Structure-Based Design, Virtual Screening, and Discovery of Novel Patulin Derivatives as Biogenic Photosystem II Inhibiting Herbicides

Computer-aided design usually gives inspirations and has become a vital strategy to develop novel pesticides through reconstructing natural lead compounds. Patulin, an unsaturated heterocyclic lactone mycotoxin, is a new natural PSII inhibitor and shows significant herbicidal activity to various weeds. However, some evidence, especially the health concern, prevents it from developing as a bioherbicide. In this work, molecular docking and toxicity risk prediction are combined to construct interaction models between the ligand and acceptor, and design and screen novel derivatives. Based on the analysis of a constructed patulin–Arabidopsis D1 protein docking model, in total, 81 derivatives are designed and ranked according to quantitative estimates of drug-likeness (QED) values and free energies. Among the newly designed derivatives, forty-five derivatives with better affinities than patulin are screened to further evaluate their toxicology. Finally, it is indicated that four patulin derivatives, D3, D6, D34, and D67, with higher binding affinity but lower toxicity than patulin have a great potential to develop as new herbicides with improved potency.


Introduction
Natural products are a key resource for developing new drugs and pesticides [1,2].However, some active ingredients face the challenge of unstable activity, toxicity to nontargeting organisms, or environmental concerns [3].Structure-based methods such as docking and molecular dynamics play a crucial role in computer-aided new drug design, enhancing our understanding of how small molecules bind to the protein targets.First of all, the strategy of such drug design is generally docking an interesting leading active ingredient or its analogues into a recognized target protein [4].The development of structural biology, in that more and more experimentally determined structures of high-resolution protein crystal structures are analyzed via X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy (cryo-EM) methods and available in databases such as the Protein Data Bank (PDB) (http://www.wwpdb.org)or AlphaFold 2 (https://alphafold.com/),which are filled with more than 200 million structures, facilitates structure-based ligand design studies [4][5][6].
Photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of plants, cyanobacteria, and algae, which is considered as the crucial photosynthetic component, catalyzing light-driven water-splitting and oxygenevolving reactions, thereby converting light energy into electrochemical potential energy and generating molecular oxygen [7][8][9].In PSII, Q A and Q B are the primary and secondary plastoquinone receptors, respectively.Many classical PSII inhibiting herbicides bind at Plants 2024, 13, 1710 2 of 16 the Q B site of the D1 protein and interfere with the photosynthetic electron transport in competition with the native electron acceptor plastoquinone, and thereby block the transfer of electrons from Q A to Q B .The Q B site is located in the connecting loop between the fourth and the fifth transmembrane helices between the amino acid residue from Phe211 to Leu275 in plants.The classical urea/triazine herbicides including atrazine and DCMU with the common functional group N-C=R (R refers to an atom of oxygen or nitrogen) can form a hydrogen bond with D1-Ser264.The phenol-type herbicides with an aromatic hydroxyl group such as dinoseb and ioxynil form a hydrogen bond towards D1-His215 [10][11][12][13].
The mycotoxin patulin, an electrophilic unsaturated heterocyclic lactone (4-hydroxy-4H-furo [3,2-c]pyran-2(6H)-one), is widely present in final products, especially in apples and apple-based products, and predominantly produces by several fungal species involving Aspergillus, Byssochlamys, and Penicillium during storage [14,15].Patulin can induce the generation of cytotoxin-involving reactive oxygen species (ROS), cytochrome c release in mitochondria, the uptake of cytosolic Ca 2+ , caspase-3 activation, cell cycle arrest, and cell apoptosis [16,17].Furthermore, patulin causes the destruction of DNA damage, chromosome abnormalities, and the appearance of micronuclei in mammalian cells [18][19][20][21].Early in the past century, it was indicated that patulin exhibited the potential not only in inhibiting seed germination but also seedling growth of wheat, indicating an interesting herbicidal activity [22].Our previous research indicates that patulin is a novel natural PSII inhibitor and shows significant herbicidal activity to various weeds, especially the invasive weed Ageratina adenophora.The unsaturated lactone containing the C=O group at the 2-position is the essential part that bounds to the Q B site through forming hydrogen bonds to D1-His252 [23].But many cell line-based and animal model-based findings are evidence that patulin has the following properties: genotoxicity, embryotoxicity, cytotoxicity, neurotoxicity, immunotoxicity, carcinogenicity, and teratogenicity.These prevent it from developing into a bioherbicide due to the health concern [24].Nevertheless, this natural product is still an interesting leading compound in discovering new active ingredients with high activity in weed control.
