Integrated Virtual Screening and Validation toward Potential HPPD Inhibition Herbicide.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the most widely studied herbicide targets and has gained significant attention. To identify potential effective HPPD inhibitors, a rational multistep virtual screening workflow was built, which included CBP models (based on the receptor-ligand interactions in the crystal complex), Hypogen models with activity prediction ability (according to the derivation of structure-activity relationships from a set of molecules with reported activity values), and a consensus docking procedure (consisting of LibDock, Glide, and CDOCKER). About 1 million molecules containing diketone or ß-keto-enol substructures were filtered by Lipinski's rules, CBP model, and Hypogen model. A total of 12 compounds with similar docking postures were generated by consensus docking. Eventually, four molecules were screened based on the specific binding pattern and affinity of the HPPD inhibitor. The biological evaluation in vivo displayed that compounds III-1 and III-2 exhibited comparable herbicidal activity to isoxaflutole and possessed superior safety on various crops (wheat, rice, sorghum, and maize). The ADMET prediction (absorption, distribution, metabolism, excretion, and toxicity) showed that compound III possessed relatively good toxicological results. This work provides a theoretical basis and valuable reference for the virtual screening and molecular design of novel HPPD inhibition herbicides.

Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa.

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to ß-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying ß-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL.

Discovery of Tetrazolamide-benzimidazol-2-ones as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is one of the most valuable herbicide targets due to its unique biological functions. In search of HPPD inhibitors with promising biological performance, we designed and synthesized a series of novel tetrazolamide-benzimidazol-2-ones using a structure-based drug design strategy. Among the synthesized compounds, 1-(2-chlorobenzyl)-3-methyl-N-(1-methyl-1H-tetrazol-5-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-4-carboxamide, 25, IC50 = 10 nM, was identified to be the most outstanding HPPD inhibitor, which showed more than 36-fold increased Arabidopsis thaliana HPPD (AtHPPD) inhibition potency than mesotrione (IC50 = 363 nM). Our AtHPPD-25 complex indicated that one nitrogen atom on the tetrazole ring and the oxygen atom on the amide group formed a classical bidentate chelation interaction with the metal ion, the benzimidazol-2-one ring created a tight π-π stacking interaction with Phe381 and Phe424, and some hydrophobic interactions were also found between the ortho-Cl-benzyl group and surrounding residues. Compound 32 showed more than 80% inhibition against all four tested weeds at 150 g ai/ha by the postemergence application. Our results indicated that the tetrazolamide-benzimidazol-2-one scaffold may be a new lead structure for herbicide discovery.

Structural and functional characterization of triketone dioxygenase from Oryza Sativa.

The transgenic expression of rice triketone dioxygenase (TDO; also known as HIS1) can provide protection from triketone herbicides to susceptible dicot crops such as soybean. Triketones are phytotoxic inhibitors of plant hydroxyphenylpyruvate dioxygenases (HPPD). The TDO gene codes for an iron/2-oxoglutarate-dependent oxidoreductase. We obtained an X-ray crystal structure of TDO using SeMet-SAD phasing to 3.16 Å resolution. The structure reveals that TDO possesses a fold like that of Arabidopsis thaliana 2-oxoglutarate­iron-dependent oxygenase anthocyanidin synthase (ANS). Unlike ANS, this TDO structure lacks bound metals or cofactors, and we propose this is because the disordered flexible loop over the active site is sterically constrained from folding properly in the crystal lattice. A combination of mass spectrometry, nuclear magnetic resonance, and enzyme activity studies indicate that rice TDO oxidizes mesotrione in a series of steps; first producing 5-hydroxy-mesotrione and then oxy-mesotrione. Evidence suggests that 5-hydroxy-mesotrione is a much weaker inhibitor of HPPD than mesotrione, and oxy-mesotrione has virtually no inhibitory activity. Of the close homologues which have been tested, only corn and rice TDO have enzymatic activity and the ability to protect plants from mesotrione. Correlating sequence and structure has identified four amino acids necessary for TDO activity. Introducing these four amino acids imparts activity to a mesotrione-inactive TDO-like protein from sorghum, which may expand triketone herbicide resistance in new crop species.

