-triazole (7m), with an IC(50) value of 0.10±0.03 µM for retardation of Arabidopsis seedling stem elongation. The compound-induced hypocotyl dwarfism was counteracted by the co-application of 10nM brassinolide, but not 1 µM GA(3), which produced seedlings that resembled BR-deficient mutants. This result suggests that 7m is a potent and specific inhibitor of BR biosynthesis.

Synthesis of 2RS,4RS-1-[2-phenyl-4-[2-(2-trifluromethoxy-phenoxy)-ethyl]-1,3-dioxolan-2-yl-methyl]-1H-1,2,4-triazole derivatives as potent inhibitors of brassinosteroid biosynthesis.

Brassinosteroids are important phytohormones that affect many aspects of plant growth and development. In order to manipulate brassinosteroid levels in plant tissues by using specific biosynthesis inhibitors, we have carried out a systemic search for specific inhibitors of brassinosteroid biosynthesis. Synthesis of triazole derivatives based on the ketoconazole scaffold revealed a series of novel brassinosteroid biosynthesis inhibitors (the YCZ series). To explore the structure-activity relationships of this synthetic series, we now report the synthesis of new triazole derivatives with different aromatic structures at position 2 of 1,3-dioxolane skeleton. We found that the variation of aromatic substituent significantly affect the inhibitory potency. Structure-activity relationships studies indicated that 4-chlorophenyl analogue is the most potent inhibitor of BR biosynthesis with an IC50 value approximately 0.12 ± 0.04 µM, while a bulky biphenyl group exhibited a great negative effect on promoting the inhibitory potency with an IC50 larger than 10 µM.

Synthesis of fluorescently labeled pyrazole derivative induceing a triple response in Arabidopsis seedlings.

A fluorescent labeled pyrazole derivative with a dansyl moiety (EH-DF) was synthesized. Design of EH-DF was carried out by using a dansyl moiety to substitute the naphthalene moiety of the parent compound (EH-1). At a concentration of 30 µM, EH-DF displayed biological activity on inducing a triple response in Arabidopsis seedlings. Compared with the non-chemical treated control, the hypocotyl length of EH-DF-treated Arabidopsis seedlings was reduced from approximately 9.2±0.7 mm to 2.4±0.2 mm. The length of the roots was reduced from 1.7±0.1 mm to 1.0±0.1 mm, and the curvature of the hook of Arabidopsis seedlings increased from 60±16 degrees to 245±35 degrees. The maxim excitation wavelength and emission wavelength of EH-DF were 350 and 535 nm, respectively. Data obtained via fluorescent microscope analysis indicated that intensive fluorescent signals of EH-DF were observed in the shoot of Arabidopsis seedlings.

Synthesis and structure-activity relationships of new pyrazole derivatives that induce triple response in Arabidopsis seedlings.

Twenty-seven analogues of pyrazole derivatives were synthesized and subjected to structure-activity relationship studies on inducing the triple response in Arabidopsis seedlings. We found that 3,4-Dichloro-N-methyl-N-[(1-allyl-3,5-dimethyl-1H-pyrazol-4-yl)methyl]benzenesulfonamide (C26) exhibits potent activity on inducing the triple response in Arabidopsis seedlings. C26 (10 µM) induced an exaggerated apical hook in Arabidopsis seedlings. The curvature of the hook of the Arabidopsis seedlings was found to be 300±23 degrees, while ethephon (10 µM), a prodrug of ethylene, and a non-chemically treated control were found to be 128±19 and 58±16 degrees, respectively. C26 also exhibited potent activity on reducing stem elongation. The hypocotyl length of Arabidopsis seedlings treated with C26 (10 µM) was found to be 0.25±0.02 cm, while those of ethephon-treated (10 µM) and treated controls were found to be 0.69±0.06 and 1.15±0.01 cm, respectively. C26 displayed potency inhibiting the root growth of Arabidopsis seedlings similar to that of ethephon.

Asymmetric synthesis and stereochemical structure-activity relationship of (R)- and (S)-8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester, a potent inhibitor of allene oxide synthase.

The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were purified by chiral high-performance liquid chromatography (HPLC) to afford pure (R)-JM-8686 and (S)-JM-8686. The inhibitory activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition potency of (R)-JM-8686 is approximately 200 times greater than that of (S)-JM-8686, with IC(50) values of approximately 5+/-0.2 nM and 950+/-18 nM, respectively. The dissociation constants of (R)-JM-8686 and (S)-JM-8686 with respect to the recombinant allene oxide synthase were approximately 1.4+/-0.3 microM and 4.8+/-0.6 microM, respectively.

Protodioscin, Isolated from the Rhizome of Dioscorea tokoro Collected in Northern Japan is the Major Antiproliferative Compound to HL-60 Leukemic Cells.

BACKGROUND: The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. METHODS: The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. RESULTS: PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. CONCLUSION: We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.

Characterization of novel imidazole derivative, JM-8686, a potent inhibitor of allene oxide synthase.

The inhibitory properties of a first synthetic jasmonic acid biosynthesis inhibitor, JM-8686, were investigated. Steady-state kinetic analysis indicates that the compound is a competitive inhibitor of allene oxide synthase (AOS) with a K(i) value of approximate 0.62+/-0.15 microM. Dialysis experiment indicates that AOS inactivation by JM-8686 is reversible. The optical difference spectroscopy analysis of JM-8686 and AOS interaction indicates that JM-8686 induced type II binding spectra with a K(d) value of approximate 1.6+/-0.2 microM, suggesting that JM-8686 binds to the prosthetic heme iron of AOS. Comparison of the inhibitory potency of the compound against HPL (CYP74B) from tomato revealed that JM-8686 was a highly selective inhibitor for AOS.