In this work, we focused on discovering novel highly bioactive and low-toxicity patulin derivatives by the strategy of structure-based ligand design.Firstly, eighty-one patulin derivatives, which are different introduced groups at 1-, C3, C4, 5-, C6, or C7 positions of patulin, were designed based on the simulated model of patulin and Arabidopsis D1 protein by computer-aided design.Subsequently, each designed derivative exhibiting higher binding affinity than patulin was screened through docking it into the D1 protein and its binding interaction energy calculation.The toxicity risks of forty-five patulin derivatives, which show a higher quantitative estimate of drug-likeness (QED) and binding affinity than patulin, were predicted by Discovery Studio software version 2016.Finally, four patulin derivatives involving D3, D6, D34, and D67 compounds with higher binding affinity but lower toxicity than patulin were used in the Q B binding site of Arabidopsis D1 protein modeling to further analyze the detail feature of molecular interaction deeply.Obviously, structure-based ligand design is an efficient and highly reliable strategy to discover novel derivatives with high herbicidal activity through the core leading scaffolds of mycotoxin patulin.Figure 1 displays a methodology with a brief description of this study.

Model-Based Ligand Design and Molecular Docking of Patulin and Its Derivatives
Previous studies indicated that patulin binds to D1 protein, which blocks PSII tron transport beyond QA.The simulated molecular modeling of patulin docking t protein of A. adenophora suggests that D1-His252 plays an important role in the doc model of patulin and D1 protein [17] (Figure 2A).In this study, the crystal structure i mation of the model plant Arabidopsis D1 protein (PDB: 5MDX) was chosen to mode position of patulin in the QB site by Discovery Studio (Figure 2B-D).In this mode residues including Phe211, Met214, Leu218, His252, Phe255, Ser264, Phe265, Le Phe274, and Leu275 in the D1 protein are identified as responsible for patulin bin The residue D1-His252 contributes to binding to patulin by forming a hydrogen bond the O2 carbonyl oxygen atom of patulin.This result is congruent with early evidence modeling of patulin binding to the D1 protein of A. adenophora [17].

Model-Based Ligand Design and Molecular Docking of Patulin and Its Derivatives
Previous studies indicated that patulin binds to D1 protein, which blocks PSII electron transport beyond Q A .The simulated molecular modeling of patulin docking to D1 protein of A. adenophora suggests that D1-His252 plays an important role in the docking model of patulin and D1 protein [17] (Figure 2A).In this study, the crystal structure information of the model plant Arabidopsis D1 protein (PDB: 5MDX) was chosen to model the position of patulin in the Q B site by Discovery Studio (Figure 2B-D).In this model, the residues including Phe211, Met214, Leu218, His252, Phe255, Ser264, Phe265, Leu271, Phe274, and Leu275 in the D1 protein are identified as responsible for patulin binding.The residue D1-His252 contributes to binding to patulin by forming a hydrogen bond with the O2 carbonyl oxygen atom of patulin.This result is congruent with early evidence from modeling of patulin binding to the D1 protein of A. adenophora [17].The technique of molecular docking is extensively employed for the prediction of ligand orientation and conformation with the active site of the target protein, as well as for virtual screening based on the analysis of ranking scores or binding interaction energy of the docking model of the ligand-receptor complex [25].On the basis of this method, two novel TeA analogues possessing higher herbicidal activity were discovered [26].Yang et al. elucidated the mode of action of mycotoxin citrinin, and furtherly screened five new citrinin derivatives by introducing different substituents at the 9-position with high herbicidal potency compared with lead compound citrinin based on a computer-aided design strategy [27].To find novel patulin derivatives with improved potency, QED values and binding interaction energy of 81 novel compounds were designed based on the modification of substituents in 1-, C3, C4, 5-, C6, or C7 positions of patulin binding to the QB site and are displayed (Tables 1-6).Each compound with negative binding energy means that a favorable