Design, Synthesis, and Bioactivity of Novel 2-(Arylformyl)cyclohexane-1,3-dione Derivatives as HPPD Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase inhibitors (Echinochloa crus-galli 1.13.11.27, HPPD) have gained significant popularity as one of the best-selling herbicides worldwide. To identify highly effective HPPD inhibitors, a rational design approach utilizing bioisosterism was employed to create a series of 2-(arylformyl)cyclohexane-1,3-dione derivatives. A total of 29 novel compounds were synthesized and characterized through various techniques, including IR, 1H NMR, 13C NMR, and HRMS. Evaluation of their inhibitory activity against Arabidopsis thaliana HPPD (AtHPPD) revealed that certain derivatives exhibited superior potency compared to mesotrione (IC50 = 0.204 µM). Initial herbicidal activity tests demonstrated that compounds 27 and 28 were comparable to mesotrione in terms of weed control and crop safety, with compound 28 exhibiting enhanced safety in canola crops. Molecular docking analyses indicated that the quinoline rings of compounds 27 and 28 formed more stable π-π interactions with the amino acid residues Phe-360 and Phe-403 in the active cavity of AtHPPD, surpassing the benzene ring of mesotrione. Molecular dynamics simulations and molecular structure comparisons confirmed the robust binding capabilities of compounds 27 and 28 to AtHPPD. This study provides a valuable reference for the development of novel triketone herbicide structures, serving as a blueprint for future advancements in this field.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Cypyrafluone, a 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor to Control Weed in Wheat Fields.

As a bleaching herbicide, cypyrafluone was applied postemergence in wheat fields for annual weed control; especially, this herbicide possesses high efficacy against cool-season grass weed species such as Alopecurus aequalis and Alopecurus japonicus. In this study, the target of action of cypyrafluone on 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition was confirmed. This herbicide caused severe foliar whitening symptoms at 5-7 days after treatment (DAT) and death of the whole plant within 10 DAT. Significant increases in phytoene content and significant decreases in kinds of carotenoid and chlorophyll pigments were observed. The content of chlorophyll pigments in cypyrafluone-treated Spirodela polyrhiza decreased upon the addition of homogentisic acid (HGA), which indicated that cypyrafluone prevents the HGA production, possibly by inhibiting the catalytic activity of 4-HPPD. Indeed, cypyrafluone strongly inhibited the catalytic activity of Arabidopsis thaliana HPPD produced by Escherichia coli, which was approximately 2 times less effective than mesotrione. In addition, overexpression of Oryza sativa HPPD in rice and A. thaliana both conferred a high tolerance level to cypyrafluone on them. Molecular docking found that cypyrafluone bonded well to the active site of the HPPD and formed a bidentate coordination interaction with the Fe2+ atom, with distances of 2.6 and 2.7 Å between oxygen atoms and the Fe2+ atom and a binding energy of -8.0 kcal mol-1.

Multivariate image analysis applied to quantitative structure-activity relationships and docking studies of recent hydroxyphenylpyruvate deoxygenase inhibitors.

BACKGROUND: Mesotrione is a triketone widely used as an inhibitor of the hydroxyphenylpyruvate deoxygenase (HPPD) enzyme. However, new agrochemicals should be developed continuously to tackle the problem of herbicide resistance. Two sets of mesotrione analogs have been synthesized recently and they have demonstrated successful phytotoxicity against weeds. In this study, these compounds were joined to form a single data set and the HPPD inhibition of this enlarged library of triketones was modeled using multivariate image analysis applied to quantitative structure-activity relationships (MIA-QSAR). Docking studies were also carried out to validate the MIA-QSAR findings and to aid the interpretation of ligand-enzyme interactions responsible for the bioactivity (pIC50 ). RESULTS: The MIA-QSAR models based on van der Waals radii (rvdW ), electronegativity (ε), and the rvdW /ε ratio as molecular descriptors were both predictive to an acceptable degree (r2 ≥ 0.80, q2 ≥ 0.68 and r2 pred ≥ 0.68). Subsequently, partial least squares (PLS) regression parameters were applied to predict the pIC50 values of newly proposed derivatives, yielding a few promising agrochemical candidates. The calculated log P for most of these derivatives was found to be higher than that of mesotrione and the library compounds, indicating that they should be less prone to leach out and contaminate groundwater. CONCLUSION: Multivariate image analysis descriptors corroborated by docking studies were capable of modeling the herbicidal activities of 68 triketones reliably. Due to the substituent effects at the triketone framework, particularly of a nitro group in R3 , promising analogs could be designed. The P9 proposal demonstrated higher calculated activity and log P than commercial mesotrione. © 2023 Society of Chemical Industry.