In vitro and in vivo evidence for the inhibition of brassinosteroid synthesis by propiconazole through interference with side chain hydroxylation.

We carried out the biochemical evaluation of the target site of propiconazole in BR biosynthesis. Applying BR biosynthesis intermediates to Arabidopsis seedlings grown in the presence of propiconazole under dark condition, we found that the target site of propiconazole in BR biosynthesis can be identified among the C22 and C23 side chain hydroxylation steps from campestanol to teasterone. Using differential spectra techniques to determine the binding affinity of propiconazole to CYP90D1, which is responsible for C23 hydroxylation of BR, we found that propiconazole induced typical type II binding spectra in response to purified recombinant CYP90D1 and the Kd value was found approximately 0.76 µM.

Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice.

Lamina joint inclination or leaf angle (the angle between the leaf blade and vertical culm) is a major trait of rice plant architecture. The plant hormone brassinosteroid (BR) is the main regulator of this trait, while other plant hormones, including ethylene, gibberellin, and auxin, also influence leaf angle. In this study, we found that methyl jasmonate (MeJA) also participates in regulating lamina joint inclination. MeJA decreased lamina joint inclination and inhibited the BR-induced increase in lamina joint inclination. Furthermore, addition of a BR synthesis inhibitor increased the extent of change in lamina joint inclination in response to treatment with a low concentration of MeJA (0.05 or 0.5mgL(-1)), but it did not alter the lamina joint inclination of plants treated with a high concentration of MeJA (5mgL(-1)). Further studies showed that MeJA treatment significantly repressed the expression of BR biosynthesis-related genes and decreased endogenous BRs levels. In addition, the lamina joint inclination in the OsBRI1 mutant d61-1 was less sensitive to MeJA compared with its wild type counterpart, and lithium chloride-induced inactivation of GSK3-like kinase, a negative regulator of BR signaling, partly rescued the MeJA-induced reduction in lamina joint inclination. Further studies showed that MeJA treatment reduced the mRNA levels of BR signaling and target genes. These results indicate that MeJA-inhibition of lamina joint inclination may depend on BR biosynthesis and the BR signaling pathway.

YCZ-18 is a new brassinosteroid biosynthesis inhibitor.

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 µM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation.

A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark.

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed.

New Compounds Induce Brassinosteroid Deficient-like Phenotypes in Rice.

Brassinosteroids (BRs) are steroidal plant hormones with potent plant growth promoting activity. Because BR-deficient mutants of rice exhibit altered plant architecture and important agronomic traits, we conducted a systemic search for specific inhibitors of BR biosynthesis to manipulate the BR levels in plant tissues. Although previous studies have been conducted with BR biosynthesis inhibitors in dicots, little is known regarding the effects of BR biosynthesis inhibition in monocot plants. In this work, we used potent inhibitors of BR biosynthesis in Arabidopsis, and we performed a hydroponic culture of rice seedlings to evaluate the effects of BR biosynthesis inhibition. Among the test compounds, we found that 1-[[2-(4-Chlorophenyl)-4-(phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (1) is a potent inhibitor that could induce phenotypes in rice seedlings that were similar to those observed in brassinosteroid deficient plants. The IC50 value for the retardation of plant growth in rice seedlings was approximately 1.27 ± 0.43 µM. The IC50 value for reducing the bending angle of the lamina joint was approximately 0.55 ± 0.15 µM.

Enzymatic synthesis of novel branched sugar alcohols mediated by the transglycosylation reaction of pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47.

Transglycosylation reactions are useful for preserving a specific sugar structure during the synthesis of branched oligosaccharides. We have previously reported a panosyl unit transglycosylation reaction by pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47 (Tonozuka et al., Carbohydr. Res., 1994, 261, 157-162). The acceptor specificity of the TVA II transglycosylation reaction was investigated using pullulan as the donor and sugar alcohols as the acceptor. TVA II transferred the α-panosyl unit to the C-1 hydroxyl group of meso-erythritol, C-1 and C-2 of xylitol, and C-1 and C-6 of d-sorbitol. TVA II differentiated between the sugar alcohols' hydroxyl groups to produce five novel non-reducing branched oligosaccharides, 1-O-α-panosylerythritol, 1-O-α-panosylxylitol, 2-O-α-panosylxylitol, 1-O-α-panosylsorbitol, and 6-O-α-panosylsorbitol. The Trp(356)→Ala mutant showed similar transglycosylation reactions; however, panose production by the mutant was 4.0-4.5-fold higher than that of the wild type. This suggests that Trp(356) is important for recognizing both water and the acceptor molecules in the transglycosylation and the hydrolysis reaction.

Design and synthesis of novel imidazole derivatives as potent inhibitors of allene oxide synthase(CYP74).

Allene oxide synthase (AOS) is a key enzyme in the oxylipin pathway in plants leading to jasmonic acid and other jasmonates (JAs), important signal mediators of defense signal networks in plants. AOS uses hydroperoxylinolenic acid as an oxygen donor as well as the substrate, thus the biochemical conversion of 13(S)-hydroperoxylinolenic acid to allene oxide can proceed in the absence of oxygen and NADPH. We have designed the synthesized of a series of novel imidazole derivatives and tested them in a bioassay as AOS inhibitors using a purified recombinant AOS enzyme isolated from Arabidopsis and expressed in E. coli. Among the derivatives prepared, heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]octanoate (k) was found to be the most potent inhibitor, with an IC(50) of 10+/-5 nM, which is 250,000-fold and 1,000,000-fold more potent than the known AOS inhibitors, acetylsalicyclic acid (2.5 mM) and ketoconazole (10 mM), respectively.