A conformationally restricted uniconazole analogue as a specific inhibitor of rice ent-kaurene oxidase, CYP701A6

Article abstract of DOI:10.1016/j.bmcl.2012.03.028

The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8′-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolis

Full text of DOI:10.1016/j.bmcl.2012.03.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3240–3243
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Bioorganic & Medicinal Chemistry Letters
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A conformationally restricted uniconazole analogue as a specific inhibitor
of rice ent-kaurene oxidase, CYP701A6
Yasushi Todoroki a, , Kumi Naiki a, Taku Muramatsu a, Toshiyuki Ohnishi b, Kotomi Ueno c,
⇑
Masaharu Mizutani c, Nobuhiro Hirai d
a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
b Division of Global Research Leaders, Shizuoka University, Shizuoka 422-8529, Japan
c Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
d Graduate School of Agriculture, Kyoto University, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of
ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA
80-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the
P450 active site by both coordinating to the heme-iron atom via an sp2 nitrogen and interacting with
surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI
may result from its small molecular size and flexible conformation that allows it to fit into active sites
differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we
examined inhibitory activities of three types of UNI analogues, which were conformationally constrained,
enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3.
Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI,
whereas it inhibited CYP707A3 less than UNI.
Received 20 January 2012
Revised 29 February 2012
Accepted 7 March 2012
Available online 24 March 2012
Keywords:
Plant growth regulator
P450 inhibitor
ent-Kaurene oxidase
Uniconazole
Ó 2012 Elsevier Ltd. All rights reserved.
S-Uniconazole (UNI) is an azole inhibitor of cytochrome P450.1,2
UNI has since been used as a plant growth regulator in agriculture
and horticulture. The main site of action of UNI is suggested to be
ent-kaurene oxidase (CYP701A), which catalyzes the three-step
oxidation of ent-kaurene to ent-kaurenoic acid (KA),3 a biosyn-
thetic precursor of the plant hormone gibberellin (GA). However,
UNI also inhibits brassinosteroid (BR) biosynthesis4,5 and alters
the level of other plant hormones, such as auxins, cytokinins,
ethylene and abscisic acid (ABA).6 In particular, UNI strongly inhib-
its ABA 80-hydroxylase (CYP707A), a key enzyme in ABA catabolism
(Fig. 1).7,8 S-Diniconazole and paclobutrazol, which have a struc-
ture very similar to S-UNI, also inhibits both CYP701A and
CYP707A.7–9 Because the selectivity for target proteins is very
important to chemical tools for biological research, these azole
compounds are not ideal inhibitors of a specific enzyme.
Azole inhibitors of P450 bind to the target P450 active site by
both coordinating to the heme-iron atom and interacting with sur-
rounding protein residues. Because heme coordination is a com-
mon property of azole inhibitors, their affinity and specificity for
individual P450 enzymes depends on structural properties other
than those of the azole group. UNI may be small and flexible en-
ough to embed itself into various substrate-binding pockets, where
it may adopt a distinct conformation and orientation for different
active sites. Based on this speculation, in previous work, we
generated selective inhibitors of CYP707A by enlarging or conform-
ationally freezing the UNI molecule (Fig. 2).10–12 Here we focused
on the inhibitory activity of these UNI analogues against CYP701A.
Although our previous studies revealed an inhibitory effect on rice
seedling growth,10–12 it did not completely parallel the inhibitory
potency against CYP701A enzymes because the dwarf phenotype
is caused by loss-of-function of various enzymes involved in
biosynthesis or catabolism of plant hormones and other bioactive
molecules. Some genes encoding members of the plant
ent-kaurene oxidase CYP701 family have been reported.13 Among
them, rice CYP701A6 (OsKO2)14 and Arabidopsis CYP701A315,16
were heterologously expressed and their three-step oxidation
activity was verified by in vitro assays. However, no direct
evidence for CYP701A inhibition of UNI and related compounds
has been reported. In this study, we examined the inhibitory effect
of UNI and structural analogues against recombinant rice
CYP701A6 produced in a baculovirus-insect cell expression system
to find a CYP701A inhibitor with higher enzyme specificity than
UNI.
SDS–PAGE analysis of the microsomal fraction prepared from
insect cells infected with baculoviruses encoding full-length
CYP701A6 showed a major band at around 50 kDa, which agrees
with the molecular mass (56 kDa) deduced from the amino acid
⇑
Corresponding author. Tel./fax: +81 54 238 4871.
E-mail address: aytodor@ipc.shizuoka.ac.jp (Y. Todoroki).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.028

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3240–3243
3241
ABA catabolism
HO
O
ABA 8'-hydroxylase
(CYP707A)
OH
OH
CO2H
CO2H
O
S-(+)-ABA
8'-hydroxy-ABA
S-UNI
UFAP2N
GA
ent-kaurene oxidase
(CYP701A)
CO2H
ent-kaurene
KA
GA biosynthesis
Figure 1. UNI is a potent inhibitor of ent-kaurene oxidase (CYP701A) and ABA 80-hydroxylase (CYP707A).
X
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
O
HO
N
O
O
HO
N
N
N
N
N
N
N
N
N
N
N
UFAP2: X = Cl
S-UNI
UFAP1
UFAP3
UFAP2N: X = NO2
Cl
Cl
O
O
N
O
O
HO
O
O
N
N
N
N
N
N
N
IFSP1
IFSP2
IFSP3 (Abz-F1)
Cl
IFAP1
Cl
O
O
HO
HO
N
HO
O
N
N
N
OH
N
N
N
N
IFAP2
DSI-201
DSI-501
DSI-504
Cl
N
N
HO
HO
N
O
HO2C
O
N
N
N
N
OTs
N
DSI-505
Abz-E1
Figure 2. Chemical structures of UNI and the analogues used in this work.

