Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein

Article abstract of DOI:10.1016/j.bmcl.2019.01.010

Strigolactones (SLs) are a class of plant hormones that regulate shoot branching as well as being known as root-derived signals for parasitic and symbiotic interactions. The physical interaction between SLs and the DWARF14 (D14) receptor family can be examined by differential scanning fluorimetry (DSF) that monitors the changes in protein melting temperature (Tm). The Tm of D14 is lowered by bioactive SLs in DSF analysis. In this report, we screened the compounds that lower the Tm of Arabidopsis D14 (AtD14) as potential candidates for SL agonists using DSF analysis. Subsequent physiological analyzes revealed that 113D10 acts as a novel SL agonist in a D14-dependent manner. Intriguingly, 113D10 has a chemical structure different from natural SLs in that it does not possess an enol ether bond that connects to a methylbutenolide moiety. Moreover, 113D10 does not stimulate seed germination of root parasitic plants. Accordingly, 113D10 can be a useful tool for SL studies and agricultural applications.

Full text of DOI:10.1016/j.bmcl.2019.01.010

Accepted Manuscript
Chemical screening of novel strigolactone agonists that specifically interact with
DWARF14 protein
Rei Yasui, Yoshiya Seto, Shinsaku Ito, Kojiro Kawada, Kaori Itto-Nakama,
Kiyoshi Mashiguchi, Shinjiro Yamaguchi
PII:
S0960-894X(19)30010-1
https://doi.org/10.1016/j.bmcl.2019.01.010
BMCL 26247
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2018
6 January 2019
10 January 2019
Please cite this article as: Yasui, R., Seto, Y., Ito, S., Kawada, K., Itto-Nakama, K., Mashiguchi, K., Yamaguchi,
S., Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein, Bioorganic
& Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.01.010
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Title
Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein
Author names and affiliations
Rei Yasuia, Yoshiya Setoa,b, Shinsaku Itoc, Kojiro Kawadac, Kaori Itto-Nakamaa, Kiyoshi
Mashiguchia, Shinjiro Yamaguchia,d*
aDepartment of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai, 980-8577, Japan
bDepartment of Agricultural Chemistry, School of Agriculture, Meiji University, 1-1-1 Higashi-mita,
Tama-ku, Kawasaki-shi, Kanagawa, 214-8571, Japan
cDepartment of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku,
Tokyo, 156-8502, Japan
dInstitute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan
*Corresponding author
Shinjiro Yamaguchi
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan
TEL: +81-774-38-3231; Fax: +81-774-38-3229
E-mail: shinjiro@scl.kyoto-u.ac.jp
Keywords: Strigolactone; Agonist; Chemical screening; Receptor.
Abstract
Strigolactones (SLs) are a class of plant hormones that regulate shoot branching as well as being
known as root-derived signals for parasitic and symbiotic interactions. The physical interaction
between SLs and the DWARF14 (D14) receptor family can be examined by differential scanning
fluorimetry (DSF) that monitors the changes in protein melting temperature (Tm). The Tm of D14 is
lowered by bioactive SLs in DSF analysis. In this report, we screened the compounds that lower the
Tm of Arabidopsis D14 (AtD14) as potential candidates for SL agonists using DSF analysis.
Subsequent physiological analyzes revealed that 113D10 acts as a novel SL agonist in a
D14-dependent manner. Intriguingly, 113D10 has a chemical structure different from natural SLs in
that it does not possess an enol ether bond that connects to a methylbutenolide moiety. Moreover,
113D10 does not stimulate seed germination of root parasitic plants. Accordingly, 113D10 can be a
useful tool for SL studies and agricultural applications.