Study of two combined series of triketones with HPPD inhibitory activity by molecular modelling.

Triketones are suitable compounds for 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition and are important compounds for eliminating agricultural weeds. We report herein quantitative structure-activity relationship (QSAR) modelling and docking studies for a series of triketone-quinoline hybrids and 2-(aryloxyacetyl)cyclohexane-1,3-diones with the aim of proposing new chemical candidates that exhibit improved performance as herbicides. The QSAR models obtained were reliable and predictive (average r2, q2, and r2pred of 0.72, 0.51, and 0.71, respectively). Guided by multivariate image analysis of the PLS regression coefficients and variable importance in projection scores, the substituent effects could be analysed, and a promising derivative with R1 = H, R2 = CN, and R3 = 5,7,8-triCl at the triketone-quinoline scaffold (P18) was proposed. Docking studies demonstrated that π-π stacking interactions and specific interactions between the substituents and amino acid residues in the binding site of the Arabidopsis thaliana HPPD (AtHPPD) enzyme support the desired bioactivity. In addition, compared to a benchmark commercial triketone (mesotrione), the proposed compounds are more lipophilic and less mobile in soil rich in organic matter and are less prone to contaminate groundwater.

Structure-Based Design of 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor as a Potential Herbicide for Cotton Fields.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is one of the most promising herbicide targets for the development of agricultural chemicals owing to its unique mechanism of action in plants. We previously reported on the co-crystal structure of Arabidopsis thaliana (At) HPPD complexed with methylbenquitrione (MBQ), an inhibitor of HPPD that we previously discovered. Based on this crystal structure, and in an attempt to discover even more effective HPPD-inhibiting herbicides, we designed a family of triketone-quinazoline-2,4-dione derivatives featuring a phenylalkyl group through increasing the interaction between the substituent at the R1 position and the amino acid residues at the active site entrance of AtHPPD. Among the derivatives, 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-(1-phenylethyl)quinazoline-2,4(1H,3H)-dione (23) was identified as a promising compound. The co-crystal structure of compound 23 with AtHPPD revealed that hydrophobic interactions with Phe392 and Met335, and effective blocking of the conformational deflection of Gln293, as compared with that of the lead compound MBQ, afforded a molecular basis for structural modification. 3-(1-(3-Fluorophenyl)ethyl)-6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethylquinazoline-2,4(1H,3H)-dione (31) was confirmed to be the best subnanomolar-range AtHPPD inhibitor (IC50 = 39 nM), making it approximately seven times more potent than MBQ. In addition, the greenhouse experiment showed favorable herbicidal potency for compound 23 with a broad spectrum and acceptable crop selectivity against cotton at the dosage of 30-120 g ai/ha. Thus, compound 23 possessed a promising prospect as a novel HPPD-inhibiting herbicide candidate for cotton fields.

Discovery of Bis-5-cyclopropylisoxazole-4-carboxamides as Novel Potential 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27; HPPD) represents a potential target for novel herbicide development. To discover the more promising HPPD inhibitor, we designed and synthesized a series of bis-5-cyclopropylisoxazole-4-carboxamides with different linkers using a multitarget pesticide design strategy. Among them, compounds b9 and b10 displayed excellent herbicidal activities versus Digitaria sanguinalis (DS) and Amaranthus retroflexus (AR) with the inhibition of about 90% at the concentration of 100 mg/L in vitro, which was better than that of isoxaflutole (IFT). Furthermore, compounds b9 and b10 displayed the best inhibitory effect versus DS and AR with the inhibition of about 90 and 85% at 90 g (ai)/ha in the greenhouse, respectively. The structure-activity relationship study showed that the flexible linker (6 carbon atoms) is responsible for increasing their herbicidal activity. The molecular docking analyses showed that compounds b9 and b10 could more closely bind to the active site of HPPD and thus exhibited a better inhibitory effect. Altogether, these results indicated that compounds b9 and b10 could be used as potential herbicide candidates targeting HPPD.

Insights into 4-hydroxyphenylpyruvate dioxygenase-inhibitor interactions from comparative structural biology.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a key role in tyrosine metabolism and has been identified as a promising target for herbicide and drug discovery. The structures of HPPD complexed with different types of inhibitors have been determined previously. We summarize the structures of HPPD complexed with structurally diverse molecules, including inhibitors, natural products, substrates, and catalytic intermediates; from these structures, the detailed inhibitory mechanisms of different inhibitors were analyzed and compared, and the key structural factors determining the slow-binding behavior of inhibitors were identified. Further, we propose four subpockets that accommodate different inhibitor substructures. We believe that these analyses will facilitate in-depth understanding of the enzymatic reaction mechanism and enable the design of new inhibitors with higher potency and selectivity.

