Mechanism of Herbicidal Action and Rice Selectivity of Iptriazopyrid: A Novel Azole Carboxamide-Based Inhibitor of 4‑Hydroxyphenylpyruvate Dioxygenase

Abstract

4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are widely used as herbicides. However, the emergence of herbicide-resistant weeds necessitates the development of new herbicides with more diverse chemical structures. Therefore, we evaluated the herbicidal and HPPD inhibitory activities of iptriazopyrid, a novel azole carboxamide compound. Phytotoxic tests on Echinochloa crus-galli demonstrated that iptriazopyrid caused chlorosis at a concentration approximately 10 times lower than that required for the common commercial HPPD inhibitor mesotrione. Furthermore, iptriazopyrid showed strong selectivity for Oryza sativa over the weed in the greenhouse-scale experiment. X-ray crystallography and in vitro inhibitory assays revealed that iptriazopyrid was bound to the enzyme active pocket of HPPD and worked as a slow-binding inhibitor. These findings indicate that this inhibitor has potent herbicidal activity with a chemical structure different from those of existing HPPD inhibitors. Thus, it has potential applications as a novel scaffold in herbicide development.

Introduction

Biochemical reactions arising from specific molecular interactions are the foundation for plant growth and response. For example, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is essential for the biosynthesis of plastoquinone (PQ), which is used in photosynthetic electron transport in plants. − This Fe­(II)-dependent nonheme oxygenase catalyzes the conversion of 4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid (HGA) in the tyrosine metabolic pathway. ,,− Moreover, HGA is a precursor for the biosynthesis of PQ and tocopherol in plants. ,,, In contrast, the inhibition of HPPD activity prevents the biosynthesis of PQ and carotenoid, and thus causes chlorosis and plant death. ,, Therefore, HPPD inhibitors are widely used as Group 27 herbicides, as determined through the Herbicide Resistance Action Committee (HRAC) Mode of Action Classification.

Drug design that enhances binding affinity to the target HPPD enzyme is essential for achieving high selectivity and enabling low dosage of the inhibitor. This can be facilitated by (1) metal-ion coordination to the active site and (2) molecular design to improve the geometric fitness of the catalytic pocket. Furthermore, drug metabolism and pharmacokinetics in plants should be considered to facilitate herbicidal activity only against weeds, not major grains. HPPD inhibitors that have been developed to date are primarily classified into three categories according to the 2024 HRAC Global Herbicide MOA Classification: − triketones such as mesotrione, , pyrazolones such as topramezone, and isoxazole such as isoxaflutole. Flusulfinam is a newly developed azole carboxamide, which has not been included in this classification. Among these HPPD inhibitors, mesotrione is one of the most commonly used in maize crops, with annual sales exceeding $400 million. , Despite the successful use of mesotrione, there remains a growing demand for herbicides with more diverse chemical structures to target a wider range of plant species and address herbicide resistance.

In this study, we aimed to evaluate the herbicidal activity of iptriazopyrid (3-[(isopropylsulfonyl)­methyl]-N-(5-methyl-1,3,4-oxadiazol-2-yl)-5-(trifluoromethyl)-[1,2,4]­triazolo­[4,3-a]­pyridine-8-carboxamide; developmental code: NC-656) (Figure ), a newly designed HPPD inhibitor, in vivo and in vitro. Iptriazopyrid, an azole carboxamide compound, is not classified into any three types of HPPD inhibitors described above. In addition to the distinct chemical framework, iptriazopyrid is being developed for the first HPPD inhibitor for paddy rice available for foliar application in contrast to all the well-known others such as pyrazolynate, benzobicyclone, tefuryltrione, and fenquinotrione used for in-water treatment. , To determine whether this compound is effective against weeds and selective for rice crops, we compared its phytotoxicity with that of mesotrione for Echinochloa crus-galli, a typical weed in paddy rice cultivation, and Oryza sativa, and analyzed compound metabolism mechanisms inside these plants. We further aimed to identify its direct binding to HPPD enzyme by X-ray crystallography and in vitro kinetic analyses. To our knowledge, this study is the first to present the crystal structure of Oryza OsHPPD and to compare it with that of Arabidopsis thaliana AtHPPD, which is commonly used for X-ray crystallography in studies on HPPD inhibitors. − ,,, Our findings demonstrate that this novel selective HPPD inhibitor designed for rice crops has a phytotoxicity profile comparable to that of mesotrione.