thermodynamic binding occurred between the ligand and D1 protein.The technique of molecular docking is extensively employed for the prediction of ligand orientation and conformation with the active site of the target protein, as well as for virtual screening based on the analysis of ranking scores or binding interaction energy of the docking model of the ligand-receptor complex [25].On the basis of this method, two novel TeA analogues possessing higher herbicidal activity were discovered [26].Yang et al. elucidated the mode of action of mycotoxin citrinin, and furtherly screened five new citrinin derivatives by introducing different substituents at the 9-position with high herbicidal potency compared with lead compound citrinin based on a computer-aided design strategy [27].To find novel patulin derivatives with improved potency, QED values and binding interaction energy of 81 novel compounds were designed based on the modification of substituents in 1-, C3, C4, 5-, C6, or C7 positions of patulin binding to the Q B site and are displayed (Tables 1-6).Each compound with negative binding energy means that a favorable thermodynamic binding occurred between the ligand and D1 protein.The technique of molecular docking is extensively employed for the prediction of ligand orientation and conformation with the active site of the target protein, as well as for virtual screening based on the analysis of ranking scores or binding interaction energy of the docking model of the ligand-receptor complex [25].On the basis of this method, two novel TeA analogues possessing higher herbicidal activity were discovered [26].Yang et al. elucidated the mode of action of mycotoxin citrinin, and furtherly screened five new citrinin derivatives by introducing different substituents at the 9-position with high herbicidal potency compared with lead compound citrinin based on a computer-aided design strategy [27].To find novel patulin derivatives with improved potency, QED values and binding interaction energy of 81 novel compounds were designed based on the modification of substituents in 1-, C3, C4, 5-, C6, or C7 positions of patulin binding to the QB site and are displayed (Tables 1-6).Each compound with negative binding energy means that a favorable thermodynamic binding occurred between the ligand and D1 protein.       1 were introduced involving different substituents at the 1-position of patulin.Compounds with a sulfur (D1), methylamino (D3), alkyl side chains (D4-D6), alkyl chloride (D7) or hydroxy group (D8) exhibit lower interaction energies than patulin in the D1 protein.Compounds D2 and D9 with an amidogen or aminomethyl group at the 1-position of patulin show lower QED and higher interaction energy than patulin, indicating that these two compounds may have lower herbicidal activity than patulin.
Compounds D10-D19 in Table 2 focused on introducing different substituents at the C3 position of patulin.Compounds with a methyl (D10), methylthio (D12), ester (D16), acylamide (D18), and chlorine group (D19) exhibit higher QED and lower interaction energies than patulin.Compounds D11, D13-D15, and D17 with a sulfhydryl, ether, aldehyde, carboxy, or amino group at the C3 position of patulin have lower QED and higher interaction energy than patulin.Compounds D1-D9 in Table 1 were introduced involving different substituents at the 1-position of patulin.Compounds with a sulfur (D1), methylamino (D3), alkyl side chains (D4-D6), alkyl chloride (D7) or hydroxy group (D8) exhibit lower interaction energies than patulin in the D1 protein.Compounds D2 and D9 with an amidogen or aminomethyl group at the 1-position of patulin show lower QED and higher interaction energy than patulin, indicating that these two compounds may have lower herbicidal activity than patulin.
Compounds D20-D40 in Table 3 focused on introducing different substituents at the C4 position of patulin.Compounds D26, D29-D37, and D39 with an acyl chloride, alkyl side chain, chlorine or methylthio group exhibit higher QED and lower interaction energies than patulin, suggesting that these compounds show higher inhibition to PSII compared with patulin.On the other hand, D20-D25, D36, D38, and D40 with an ether, hydroxy, aldehyde, carboxy, chloride, sulfhydryl, or amino group introduced to the C4 position of patulin show a lower value of QED and higher interaction energy than patulin.