3242
Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3240–3243
m/z 316
Me KA
a
b
c
d
e
no inhibitor
+UNI
+UFAP2
−NADPH
Me KA standard
8.0
8.5
9.0
9.5
10.0
10.5
retention time (min)
Figure 3. Selected ion chromatograms (m/z 316) from GC–MS analysis of the recombinant CYP701A6 conversion of ent-kaurene into KA: (a) in the absence of inhibitors; (b) in
the presence of 0.1 lM UNI; (c) in the presence of 1 lM UFAP2; and (d) in the absence of NADPH; (e) a standard sample of Me KA.
Table 1
Inhibitory activity of UNI and its analogues against recombinant CYP701A6 and CYP707A3
Compound
CYP701A6
CYP707A3
Rice growth
IC50 M)
0.16 0.03
Inhibitiona (%)
IC50 (nM)
58
231 56
153 45
264 26
68 27
Inhibitionb (%)
100
31 10c
KI (nM)
(l
S-UNI
100
100
100
100
100
0
0
0
0
0
9
0
9.9 0.4
d
UFAP2
UFAP2N
IFAP2
—
21
3
3
8
7
40150 18300
161 42
156 80
22
100
0
9.0c
5.4e
DSI-501
95e
The values are averages of at least three independent experiments.
a
Inhibition ratio of compounds (1
Inhibition ratio of compounds (10
See Ref. 10.
l
l
M) in the presence of ent-kaurene (10
lM).
b
c
M) in the presence of S-ABA (5 M).
l
d
e
Not measured.
See Ref. 11.
sequence (Supplementary Fig. S1). The CO difference spectrum of
the reduced microsomes exhibited a prominent peak at 450 nm
(Supplementary Fig. S2). Incubation of the microsomal fraction
with ent-kaurene in the presence of the Arabidopsis P450 reductase
ATR2 and NADPH supplementation yielded KA, detected as the
methyl ester (Me KA), which has a molecular ion of m/z 316 in
153 nM, respectively, which are equivalent to that of UNI
(58 nM) (Table 1). IFAP2 and DSI-501 are relatively potent inhibi-
tors of CYP707A3, although their activity is much less than that
of UNI, whereas UFAP2 and UFAP2N are very poor inhibitors of
CYP707A3; especially, UFAP2N is less effective than UNI by a factor
of 4000 on the basis of the KI values. These results suggest that
UFAP2 and UFAP2N are more specific CYP701A inhibitors than
UNI. Abz-F1 and Abz-E1, which were developed as specific inhibi-
tors of ABA 80-hydroxylase CYP707A, were much less effective than
UNI (Fig. 4), as expected from their poor activity in a rice growth
assay.10,11
UFAP2 and UFAP2N were designed as a conformationally re-
stricted analogue of UNI, based on the speculation that introduc-
tion of rigidity to the structure of UNI may cause an increase in
enzyme specificity if the low enzyme specificity of UNI depends
on a flexible structure.10 Although UFAP2 and UFAP2N inhibited
the elongation of rice seedlings, its activity was much lower than
GC–MS analysis (Fig. 3a). When 0.1
lM UNI was added in the pres-
ence of 10 M ent-kaurene in the same assay system, Me KA was
l
not detected (Fig. 3b). Dose–response analysis revealed that the
IC50 value of UNI was 58 nM (Table 1). This is the first direct evi-
dence that UNI functions as a potent inhibitor of CYP701A activity.
The 14 structural UNI analogues, that were synthesized in our
previous work10–12 except for UFAP2N which is a new compound,
were tested in the CYP701A6 inhibition assay. UFAP2N was pre-
pared in a similar manner10 to UFAP2 (Supplementary Scheme
S1). Activity was evaluated based on the decrease in the amount
of the enzymatic product, KA, caused by addition of a test com-
pound at a concentration one-tenth (1
lM) that of the substrate
that of UNI. The IC50 values were ca. 20
lM for UFAP2 and UFAP2N,
ent-kaurene (10 M). Four compounds, IFAP2, UFAP2, UFAP2N
l
whereas it was 0.16 M for UNI (Table 1), meaning that UFAP2 and
l
and DSI-501, showed 100% activity, whereas the activity of other
analogues was less than 50% (Fig. 4). The IC50 values of UFAP2
and UFAP2N in the CYP701A6 inhibition assay were 231 and
UFAP2N are 100-fold less effective than UNI. It is not clear why
there is a discrepancy in the activity in vitro and in vivo. The very
strong retardant effect of UNI might be caused from combined

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3240–3243
3243
(No. 22580118) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
S-UNI
UFAP1
UFAP2
Supplementary data
UFAP2N
UFAP3
Supplementary data (the procedures for preparation of a new
compound UFAP2N, enzyme expression, enzyme assay, and biolog-
ical assays) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.028.
IFSP1
IFSP2
NI
NI
IFSP3 (Abz-F1)
IFAP1
References and notes
IFAP2
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Inhibition ratio (%)
Figure 4. Activity of UNI analogues (1
lM) in the CYP701A6 inhibition assay
(10 M ent-kaurene). NI: no measurable inhibition at the tested concentration.
l
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Acknowledgments
We thank Professor Tomonobu Toyomasu (Yamagata Univer-
sity) for kindly providing ent-kaurene. We thank Professor Akira
Kitajima (Kyoto University) for kindly providing rice (Nipponbare)
seeds. We thank Toray Industries Inc., Tokyo, Japan, for a 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