Strigolactones (SLs) were originally identified from plant root exudates as seed germination
stimulants of root parasitic plants (Fig. 1)1. Subsequently, SLs have been shown to be symbiotic
signals for arbuscular mycorrhizal fungi which allow land plants to effectively absorb mineral
nutrition, such as inorganic phosphate, from the soil2. In addition to these functions as
allelochemicals in the rhizosphere, now SLs are known as endogenous plant hormones that regulate
diverse aspects of plant development such as outgrowth of axillary shoots, root development and leaf
senescence3-7.
In plants, SLs are perceived by the DWARF14 (D14) family protein [rice; Oryza sativa D14
(OsD14), Arabidopsis; Arabidopsis thaliana D14 (AtD14), petunia; DECREASED APICAL
DOMINANCE2 (DAD2), pea; RAMOSUS3 (RMS3)]8-11. This family belongs to the /-hydrolase
superfamily and possesses a conserved catalytic triad (Ser, His, and Asp) necessary for catalyzing the
hydrolysis reaction12. In fact, D14 can hydrolyze SLs into ABC-ring (ABC-formyltricyclic lactone:
ABC-FTL) and D-ring (hydroxymethylbutenolide: HMB) parts in vitro9,11,13-15. On the other hand,
both biochemical and genetic studies have shown that D14 plays a critical role in the SL perception
in several plant species8,9. Until now, the physiological importance of the hydrolase activity of D14
has been discussed. More recently, two groups reported that a covalently linked hydrolytic
intermediate, which is derived from the D-ring of SLs, may have an important role to trigger SL
signaling11,15
.
As for the interaction between D14 and SLs, a significant finding has been reported using
differential scanning fluorimetry (DSF) that enables us to evaluate the protein-ligand interaction by
measuring the changes in the protein melting temperature (Tm)16. Generally, receptor proteins are
stabilized in the presence of their ligands, and their Tm values shift to a higher temperature16. In
contrast, the Tm of D14 is lowered by the addition of bioactive SLs in DSF analysis, suggesting that
SLs induce the structural destabilization of D149,11,14,17. This transition of protein state is considered
to be important for the SL signal transduction. Based on these findings, it was presumed that the SL
agonists showing SL-like biological activity can reduce the Tm of D14 as does SLs. Therefore, in
this study, we performed the initial screening using DSF analysis to find novel SL agonists.
Most of the SL agonists which have been synthesized so far contain the D-ring or a D-ring-like
moiety18,19. Here, a small-molecule chemical library consisting of 10,000 compounds was used as the
chemical resource to obtain novel SL agonists which do not have such structures. If we obtain novel

SL agonists that are not hydrolyzed by D14, it may help to elucidate the mechanism for SL
perception and signaling. First, the interaction between each compound from the chemical library
and the recombinant AtD14 protein was examined using DSF analysis (Fig. 2; 1st step). Through this
screening, we visually evaluated the DSF data and selected 111 compounds that are capable of
lowering the Tm of AtD14. Subsequently, we examined whether they competitively inhibit
hydrolysis activity of AtD14 in order to confirm that these compounds can interact with the binding
pocket of AtD14 in which the catalytic residues are located (Fig. 2; 2nd step). 5-deoxystrigol (5DS,
Fig. 1), a naturally occurring SL, was incubated together with each of 111 compounds, and then
hydrolysis of 5DS was evaluated by quantifying a hydrolytic product, ABC-FTL, using LC-MS/MS.
In this step, in order to avoid excluding promising candidates, compounds with weak inhibitory
activity were also selected for further analysis. As a result, we found that 38 compounds decreased
the ABC-FTL production (20% to 85%). In the next screening, we checked whether these 38
compounds lowered the Tm of AtD14 in a concentration-dependent manner to further narrow down
the candidates (Fig. 2; 3rd step). As a result, 19 compounds were confirmed to lower the Tm of
AtD14 in a concentration-dependent manner. These compounds do not have a common or SL-like
structure, suggesting that they may interact with AtD14 in various manners. The list of 19
compounds that passed the third screening step is shown in Fig. S1.
SLs are known to inhibit hypocotyl elongation of Arabidopsis20,21. Both AtD14 and its paralog,
HYPOSENSITIVE TO LIGHT (HTL)/KARRIKIN INSENSITIVE2 (KAI2), are independently involved
in this inhibition by SLs because the atd14 and kai2 mutants are sensitive to exogenously applied
SLs, but the atd14 kai2 double mutant is completely resistant to the exogenously applied SLs10,22,23
Thus, we finally examined whether 19 candidates inhibit hypocotyl elongation in Arabidopsis (Fig.
2; 4th step). Each compound was exogenously applied to the seedlings of wild type (WT) and the
atd14-2, kai2-4 and atd14-2 kai2-4 mutants. In this assay, a synthetic SL analog, GR24 (a racemic
mixture, Fig. 1), inhibited hypocotyl elongation of WT and the atd14-2 and kai2-4 mutants, but not
the atd14-2 kai2-4 double mutant as was reported previously10,22. Among 19 compounds, we found
that only one compound, N-(3,4-dichlorophenyl)-4-trifluoromethyl-2-thiazolamine (113D10, Fig.
3A), inhibited hypocotyl elongation of WT and the kai2-4 mutant, but not the atd14-2 and atd14-2
.