1.

Phytotoxic effects of mesotrione and iptriazopyrid in E. crus-galli in test tubes. The chemical structures of mesotrione and iptriazopyrid are indicated in the upper row.

Materials and Methods

Chemicals

Iptriazopyrid (NC-656) was synthesized by Nissan Chemical Corporation following the synthetic scheme in the patent WO2014192936. The plant metabolites of iptriazopyrid, namely, 656Ox-1 [5-methyl-1,3,4-oxadiazol-2-amine], HGA, and mesotrione, were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Plant Materials

O. sativa “Koshihikari” and E. crus-galli seeds were sown and grown inside a greenhouse (natural light, 35 °C/25 °C; relative humidity, approximately 80%) on paddy soil in a 15 cm² plastic pot with an appropriate amount of water.

Phytotoxic Effects of Iptriazopyrid in E. crus-galli and Reversal by HGA

Iptriazopyrid, mesotrione, and HGA were prepared in acetone solution and added to 0.5% agarose without sucrose in glass tubes. The concentrations of iptriazopyrid and mesotrione were 10^–5, 10^–6, and 10^–7 M and 10^–4, 10^–5, and 10^–6 M, respectively. HGA (10^–3, 5 × 10^–4, and 10^–4 M) was added to analyze the reversal of phytotoxic effects. Ten E. crus-galli seeds were sown on the agarose medium, covered with aluminum lids, and incubated under continuous light (photon flux density 400 μmol·m^–2·s^–1) in a growth chamber at 25 °C. Pictures of the test tubes were taken 7 days after treatment.

Absorption and Metabolism

Treatments were applied at the three-leaf growth stage to both plant species. The application solution was adjusted by diluting an iptriazopyrid 10% suspension concentrate (SC) formulation with water to 100 ng μL^–1. Ten droplets (1 μL each) of this solution were applied using a micropipette onto the second leaves of O. sativa and E. crus-galli. The doses of iptriazopyrid were equivalent to a field rate of approximately 15 g a.i. ha^–1 in a 150 L carrier. In all cases, the solutions contained the adjuvant Tween20 (400 ng μL^–1). Furthermore, the plants were harvested 6, 24, and 48 h after treatment. Unabsorbed iptriazopyrid was collected by rinsing treated leaves with 10 mL acetonitrile. The rinse and plant materials were then stored at −20 °C for subsequent analysis.

The rinse was filtrated with a 0.45 μm PTFE membrane filter for liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plant materials were homogenized in 10 mL of acetonitrile/water (80/20, by volume) using a POLYTRON (KINEMATICA) and centrifuged at 4000 rpm for 5 min. Supernatants were separated, and the pellets were re-extracted with 10 mL of acetonitrile/water (80/20, by volume) on a shaker for 30 min at room temperature (25 °C). After centrifugation, the first and second extracts were pooled and filtered with a 0.45 μm PTFE membrane filter for LC-MS/MS analysis.

LC-MS/MS analyses were performed on a Prominence Modular HPLC (Shimadzu Corporation) coupled with an LSMS-7060 (Shimadzu Corporation). Next, samples were loaded on a Waters ACQUITY UPLC HSS T3 (1.8 μm; 2.1 × 100 mm) reverse phase column for iptriazopyrid analysis. The column was held at 40 °C and a flow rate of 0.25 mL min^–1. The mobile phase for the T3 column was 0.1% formic acid/acetonitrile (component A/component B) and gradient elution was performed under the following conditions: A/B = 80:20 (initial), 50/50 (3 min), 5:95 (3–4 min), and 80:20 (4–7 min). Each sample was loaded on a TOSO H TSK gel amide (3 μm; 2.0 × 150 mm) column (40 °C; flow rate = 0.3 mL min^–1) to analyze metabolite 656Ox-1. The mobile phase for the amide column was 5 mM ammonium formate aq./acetonitrile (3/97, isocratic). Multiple reaction monitoring methods in positive ion mode were used to detect iptriazopyrid (m/z 432.80 > 326.15) and 656Ox-1 (m/z 99.90 > 83.05). The lower limit of quantification was fixed at 0.1% of the treatment, i.e., 1 ng, regardless of plant weight.