Discovery of Subnanomolar Inhibitors of 4-Hydroxyphenylpyruvate Dioxygenase via Structure-Based Rational Design.

High-potency 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are usually featured by time-dependent inhibition. However, the molecular mechanism underlying time-dependent inhibition by HPPD inhibitors has not been fully elucidated. Here, based on the determination of the HPPD binding mode of natural products, the π-π sandwich stacking interaction was found to be a critical element determining time-dependent inhibition. This result implied that, for the time-dependent inhibitors, strengthening the π-π sandwich stacking interaction might improve their inhibitory efficacy. Consequently, modification with one methyl group on the bicyclic ring of quinazolindione inhibitors was achieved, thereby strengthening the stacking interaction and significantly improving the inhibitory efficacy. Further introduction of bulkier hydrophobic substituents with higher flexibility resulted in a series of HPPD inhibitors with outstanding subnanomolar potency. Exploration of the time-dependent inhibition mechanism and molecular design based on the exploration results are very successful cases of structure-based rational design and provide a guiding reference for future development of HPPD inhibitors.

Assessing the effects of ß-triketone herbicides on HPPD from environmental bacteria using a combination of in silico and microbiological approaches.

4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of ß-triketone herbicides in plants. This enzyme, involved in the tyrosine pathway, is also present in a wide range of living organisms, including microorganisms. Previous studies, focusing on a few strains and using high herbicide concentrations, showed that ß-triketones are able to inhibit microbial HPPD. Here, we measured the effect of agronomical doses of ß-triketone herbicides on soil bacterial strains. The HPPD activity of six bacterial strains was tested with 1× or 10× the recommended field dose of the herbicide sulcotrione. The selected strains were tested with 0.01× to 15× the recommended field dose of sulcotrione, mesotrione, and tembotrione. Molecular docking was also used to measure and model the binding mode of the three herbicides with the different bacterial HPPD. Our results show that responses to herbicides are strain-dependent with Pseudomonas fluorescens F113 HPPD activity not inhibited by any of the herbicide tested, when all three ß-triketone herbicides inhibited HPPD in Bacillus cereus ATCC14579 and Shewanella oneidensis MR-1. These responses are also molecule-dependent with tembotrione harboring the strongest inhibitory effect. Molecular docking also reveals different binding potentials. This is the first time that the inhibitory effect of ß-triketone herbicides is tested on environmental strains at agronomical doses, showing a potential effect of these molecules on the HPPD enzymatic activity of non-target microorganisms. The whole-cell assay developed in this study, coupled with molecular docking analysis, appears as an interesting way to have a first idea of the effect of herbicides on microbial communities, prior to setting up microcosm or even field experiments. This methodology could then largely be applied to other family of pesticides also targeting an enzyme present in microorganisms.

Synthesis, Crystal Structure and Biological Activity of Two Triketone-Containing Quinoxalines as HPPD Inhibitors.

Two new triketone-containing quinoxaline derivatives were designed by fragment splicing strategy and synthesized using 3,4-diaminobenzoic acid and substituted cyclohexanedione as starting materials. Both compounds were characterized by IR, 1H and 13C NMR, HRMS and X-ray diffraction. 3-Hydroxy-5-methyl-2-(quinoxaline-6-carbonyl)cyclohex-2-en-1-one (6a) crystallized in the triclinic system, space group Pi, a = 7.9829(2) Å, b = 8.1462(2) Å, c = 10.7057(3) Å, α = 84.3590(10)°, ß = 89.7760(10)°, γ = 87.4190(10)°, Z = 2, V = 692.12(3) Å3, F(000) = 296, Dc = 1.335 Mg/m3, m(MoKa) = 0.095 mm-1, R = 0.0683 and wR= 0.1983. 3-Hydroxy-5,5-dimethyl-2-(3-ethoxyquinoxaline-6-carbonyl)cyclohex-2-en-1-one (6b) crystallized in the monoclinic system, space group P21/c, a = 10.1554(6) Å, b = 9.6491(6) Å, c = 17.7645(10) Å, ß = 90.784(2)°, Z = 4, V = 1740.59(18) Å3, F(000) = 720, Dc = 1.299 Mg/m3, m(MoKa) = 0.092 mm-1, R = 0.0462 and wR = 0.1235. Physicochemical property comparison and ADMET prediction showed that compound 6a had similar properties to the commercial herbicide mesotrione. Molecular docking results showed that the interactions between 6a and AtHPPD were similar to mesotrione. Moreover, the extended aromatic ring system and the additional alkyl form more interactions with the surrounding residues. Examination of AtHPPD inhibition and herbicidal activity showed that 6a had similar inhibition values to mesotrione and had a superior inhibitory effect on Echinochloa crus-galli.