The QED and binding interaction energy of different substituents introduced at the 5-position of patulin were displayed in Table 4. Compounds D41 and D43-D46 with hydrophobic groups including sulfur, methylamino, or alkyl side chain groups exhibit a higher QED value and lower interaction energies than patulin.D42, D44, and D47-D49 with a hydrophilic group, such as an amino, chloride, or hydroxy group introduced to the 5-position of patulin, show a lower value of QED and higher interaction energy than patulin.
The QED and binding interaction energy of different substituents introduced at the C6 position of patulin were shown in Table 5. Compounds with an alkyl side chain (D50-D53) and a chloride (D54), ether (D56 and D57), carbonyl (D59), and methylthio (D65 and D66) group exhibit a higher QED value and lower interaction energies than patulin.D55, D58, D60-D63, and D68 with a hydroxy, aldehyde, carboxy, sulfhydryl, ester, or amino group at the C6 position of patulin have a lower value of QED and higher interaction energy relative to patulin.
Compounds D69-D81 in Table 6 focused on the modification of different substituents at the C7 position.Compounds with an alkyl side chain (D69 and D70) and chloromethyl (D71), ether (D73), carbonyl (D75), ester (D77), or methylthio (D79) group exhibit a higher QED value and lower interaction energies than patulin.D72, D74, D76, D78, and D80-D81 with a hydroxy, aldehyde, carboxy, sulfhydryl, amino, or chloride group at the C7 position of patulin show a lower value of QED and higher interaction energy with comparison to patulin.Compounds D20-D40 in Table 3 focused on introducing different substituents at the C4 position of patulin.Compounds D26, D29-D37, and D39 with an acyl chloride, alkyl side chain, chlorine or methylthio group exhibit higher QED and lower interaction energies than patulin, suggesting that these compounds show higher inhibition to PSII compared with patulin.On the other hand, D20-D25, D36, D38, and D40 with an ether, hydroxy, aldehyde, carboxy, chloride, sulfhydryl, or amino group introduced to the C4 position of patulin show a lower value of QED and higher interaction energy than patulin.
The QED and binding interaction energy of different substituents introduced at the 5-position of patulin were displayed in Table 4. Compounds D41 and D43-D46 with hydrophobic groups including sulfur, methylamino, or alkyl side chain groups exhibit a higher QED value and lower interaction energies than patulin.D42, D44, and D47-D49 with a hydrophilic group, such as an amino, chloride, or hydroxy group introduced to the 5-position of patulin, show a lower value of QED and higher interaction energy than patulin.
The QED and binding interaction energy of different substituents introduced at the C6 position of patulin were shown in Table 5. Compounds with an alkyl side chain (D50-D53) and a chloride (D54), ether (D56 and D57), carbonyl (D59), and methylthio (D65 and D66) group exhibit a higher QED value and lower interaction energies than patulin.D55, D58, D60-D63, and D68 with a hydroxy, aldehyde, carboxy, sulfhydryl, ester, or amino group at the C6 position of patulin have a lower value of QED and higher interaction energy relative to patulin.
Compounds D69-D81 in Table 6 focused on the modification of different substituents at the C7 position.Compounds with an alkyl side chain (D69 and D70) and chloromethyl (D71), ether (D73), carbonyl (D75), ester (D77), or methylthio (D79) group exhibit a higher QED value and lower interaction energies than patulin.D72, D74, D76, D78, and D80-D81 with a hydroxy, aldehyde, carboxy, sulfhydryl, amino, or chloride group at the C7 position of patulin show a lower value of QED and higher interaction energy with comparison to patulin.