Plasmid Construction

DNA fragments for HPPD genes were amplified from the genomic DNA of Arabidopsis thaliana and O. sativa using PCR to construct expression plasmids for the HPPD proteins. Then, DNA fragments were inserted into the plasmid pETGS carrying poly histidine-tag, green fluorescent protein, and bdSUMO at the N-terminus. Sequences are listed in the Supporting Information.

Protein Expression and Purification

AtHPPD and OsHPPD were expressed in the transformed Escherichia coli strain BL21­(DE3) with pETGS-AtHPPD and pETGS-OsHPPD plasmids, respectively. The overnight starter cultures were diluted 100-fold into LB broth (Lennox; Merck, Darmstadt, Germany) medium supplemented with 10 μg mL^–1 kanamycin. The cells were incubated at 37 °C until A₆₀₀ reached 0.7, and protein expression was induced with 0.1 mM isopropyl β-d-thiogalactopyranoside. They were then incubated overnight at 18 °C, harvested, and lysed with Lysis10 buffer (50 mM Tris (pH 8.0), 500 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT, and 1 mM AEBSF protease inhibitor) using the Digital Sonifier 250D Advanced homogenizer (Branson, CT). Next, the homogenized lysates were centrifuged. The supernatants were applied to a Ni-NTA column (QIAGEN N. V., Venlo, Netherlands) and eluted with Lysis10 buffer supplemented with 500 mM imidazole. The buffer of eluted protein solutions was exchanged with BufferA (10 mM Tris–HCl (pH8.0) and 500 mM NaCl) using gel filtration chromatography (Akta pure 25, Cytiva, MA). The N-terminus of SUMO tag was cleaved off through incubation with 0.2 μM poly histidine-tagged SENP1 overnight at 4 °C. The untagged HPPD proteins were collected from the flow-through fraction of the Ni-NTA column and further purified using a gel filtration column (HiLoad 16/600 Superdex 200 pg; GE Healthcare, Chicago, IL) with BufferA. The fraction containing HPPD was diluted with 10 mM Tris-HCl (pH 8.0) buffer and further concentrated with Amicon Ultra 10 kDa. The purified HPPD proteins were stored at −80 °C.

To prepare human homogentisate 1,2-dioxygenase (HGD), the Lenti-X 293T (Takara Bio, Shiga, Japan) human cell line was grown to 50% confluency in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum in a 6-well plate and transfected with 2.5 μg pCMV6-HGD (OriGene, Rockville, MD) using TransIt-X2 (Takara Bio) according to the manufacturer’s instructions. The cells were collected after 2 days, washed with PBS, and lysed with 100 μL of RIPA buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40). We subsequently collected supernatants after centrifuging the cell lysate at 4 °C for 10 min at 15,000 rpm. The supernatants estimated to contain HGD > 100 ng μL^–1 were stored as an HGD mixture at −80 °C immediately before use.

Crystallization and Structure Determination

The crystals of AtHPPD-iptriazopyrid, OsHPPD, and OsHPPD-iptriazopyrid grew via hanging drop vapor diffusion at 20 °C for 1–5 weeks. Moreover, a protein mixture containing 10 mg mL^–1 (210 μM) HPPD, 2 mM iptriazopyrid, and 2 mM CoCl₂ was mixed with an equal volume of the reservoir solution; 0.1 M sodium citrate (pH 5.0), 32 or 28 vol % poly­(ethylene glycol) (PEG) 400, and 15 or 10 vol % 2-propanol for AtHPPD-iptriazopyrid; 0.1 M sodium acetate (pH 4.5), 20 vol % PEG1000, and 0.2 M zinc acetate for OsHPPD; and 0.1 M sodium citrate (pH 5.0), 12 weight% PEG6000, and 50 mM sodium iodide for OsHPPD-iptriazopyrid. Iptriazopyrid and CoCl₂ were added into the protein solution at ten times the molar concentration of proteins.