kai2-4 mutants. On the other hand, other 18 compounds did not inhibit hypocotyl elongation in
Arabidopsis. These results suggested that 113D10 seems to act specifically in the AtD14 pathway.
Moreover,113D10 inhibited Arabidopsis hypocotyl elongation in a concentration-dependent manner.
A possible reason for the difference in the inhibitory activity between GR24 and 113D10 would be
that 113D10 is only accepted by AtD14, while both enantiomers of GR24 are independently
perceived by AtD14 or KAI222. Through these screenings, 113D10 was chosen for the subsequent
analyzes as a good candidate for a novel SL agonist. Interestingly, 113D10 does not have the D-ring
moiety found in natural SLs (Fig. 1 and 3A). The characteristics of 113D10 are summarized in Fig.
3.

Next, we examined whether 113D10 inhibits shoot branching of Arabidopsis. MORE AXILLARY
GROWTH4 (MAX4) encodes CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) which is
involved in SL biosynthesis. The Arabidopsis CCD8-defective max4-8 mutant shows an increased
shoot branching phenotype that is rescued by SL treatment3,4. As shown in Fig. 4A, exogenously
applied 113D10 slightly but significantly decreased the number of axillary branches of the max4-8
mutant. The activity of 113D10 was lower than that of GR24 as observed in the inhibition assay of
hypocotyl elongation. Moreover, we found that the atd14-2 mutant is resistant to 113D10 also in this
assay (Fig. 4B), supporting the idea that 113D10 is a D14-specific SL agonist in Arabidopsis.
Next, we biochemically characterized 113D10. At first, we performed a fluorescence-based
competition assay using a pro-fluorescent SL analog, Yoshimulactone Green (YLG)24. When YLG is
hydrolyzed by D14, the fluorescent molecule is released, and this reaction is competitively inhibited
by SLs24. In this assay, the half maximal inhibitory concentration (IC50) of GR24 and 113D10 for
AtD14-mediated YLG hydrolysis was calculated to be 1.5 M and 2.4 M, respectively (Fig. 5A).
We also tested whether 113D10 is hydrolyzed by D14. GR24 or 113D10 was added to the reaction
solution containing AtD14 protein, and their hydrolysis was examined. The hydrolysis of 113D10
was not observed even though the equimolar of GR24 was almost completely hydrolyzed after 4h
(Fig. 5B). Although no significant difference was observed between IC50GR24 and IC50113D10 (Fig. 5A),
113D10 shows lower biological activities than GR24 in the Arabidopsis assays (Fig. 3D and Fig. 4).
It is possible that 113D10 cannot fully induce the structural change of AtD14 responsible for the
formation of the signaling complex with other components such as MAX2 and SMAX1-LIKE
proteins though it can interact with AtD14 effectively25. Another possibility is that 113D10 cannot be