Rational Redesign of Enzyme via the Combination of Quantum Mechanics/Molecular Mechanics, Molecular Dynamics, and Structural Biology Study.

Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering α-ketoglutarate-dependent oxygenases is to develop a rational strategy which can be widely used for directly evolving the desired mutant to generate new products. Herein, we report a strategy for rational redesign of a model enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD), based on quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamic simulations. This strategy enriched our understanding of the HPPD catalytic reaction pathway and led to the discovery of a series of HPPD mutants producing hydroxyphenylacetate (HPA) as the alternative product other than the native product homogentisate. The predicted HPPD-Fe(IV)═O-HPA intermediate was further confirmed by the crystal structure of Arabidopsis thaliana HPPD/S267W complexed with HPA. These findings not only provide a good understanding of the structure-function relationship of HPPD but also demonstrate a generally applicable platform for the development of biocatalysts.

Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Structure-Guided Discovery of Silicon-Containing Subnanomolar Inhibitor of Hydroxyphenylpyruvate Dioxygenase as a Potential Herbicide.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been recognized as one of the most promising targets in the field of herbicide innovation considering the severity of weed resistance currently. In a persistent effort to develop effective HPPD-inhibiting herbicides, a structure-guided strategy was carried out to perform the structural optimization for triketone-quinazoline-2,4-diones, a novel HPPD inhibitor scaffold first discovered in our lab. Herein, starting from the crystal structure of Arabidopsis thaliana (At)HPPD complexed with 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-(o-tolyl)quinazoline-2,4(1H,3H)-dione (MBQ), three subseries of quinazoline-2,4-dione derivatives were designed and prepared by optimizing the hydrophobic interactions between the side chain of the core structure at the R1 position and the hydrophobic pocket at the active site entrance of AtHPPD. 6-(2-Hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-(3-(trimethylsilyl)prop-2-yn-1-yl)quinazoline-2,4(1H,3H)-dione (60) with the best inhibitory activity against AtHPPD was identified to be the first subnanomolar-range AtHPPD inhibitor (Ki = 0.86 nM), which significantly outperformed that of the lead compound MBQ (Ki = 8.2 nM). Further determination of the crystal structure of AtHPPD in complex with compound 60 (1.85 Å) and the binding energy calculation provided a molecular basis for the understanding of its high efficiency. Additionally, the greenhouse assay indicated that 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,5-dimethyl-3-propylquinazoline-2,4(1H,3H)-dione (28) and compound 60 showed acceptable crop safety against peanut and good herbicidal activity with a broad spectrum. Moreover, compound 28 also showed superior selectivity for wheat at the dosage of 120 g ai/ha and favorable herbicidal efficacy toward the gramineous weeds at the dosage of as low as 30 g ai/ha. We believe that compounds 28 and 60 have promising prospects as new herbicide candidates for wheat and peanut fields.

Improved Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19 through Directed Evolution.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors' resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.

Based on the Virtual Screening of Multiple Pharmacophores, Docking and Molecular Dynamics Simulation Approaches toward the Discovery of Novel HPPD Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an iron-dependent non-heme oxygenase involved in the catabolic pathway of tyrosine, which is an important enzyme in the transformation of 4-hydroxyphenylpyruvic acid to homogentisic acid, and thus being considered as herbicide target. Within this study, a set of multiple structure-based pharmacophore models for HPPD inhibitors were developed. The ZINC and natural product database were virtually screened, and 29 compounds were obtained. The binding mode of HPPD and its inhibitors obtained through molecular docking study showed that the residues of Phe424, Phe381, His308, His226, Gln307 and Glu394 were crucial for activity. Molecular-mechanics-generalized born surface area (MM/GBSA) results showed that the coulomb force, lipophilic and van der Waals (vdW) interactions made major contributions to the binding affinity. These efforts will greatly contribute to design novel and effective HPPD inhibitory herbicides.