The crystal was mounted into a cryo-loop and flash-frozen in liquid nitrogen, with cryoprotectant comprising 10% glycerol for OsHPPD-iptriazopyrid or without cryoprotectant for the others. X-ray diffraction was measured at the BL-1A beamline in the Photon Factory in KEK (Tsukuba, Japan). The diffraction data were obtained using XDS (Table S2). The initial phase was determined through molecular replacement with PHASER module of the CCP4 program package using 1SP9 as the search model. Refinement and model building were performed using the REFMAC5 and COOT modules in CCP4 (Refinement statistics are shown in Table S2). The coordinates and structure factor data were deposited in PDB under accession ID 9KOY, 9KOZ, and 9KP0. Structure figures were drawn using PyMol.

HPPD Activity Assay and Inhibitory Kinetics

The coupled enzyme assays used to evaluate the inhibitory activity of iptriazopyrid and mesotrione on HPPD were performed as previously described. , Inhibitors were dissolved in DMSO at 1 mM to prepare the stock solution. The assay was carried out in a final volume of 50 μL in a 96-well microplate (UV-Star, Half Area, Greiner Bio-One GmbH, Austria) at 25 °C. The reaction mixture contained 50 mM HEPES (pH 7.0), 100 μM FeCl₂, 2 mM sodium ascorbate, HPPA (Tokyo Chemical Industry Co., Ltd., Japan), and excess HGD mixture (2% in volume). The inhibitor solutions were added to the reaction mixture at a final concentration of 0–800 nM. Absorbance was measured at 318 nm using an Infinite M Plex (Tecan Group Ltd., Switzerland) to monitor maleylacetoacetate (MAA) synthesis. , The absorbance of the reaction mixture containing inhibitors was measured for 5 min to obtain a background absorbance. Subsequently, HPPD was added at a final concentration of 100 nM to initiate the reaction. The time courses of the absorbance were monitored for 25 min until all curves reached a steady state. We confirmed that neither iptriazopyrid nor mesotrione affected the activity of HGD. The absorbance was converted to MAA concentration based on the Beer–Lambert law, e ₃₃₀ = 13500 M^–1 cm^–1 and the light path length (0.318 cm). ,

Equation shows the reaction scheme of competitive slow-binding HPPD inhibitors. Based on the literature, ,, we obtained the kinetic parameters from the experimental data by fitting them to eqs – based on a nonlinear regression analysis, step by step. The concentration of the product (P) can be described by eq

$$[\mathrm{P}]=v_{\mathrm{s}}t+\frac{v_0-v_{\mathrm{s}}}{k_{\mathrm{obs}}}(1-{\mathrm{e}}^{-k_{\mathrm{obs}}t})$$ 1

where S, E, and I are the substrate, enzyme, and inhibitor, respectively. v ₀ and v _s are the initial and steady-state reaction velocities. K _obs is the observed first-order rate constant and shows a linear response to the concentrations of inhibitors, as described in eq

$$k_{\mathrm{o}\mathrm{b}\mathrm{s}}=A[\mathrm{I}]+B$$ 2

A and B are the apparent rate constants. The association and dissociation rate constants K ₊₀ and K _–0 can be calculated from these constants using eqs and

$$A=\frac{K_{+0}{K}_{\mathrm{M}}}{K_{\mathrm{m}}+[\mathrm{S}]}$$ 3

$$B=K_{-0}$$ 4

The Michaelis–Menten constants (K _m) obtained using the Lineweaver–Burk plots (eq ) were 44.04 and 40.02 μM for AtHPPD and OsHPPD, respectively.

$$\frac{1}{v_0}=\left(\frac{K_{\mathrm{m}}}{v_{\max }}\right)\frac{1}{[\mathrm{S}]}+\frac{1}{v_{\max }}$$ 5

The time course of MAA synthesis was monitored at various concentrations of HPPDs in the absence of inhibitors for 5 min, with a 10-s interval, to obtain v ₀ by linear fitting. We obtained the inhibitory constant (K _i) of mesotrione and iptriazopyrid on AtHPPD and OsHPPD, respectively, as an average of three individual measurements. These are listed in Table with standard deviation.

