Structure–activity relationship of uniconazole, a potent inhibitor of ABA 8′-hydroxylase, with a focus on hydrophilic functional groups and conformation

doi:10.1016/j.bmc.2007.12.019

Bioorganic & Medicinal Chemistry

Volume 16, Issue 6, 15 March 2008, Pages 3141-3152

https://doi.org/10.1016/j.bmc.2007.12.019 Get rights and content

Abstract

The plant growth retardant S-(+)-uniconazole (UNI-OH) is a strong inhibitor of abscisic acid (ABA) 8′-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, stomatal closure, flowering, seed dormancy, and other physiological events. In the present study, we focused on the two polar sites of UNI-OH and synthesized 3- and 2″-modified analogs. Conformational analysis and an in vitro enzyme inhibition assay yielded new findings on the structure–activity relationship of UNI-OH: (1) by substituting imidazole for triazole, which increases affinity to heme iron, we identified a more potent compound, IMI-OH; (2) the polar group at the 3-position increases affinity for the active site by electrostatic or hydrogen-bonding interactions; (3) the conformer preference for a polar environment partially contributes to affinity for the active site. These findings should be useful for designing potent azole-containing specific inhibitors of ABA 8′-hydroxylase.

Graphical abstract

Introduction

Uniconazole [S-(+)-E-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazo-1-yl)-1-penten-3-ol, UNI-OH] is an azole-containing P450 inhibitor developed as a plant growth retardant in the 1980s.1, 2 UNI-OH has since been used as a plant growth regulator in agriculture and horticulture. The main site of action of UNI-OH is suggested to be ent-kaurene oxidase, which catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid,³ biosynthetic precursors of the plant hormone gibberellin (GA). This has prompted researchers to use UNI-OH as a chemical tool inhibiting GA biosynthesis. However, UNI-OH also inhibits brassinosteroid (BR) biosynthesis,4, 5 and alters the level of other plant hormones, such as auxins, cytokinins, ethylene, and abscisic acid (ABA).⁶ Recently, Kitahata et al.⁷ and Saito et al.⁸ revealed that UNI-OH strongly inhibits ABA 8′-hydroxylase, a key enzyme in ABA catabolism (Fig. 1).

ABA is the classical plant hormone involved in stress tolerance, stomatal closure, flowering, seed dormancy, and other physiological events.9, 10, 11 In addition, it was reported recently that ABA functions as an endogenous proinflammatory cytokine in humans.¹² Proper endogenous levels of ABA in plants are cooperatively controlled by biosynthesis, transportation, and catabolic inactivation in response to environmental changes.9, 10, 11 A natural or artificial chemical that perturbs this highly controlled system is promising not only as a chemical probe for the mechanism of ABA action,¹³ but also for its potential agricultural, horticultural, and clinical use. Catabolic inactivation of ABA is mainly controlled by ABA 8′-hydroxylase, which is the cytochrome P450 catalyzing the C8′-hydroxylation of ABA into 8′-hydroxy-ABA and its more stable tautomer phaseic acid, which has much lower hormonal activity than ABA.¹⁴ ABA 8′-hydroxylase was identified as CYP707A1-4 in the model plant Arabidopsis thaliana in 2004,15, 16 and since then CYP707A isozymes have been found in various plants.17, 18, 19, 20 Gene knockdown and overexpression studies suggest that ABA 8′-hydroxylase is a key enzyme controlling ABA concentration during water deficit stress and dormancy.21, 22 UNI-OH inhibits ABA 8′-hydroxylase at a very low concentration: its K_I value was 8 nM in an in vitro assay.⁸ UNI-OH-treated plants have enhanced drought tolerance due to twofold higher accumulation of endogenous ABA than untreated plants.⁸ Thus, UNI-OH is a good inhibitor of ABA 8′-hydroxylase, though it has side effects such as inhibition of GA and BR biosynthesis.

Azole-type inhibitors bind to P450 active sites by both coordinating to the heme-iron atom and interacting with surrounding protein residues. Because heme coordination is a common property of azole-containing inhibitors, their affinity and specificity for individual P450 enzymes depends on structural properties other than the azole group. To develop a specific inhibitor of ABA 8′-hydroxylase by structural modification of UNI-OH, the structure–activity relationship of UNI-OH should be revealed in detail. Kitahata et al. investigated the structure–activity relationship of UNI-OH by focusing on the two lipophilic substituents, the tert-butyl at the 3-position and 4-chlorophenyl group at the 1-position,⁷ revealing that these functional groups are essential for inhibiting ABA 8′-hydroxylase and should not be modified in the lead optimization phase. Other functional groups have not been examined.