Among these 81 derivatives, forty-five derivatives as possible candidates show a higher QED value and binding affinity than leading compound patulin.The QED value, which integrated eight key physicochemical properties of a drug candidate, provides an efficient approach to assessing drug-likeness and chemical attractiveness [28].A greater QED value means a higher drug-likeness, representing a compound as more bioactive [29].On the basis of the analysis, we found that the substituents including an alkyl side chain and methylthio group introduced to the patulin can increase the QED value of the new compound, such as compounds D4-D6 (1-), D10 (C3), D12 (C3), D29-D35 (C4), D39 (C4), Plants 2024, 13, 1710 8 of 16 D44-D46 (5-), D50-D53 (C6), D65-D67 (C6), D69 (C7), D70 (C7), and D79 (C7).Methylamino and acylamide groups introduced to the patulin at the 1-, C3, or 5-position, such as compounds D3, D18, and D43, can increase the QED value.The QED value of the compounds will be increased while the sulfur is introduced in the patulin at the 1-or 5-position, e.g., compounds D1 and D41.Chloride and alkyl chloride groups are introduced in the patulin at the C3, C4 (CH 2 Cl), or C6 (CH 2 Cl) position involving compounds D19, D37, and D54 and can increase the QED value.The introduction of an ether group at the C6 or C7 position can also increase the QED value including compound D56, D57, and D73.The QED values of compounds are increased when an ester group is introduced at the C3 or C7 position.However, the introducing of hydrophilic groups involving hydroxy, aldehyde, carboxy, sulfhydryl, or amino leads to a decrease in the QED value and the binding affinity.We expect that these introduced groups, which can increase the QED value of new compounds, may donate electrons by resonance to the C=O bond at the 2-position, or to the double bonds at C7 up to the carboxyl bond, resulting in increasing charge transfer between the proton donor and acceptor molecules and reinforcing hydrogen bond formation with D1-His252 [30].

Prediction of Toxicity of Patulin's Derivatives by Computational Analysis
However, to ensure safe use of herbicides, strategies to assess the possible risks need to be developed.The characterization of the risk for non-target organisms is essential.The AAL toxin produced by A. alternata f. sp.Lycopersici has been known as an excellent herbicidal compound for a long time.However, its toxicity to cultured mammalian cells prevents it from developing as a bioherbicide.Recently, a few AAL toxin derivatives were designed and synthesized.Based on a series of experiments, it was indicated that one of them exhibited higher significant phytotoxicity but lower mammalian toxicity than the lead compound, which exhibited a potential for being developed as a safe and effective herbicide [31].So, some toxicity risk parameters including the median lethal dose (LD 50 ) of rat oral and rat inhalational LD 50 , skin irritancy, skin sensitization, ocular irritancy, or Ames mutagenicity of patulin and its forty-five derivatives were also predicted through the tools in Discovery studio.To validate the reliability of prediction, the values of rat oral LD 50 and Ames mutagenicity were successfully obtained from PubChem Database (https:// pubchem.ncbi.nlm.nih.gov/source/hsdb/3522#section=Animal-Toxicity-Studies,accessed on 15 April 2024) and previous studies.As shown in Table 7, patulin has the rat oral LD 50 of 55 mg/kg and no mutagenicity.Based on the Data Requirements for Pesticide Registration, the rating of rat oral LD 50 of patulin is a moderate toxicity level (50-500 mg/kg).Here, the predicted rat oral LD 50 of patulin is 139 mg/kg; moreover, it also does not have a mutagenic effect.Undoubtedly, our result is consistent with the existing data, showing high reliability and accuracy.On the basis of these predicted data, there are 39 compounds exhibiting a higher rat oral value than patulin, indicating lower oral toxicity.Especially, compounds D1, D3, D4, D6, D8, and D34 have an oral LD 50 of over 500 mg/kg, which are of low toxicity.Among the predicting data of rat inhalational LD 50 , only the value of compound D54 is lower than 2000 mg/m 3 , which is of moderate toxicity (200-2000 mg/m 3 ) in rats.The inhalational LD 50 values of other patulin derivatives are higher than 2000 mg/m 3 , which is of low toxicity (2000-5000 mg/m 3 ) or mild toxicity (>5000 mg/m 3 ) in rats.Based on the data of skin irritancy, D31 shows severe irritancy to skin, and compounds D1, D32, D33, and D35 show moderate irritancy to skin.Other patulin derivatives are of mild irritancy to skin.According to the predicting data of skin sensitization, only D1 shows moderate sensitization to skin.The other 44 patulin's derivatives are of weak or no sensitization to skin.More than 73% of patulin derivatives in Table 7 exhibit severe ocular irritancy.Compounds D16, D19, D56, D57, D59, and D77 show moderate ocular irritancy.Seven patulin derivatives involving D3, D6, D18, D34, D35, D39, and D67 exhibit mild ocular irritancy.The data about Ames mutagenicity indicate that all derivatives of patulin are non-mutagen.Table 7. Toxicity risks of patulin and its derivatives, which show higher QED and lower interaction energy, were predicted by Discovery Studio.Rat oral lethal dose (LD 50 ), rat inhalational LD 50 , skin irritancy, skin sensitization, ocular irritancy, and Ames mutagenicity were evaluated.The LD 50 experimental data (exp.) of rat oral LD 50 and Ames mutagenicity are also exhibited according to previous reports to validate the reliability of the predicted toxicity in this study.All things considered, four derivatives, D3, D6, D34, and D67, among forty-five candidates were screened out as real candidates since they possess a high QED value and binding affinity as well as reasonable low animal toxicity.