1. Inhibitory Constant (K _i) of Mesotrione and Iptriazopyrid on AtHPPD and OsHPPD.

		Ki/nM
AtHPPD	mesotrione	22.1 ± 4.5
	iptriazopyrid	24.3 ± 0.3
OsHPPD	mesotrione	26.5 ± 10.8
	iptriazopyrid	33.3 ± 3.3

Results and Discussion

Phytotoxic Effects in E. crus-galli

We first compared the phytotoxic effects of iptriazopyrid and mesotrione against E. crus-galli, a monocotyledonous weed typically found in paddy rice cultivation. The top row of Figure shows photos of E. crus-galli 7 days after treatment with various concentrations of iptriazopyrid or mesotrione in test tubes. Iptriazopyrid caused chlorosis at 1 μM, which is approximately 10-times lower than the mesotrione concentration (Figure ). To quantitatively compare the phytotoxicity of iptriazopyrid and mesotrione to E. crus-galli, IC₅₀ was determined in greenhouse-scale experiments (Figure S1 and Table S1). The IC₅₀ of iptriazopyrid was 6.3 g ai ha^–1, 15-times lower than that of mesotrione (100 g ai ha^–1). These results indicate that iptriazopyrid is more phytotoxic to E. crus-galli than mesotrione. In addition, we compared the IC₅₀ of iptriazopyrid and mesotrione to O. sativa. Iptriazopyrid showed high selectivity to O. sativa, compared to E. crus-galli, with a selectivity index (Si) > 64 (Table S1). This selectivity means lower herbicidal activity on specific crops. In contrast, mesotrione was less selective for O. sativa, with an Si of 4, and inhibited the growth of O. sativa at higher concentrations than 200 g ai ha^–1. This suggests that the herbicidal activity of iptriazopyrid is highly selective for O. sativa. These characteristics are expected to offer a new approach to weed management.

We further evaluated the recovery from phytotoxicity by adding various concentrations of HGA, the product of HPPD, to confirm that the herbicidal effect of iptriazopyrid is attributed to HPPD inhibition. As shown in Figure , 1000 μM HGA considerably restored E. crus-galli growth, even with 10-times higher iptriazopyrid or mesotrione concentrations than their effective chlorosis-causing concentrations. These results indicate that iptriazopyrid induces phytotoxicity by inhibiting the HPPD pathway even at concentrations an order of magnitude lower than those of mesotrione in E. crus-galli.

Metabolite Analysis of Iptriazopyrid in O. sativa and E. crus-galli

We analyzed the metabolites of iptriazopyrid after treatment of O. sativa and E. crus-galli to clarify why the herbicidal activity of iptriazopyrid is highly selective for O. sativa. Figure shows the residual amounts of iptriazopyrid (b) on the leaf surface or (c) within the plants, along with (d) the amounts of 656Ox-1 in the plants. 656Ox-1 was not detected from the leaf surface of either plant. As shown in Figure b, the residual amounts of iptriazopyrid on the O. sativa leaf surface were comparable to those in E. crus-galli. Contrastingly, the residual amounts of iptriazopyrid within O. sativa were approximately 10-times lower than those in E. crus-galli (Figure c). In addition, 50–100 ng of 656Ox-1 was detected within O. sativa, whereas none was detected within E. crus-galli at any time points (Figure d). These results indicate that O. sativa has a high drug-metabolizing activity and promptly detoxifies iptriazopyrid, resulting in the high selectivity of iptriazopyrid to this plant.

2.