In the present study, we focused on the two hydrophilic functional groups of UNI-OH, the aza-nitrogen (N2″) and the hydroxy group at the 3-position. The electrostatic or hydrogen-bonding interactions mediated by these groups can affect not only electrostatic interactions with the active site but also conformational properties. In fact, Katagi et al. reported, based on ¹H NMR and IR analyses, that UNI-OH adopts conformer A in nonpolar or weakly polar solvents (Fig. 2) by the formation of an intramolecular hydrogen bond between the 3-hydroxy and N2″.²³ We designed UNI-X and IMI-X (X = OH, OMe, F, and H) (Fig. 3) as probes to explore the role of the 3-hydroxy and N2″ of UNI-OH in binding to ABA 8′-hydroxylase. The hydroxy group can act as both a donor and an acceptor in a hydrogen bond, whereas the methoxy group can act only as an acceptor. Fluorine can act only as a weaker acceptor than hydroxy and methoxy groups. The aliphatic hydrogen can be neither donor nor acceptor in a conventional hydrogen bond. The introduction of a carbon at the 2″-position can eliminate the intramolecular hydrogen bond. Although UNI-OMe,² UNI-H,²⁴ and IMI-OH²⁵ are known compounds, the detailed effects of all optically pure UNI-X and IMI-X containing these known compounds on ABA 8′-hydroxylase have never been reported. These analogs are expected to have different conformational profiles and different interactions with the active site; thus, a comparison between their structure and inhibitory activity against ABA 8′-hydroxylase can reveal the significance of the hydrophilic moieties of UNI-OH in binding to the active site.

Section snippets

Synthesis of UNI-OH analogs

Commercially available UNI-OH, uniconazole-P (Wako Pure Chemical Industries, Ltd), was a mixture of S-(+)- and R-(−)-UNI-OH at a ratio of 89:11, based on chiral HPLC analysis; that is, the enantiomeric excess was 79%. Optically pure S-(+)-UNI-OH (>99% ee) was obtained by semi-preparatory HPLC with a chiral stationary phase. Only the S-enantiomers were used in this study because S-UNI-OH and its derivatives (e.g., diniconazole) are effective in plants, whereas the R-enantiomers are ineffective

Conclusions

In the present study, we focused on the two polar sites of S-(+)-uniconazole (UNI-OH), a strong inhibitor of ABA 8′-hydroxylase, and synthesized 3- and 2″-modified analogs. Conformational analysis and in vitro enzyme inhibition assays yielded new findings about the structure–activity relationship of UNI-OH: (1) by substituting imidazole for triazole, which increases affinity to the heme iron, we identified a more potent compound, IMI-OH; (2) the polar group at the 3-position increases the

General

Uniconazole-P was purchased from Wako Pure Chemical Industries, Ltd, Osaka, Japan. (+)-ABA was a gift from Toray Industries Inc., Tokyo, Japan. ¹H NMR spectra were recorded with tetramethylsilane as the internal standard using a JEOL JNM-EX270 (270 MHz) and JNM-LA500 (500 MHz) NMR spectrometer. ¹³C NMR and 2D-correlation NMR experiments were recorded using JNM-LA500 (500 MHz) NMR spectrometer. ¹⁹F NMR spectra were recorded with trifluorotoluene as the internal standard using a Bruker Avance 400

Acknowledgments

We thank Toray Industries Inc., Tokyo, Japan, for the gift of (+)-ABA. Part of this research was carried out using an instrument at the Center for Instrumental Analysis of Shizuoka University. This research was supported by a Grant-in-Aid for Scientific Research (No. 18380192) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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^†: Present address: Sagami Chemical Research Center, 2743-1 Hayakawa, Ayase, Kanagawa 252-1193, Japan.

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Structure–activity relationship of uniconazole, a potent inhibitor of ABA 8′-hydroxylase, with a focus on hydrophilic functional groups and conformation

Abstract

Graphical abstract

Introduction

Section snippets

Synthesis of UNI-OH analogs

Conclusions

General

Acknowledgments

J. Biol. Chem.

Bioorg. Med. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Adv. Heterocycl. Chem.

J. Pesticide Sci.

Plant Cell Physiol.

Plant Cell Physiol.

Plant Cell Physiol.

Biosci. Biotechnol. Biochem.

Abscisic acid

Abscisic Acid

Annu. Rev. Plant Physiol. Plant Mol. Biol.

Proc. Natl. Acad. Sci. U.S.A.

Plant J.

Annu. Rev. Plant. Biol.