transported to the site of action efficiently unlike naturally occurring SLs, or is immediately
metabolized to inactive form(s) in plants.
We further synthesized 113D10 derivatives in order to increase its biological activity. Eighteen
derivatives were synthesized and their inhibitory effect on Arabidopsis WT hypocotyl elongation was
evaluated (Fig. S2). Most of the derivatives lost the biological activity, but compound 8 and 9, which
have mono- and dibromo substituents in the aromatic ring, respectively, inhibited hypocotyl
elongation (Fig. S3). The inhibitory activity of compound 8 for hypocotyl elongation was
AtD14-dependent, and slightly stronger than 113D10 (Fig. 6A, B and S4A). In addition, compound 8
also suppressed shoot branching of the max4-8 mutant, but not the atd14-2 mutant (Fig. 6C and S4B).
These results suggest that the introduction of Br at the 3-position of benzene ring of 113D10 slightly
affects its activity.
Finally, we examined whether 113D10 acts as an SL agonist in rice, a model plant of monocots.
113D10 clearly lowered the Tm of OsD14 as well as that of AtD14 in a concentration-dependent
manner in DSF analysis (Fig. S5A). However, when 113D10 was exogenously applied to dwarf10-2
(d10-2), a rice CCD8-defective mutant, it could not rescue its excess branching phenotype (Fig. S5B).
In rice, SLs are also known to inhibit mesocotyl elongation dependently on both OsD14 and the
F-box protein, DWARF3 (D3)26,27. On the other hand, D14LIKE, a rice KAI2 ortholog, is supposed
to regulate mesocotyl length independently of the SL pathway28. When the rice WT and the d14-1N
and d3-2 mutants were grown in the presence of GR24 or 113D10, GR24 inhibited mesocotyl
elongation of WT and the d14-1N mutant, but not the d3-2 mutant. Because a relatively high
concentration of rac-GR24 was used in this assay, it is likely that GR24 inhibited mesocotyl
elongation partially via the D14LIKE pathway in the d14-1N mutant. On the other hand, 113D10
inhibited mesocotyl elongation of only WT, but not the d14-1N mutant (Fig. S5C). These results
suggest that 113D10 inhibits mesocotyl elongation in an OsD14- and D3-dependent manner.

Biological activity-based chemical screening is often time-consuming, while DSF analysis which
was used for the initial screening in this study is convenient and high throughput because
approximately 100 samples can be analyzed within 1 h at the same time. After the initial screening,
through several biological and biochemical assays, we successfully discovered a novel SL agonist,
113D10. Moreover, in a structure-activity relationship study, we found its active derivatives,
compound 8 and 9. These three compounds may be used to reveal the SL signaling mechanism by
D14 because 113D10 is stable and is hardly hydrolyzed by AtD14. Recently, it was proposed that the
formation of a covalent link between the hydrolyzed D-ring part and the catalytic residues of AtD14
protein is a key step in transducing the SL signal15. Because 113D10 was not hydrolyzed by AtD14
(Fig. 5B), our findings suggest that the SL signal can be at least weakly transmitted through AtD14
without hydrolysis of the ligand. In addition, intriguingly, 113D10 and compound 8 did not show
seed germination-stimulating activity against the root parasitic plants, Striga hermonthica and
Orobanche minor, even at 100 M (Fig. 7). Therefore, these novel SL agonists may be used for

agricultural applications, for example, to improve crop yield by controlling the branch number
without stimulating seed germination of root parasites.
Acknowledgments
We thank Prof. Hiroyuki Kasahara for providing chemicals. We also thank Prof. Hirokazu Arimoto
for his help in preparation of 113D10 derivatives. This work was supported by Grant-in-Aid for JSPS
Research Fellow Grant Number JP17J00463, MEXT KAKENHI (grant number 17H06474) and the
Core Research for Evolutional Science and Technology (CREST) by JST (grant number
JPMJCR13B1).
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The melting temperature curves of AtD14
178
28
― control
― addition of a compound
-122
Reducing the melting temperature
-272
25
45
65
85
H
Cl
Cl
N
Temperature (oC)
S
10,000 compounds
Chemical library
N
Screening
CF3
Novel strigolactone agonist