Time courses of iptriazopyrid and metabolite 656Ox-1 amounts in E. crus-galli and O. sativa. Plants at the third leaf stage were foliar-treated with 1000 ng iptriazopyrid (SC formulation). (a) The total amounts of iptriazopyrid remaining on the leaf surface, iptriazopyrid within the plant, and 656Ox-1 within the plant. (b) The amount of iptriazopyrid remaining on the leaf surface. (c) The amount of iptriazopyrid within the plant. (d) The amount of 656Ox-1 within the plant. The amount of 656Ox-1 was converted to iptriazopyrid equivalents based on the molecular weight ratio of iptriazopyrid to 656Ox-1. All data are expressed as the mean ± standard deviation (N = 3).

Structural Basis of Iptriazopyrid Inhibition

We performed X-ray crystallography to further validate the effect of iptriazopyrid on HPPDs at the atomic resolution. We first determined the crystal structures of AtHPPD-iptriazopyrid complexes at 2.15 Å resolution (Figure a). Co-crystal structures of AtHPPD have been determined with various herbicides, including mesotrione. − ,,, In the absence of substrates or inhibitors, the Fe ion required for HPPD enzymatic reactions is located at the catalytic center and coordinates with glutamine, glutamate, and two histidine residues. , In addition, the a-helix at the C-terminus lids the cavity to introduce substrates from solvent for the oxidative reaction. ,

3.

Structural comparison of the AtHPPD-iptriazopyrid and OsHPPD-iptriazopyrid complexes. (a, b) Molecular structure of iptriazopyrid and its corresponding omit map (contoured at 2.0 σ). (c, d) Key residues surrounding iptriazopyrid.

In the AtHPPD-mesotrione structure, two oxygens on the diketocyclohexane-triketone moiety coordinate to the metal ion replaced with an inert cobalt­(II) in the crystal. This structure further shows strong hydrophobic bonds via the aromatic ring, and the α-helix lid is in an open state (Figure S2). The crystal structure of the AtHPPD-iptriazopyrid complex revealed that iptriazopyrid occupies the catalysis-active pocket of AtHPPD, similar to other HPPD inhibitors, although it has a different scaffold from others. − ,,, One of the nitrogen atoms in the azole carboxamide (N5), the oxygen in the amide (O2), a water molecule, and three amino acid residues (H226, H308, and E394) coordinated to the metal ion to form a typical six-coordinate octahedral geometry (Figure c). The exocyclic methyl group of iptriazopyrid interacted with P280 and F419 at 3.3 Å, and the endocyclic nitrogen that was not coordinated with the Co was located within close distance (3.7 Å) from H226. The π−π stacking interactions of the triazolopyridine bicyclic-ring with F381 and F424 were observed in this study.

In the AtHPPD-mesotrione structure, F424 was located >4 Å from the monocyclic ring because of its structure (Figure S2b). This suggests that the bicyclic ring contributes to the stable interaction. Furthermore, the exocyclic CF3-group contacted with CD2 of L368 within 3.7 Å. The CF3 structure may work as a hydrophobic patch. In contrast, electrostatic interaction (or hydrogen bond) between one oxygen on the (isopropylsulfonyl)­methyl and Q293 (3.7A) is not likely to have contributed to the stability (see below). Additionally, the isopropyl moiety is located in parallel to F392 and forms the π-sigma hydrophobic bond. Overall, these results suggest that iptriazopyrid binds to the active pocket of AtHPPD and inhibits enzymatic activity. The results further demonstrate that the structural features of iptriazopyrid, including the bicyclic ring, contribute to its interaction with AtHPPD. In contrast, flusulfinam, possessing a monocyclic ring with chirality, has not shown stacking interactions with F424 in the simulation model.

We also compared the effect of iptriazopyrid on the crystal structures of OsHPPD and AtHPPD. To shed light on the structural changes of OsHPPD caused by iptriazopyrid, we determined the crystal structures of OsHPPD and OsHPPD-iptriazopyrid complexes at 2.00 Å and 2.90 Å resolution, respectively (Figures S3 and d). As OsHPPD has a considerably similar amino acid sequence to that of AtHPPD (Figure S4), we assumed that the two proteins had similar conformational features. No major structural change was observed between the OsHPPD and OsHPPD-iptriazopyrid structures, except in the C-terminal π-helix, which showed an open conformation with iptriazopyrid similar to that of AtHPPD. Figure shows the detailed structural comparison of (b) AtHPPD- and (d) OsHPPD-iptriazopyrid. A lot of common characteristics were observed in the interaction of both cocrystal structures.

The interactions of iptriazopyrid with P277 (P280 in At), F416 (F419), H223 (H226), F378 (F381), F421 (F424), L365 (L368), and Q290 (Q293) on OsHPPD and the coordination with the Co ion were similar to the interaction between AtHPPD and iptriazopyrid. (Note that all residues are conserved in two enzymes.) The detailed contact distances are summarized in Table S3. However, the isopropyl moiety located far from the metal rotated c.a. 120° around the sulfonyl group in the OsHPPD-iptriazopyrid structure. The isopropyl moiety was distant from L332­(M335) in OsHPPD but was located near M335 in AtHPPD. This leucine replacement was also found in Echinochloa (Figure S4). Consequently, the isopropyl moiety appears to have rotated to be closer to Q290 (Q293) rather than F389 (F392). Furthermore, F392 but not Q293 interacts with the isopropyl moiety in AtHPPD. In addition, the oxadiazole of iptriazopyrid was located at 3.5 Å from F250 (F253) but far from K418­(K421) in OsHPPD and was oppositely located at 3.3 Å from K421 but far from F253 in AtHPPD. This suggests that the two interactions are interchangeable and mutually compensate for each other. Therefore, it became clear that the interaction between AtHPPD- and OsHPPD-iptriazopyrid was remarkably similar, despite the differences observed.

Inhibitory Activity of Iptriazopyrid against HPPD In Vitro

The major interactions in the crystal structures of AtHPPD– iptriazopyrid and OsHPPD– iptriazopyrid were essentially the same, although iptriazopyrid showed selectivity to O. sativa in vivo. Thus, we performed in vitro inhibition assays based on the coupled enzyme method and compared the inhibitory activity of iptriazopyrid against AtHPPD and OsHPPD with that of mesotrione to elucidate the inhibitory mechanisms of iptriazopyrid against HPPD at the molecular level. Figure shows the progress curves of MAA synthesis from 100 μM HPPA in the presence of various concentrations of inhibitors.

4.

Inhibitory kinetics of AtHPPD and OsHPPD by mesotrione and iptriazopyrid. Progress curves of MAA synthesis from 100 μM HPPA in the presence of various concentrations of inhibitors. The plots and solid lines indicate data and fitting curves, respectively.

As the reaction progress curves for AtHPPD and OsHPPD with iptriazopyrid displayed typical curvilinear functions of slow-binding inhibitors, including mesotrione (Figure ), we evaluated the inhibitory constant K _i using nonlinear regression analysis (Table ). The assay was carried out in triplicates with 100, 125, 150, and 175 μM HPPA (Figure and S5). The K _i value of iptriazopyrid against AtHPPD was 24.3 nM and was comparable to that of mesotrione (22.1 nM). In addition, the K _i values of iptriazopyrid and mesotrione against OsHPPD were at the same level (33.3 and 26.5 nM, respectively). However, no remarkable differences were observed in the inhibitory activity of iptriazopyrid between AtHPPD and OsHPPD. These results suggest that iptriazopyrid has a comparable HPPD inhibitory activity to that of mesotrione despite its unique chemical structure.

Binding affinity attributed to the association and dissociation rates in the active pocket of a target enzyme is important in herbicide design. Therefore, numerous commercialized herbicides that work as slow-binding inhibitors have been developed. However, the emergence of weeds resistant to existing herbicides is an inevitable problem, resulting in a growing demand for new herbicides with more diverse chemical structures. Despite its chemical structure being completely different from those of previously reported HPPD inhibitors, iptriazopyrid worked as a slow-binding inhibitor against both AtHPPD and OsHPPD, and the in vitro K _i was comparable with that of mesotrione.

In summary, we showed that iptriazopyrid, a novel azole carboxamide compound, acted as an inhibitor of HPPD both at a molecular and plant level. We determined the cocrystal structures of AtHPPD-iptriazopyrid and OsHPPD-iptriazopyrid and found that iptriazopyrid occupies the catalytic pocket of AtHPPD, which is similar to the behavior of other HPPD inhibitors (Figure ). − ,,, Comparisons with OsHPPD revealed that many interactions are species-independent. To our knowledge, this is the first study showing the crystal structure of OsHPPD, which may also provide insights into the selectivity. In vitro inhibitory kinetics of iptriazopyrid against AtHPPD- and OsHPPD revealed that the progress curve of MAA synthesis displayed typical curvilinear function of slow-binding inhibitors ,, (Figure ). In addition, calculated K _i values were comparable to those of mesotrione (Table ). These results indicate that iptriazopyrid acts as a promising slow-binding HPPD inhibitor comparable to mesotrione.

At the plant level, iptriazopyrid caused chlorosis of E. crus-galli at a concentration 10-times lower than that required for mesotrione (Figure ). IC₅₀ measurements in greenhouse-scale experiments showed that iptriazopyrid was highly selective for O. sativa (Table S1). Metabolite analysis revealed that O. sativa rapidly degraded iptriazopyrid, in contrast to E. crus-galli (Figure ). These results imply that the selectivity of iptriazopyrid for O. sativa is due to the differences in metabolism rather than the difference in molecular structure of HPPDs. Several studies about degradation products of HPPD inhibitors, including flusulfinam, have been reported; this study is the first to report on the metabolites of azole carboxamide HPPD inhibitor inside plants. −

Our findings indicate that iptriazopyrid is an effective herbicide with a high selectivity to O. sativa and a well-defined mechanism, despite its unique azole carboxamide structure. These findings demonstrate the potential use of azole carboxamide as a scaffold to explore a new class of herbicides in a broad range of weeds. However, details of the metabolism of azole carboxamide compound in plants remain unclear, given the novel chemotype of this HPPD inhibitor. Further studies using techniques such as transcriptomics and proteomics will provide further insights into understanding the characteristics of the newer class of herbicides. ,

Glossary

Abbreviations

HPPD

4-hydroxyphenylpyruvate dioxygenase

PQ

plastoquinone

HPPA

4-hydroxyphenylpyruvic acid

HGA

homogentisic acid

HGD

human homogentisate 1,2-dioxygenase

MAA

maleylacetoacetate

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c11831.

• Comparison of phytotoxic effects of mesotrione and iptriazopyrid on (a) E. crus-galli and (b) O. sativa in a planter (Figure S1); the crystal structure of (a) AtHPPD-iptriazopyrid and (b) AtHPPD-mesotrione (PDB: 5YWG); inhibitors and their key surrounding residues are shown as sticks (Figure S2); structural comparison of OsHPPD in the presence (pink) and absence (blue) of iptriazopyrid (Figure S3); alignment of amino acid sequences from representative plants, the red highlights indicate residues contacting iptriazopyrid within 4 Å (Figure S4); inhibitory kinetics of AtHPPD and OsHPPD by mesotrione and iptriazopyrid (Figure S5); effects of iptriazopyrid on the growth of E. crus-galli and O. sativa (Table S1); data collection and refinement statistics for the crystal structures of AtHPPD-iptriazopyrid, OsHPPD, and OsHPPD-iptriazopyrid (Table S2); interactions between HPPDs and iptriazopyrid. Interactions within 3.5 Å are listed, with important residues mentioned in the main text also included (Table S3); amino acid sequences for HPPD proteins (Table S4) (PDF)

This work was supported by funding from Nissan Chemical Corporation (to T.N., N.N., Y.F., T.K., and Y.K.).

The authors declare the following competing financial interest(s): T.N., N.N., Y.F., T.K., and Y.K (National Institute of AIST). report no potential conflicts of interest. Y.N., H.P., and T.F. are employees of Nissan Chemical Corporation.