Verification of the Molecular Models of Real Candidates' Binding to D1 Protein
Furthermore, the molecular models of compound D3, D6, D34, and D67, which bind to the Q B site of Arabidopsis D1 protein (5MDX), were constructed in order to investigate the properties of these four real candidates further.The binding energies of each docked pose are −26.871kal•mol −1 (D3), −34.373 kal•mol −1 (D6), −33.048 kal•mol −1 (D34), and −35.601 kal•mol −1 (D67), which were displayed in Table 8.There is a single hydrogen bond between the residue His252 of D1 protein and the O2 carbonyl oxygen atom of these four compounds, while several kinds of hydrophobic interactions also occur with other residues of D1 protein (Figure 3).The modeled distance of their hydrogen bonds is 2.27 Å (D3), 2.13 Å (D6), 2.18 Å (D34), and 2.06 Å (D67), respectively, which is less than that of D0 (patulin), which is 2.38 Å.A shorter hydrogen bond distance may reinforce these candidates' binding stability in the D1 protein.The molecular physicochemical properties of a compound play a crucial role in the determination of its binding affinity for the target protein [32].Our results show a positive relationship between QED values and the binding affinity, and a negative relationship between QED values and the distance of a hydrogen bond.It was concluded that compounds D3, D6, D34, and D67 with a higher QED value improved the binding affinity of D1 protein due to having shorter hydrogen bond distances compared with lead compound patulin.As a consequence, these four derivatives, D3, D6, D34, and D67, have potential to be developed into biogenic photosynthetic herbicides.

Software
Molecular modeling studies are carried out through Discovery Studio (version 2016, BIOVIA, San Diego, CA, USA).The structures of various candidate ligands are created using ChemDraw 18.0 (Cambridge Soft, Cambridge, MA, USA).The ligand structures are optimized using MM2 energy minimizations by Chem3D Pro 14.0 (Cambridge Soft, USA).

The Preparation of Ligands
After energy minimization and the transformation of each ligand into the Discovery Studio program, the QED value was calculated according to our previous research [26].

The Preparation of Protein Data
Due to the available data of the crystal structure of Arabidopsis D1 protein, its highresolution crystal structures were obtained from the database of Protein Data Bank (https: //rcsb.org,PDB code: 5MDX, resolution: 5.30 Å), which was used as a template for the Dl protein according to our previous research [26].Its dimeric structure was simplified to a monomer and optimized using Discovery Studio's tools.

Interaction Model of Patulin Binding to D1 Protein of A. thaliana
The automation of the preliminary docking process was performed through Discovery Studio according to our previous research [26].After the docking was finished, it was indicated that six residues, Met214, His252, Phe255, Ser264, Phe265, and Leu271, in the D1 protein might provide bonds to patulin molecules.Thus, the binding site for the accuracy of docking of patulin and these six residues is focused on through the CDocker tool of Discovery Studio.These six amino acid residues create a binding site sphere with a radius of 12.34 Å and coordinate axes of x = 301.43Å, y = 242.62Å, and z = 239.58Å.Furthermore, the optimal binding pose, with the lowest interaction energy, was selected as the favorable binding configuration between patulin and the Q B binding site of the A. thaliana D1 protein.The 2D and 3D diagrams were opted for to illustrate the ligand binding site atoms in the molecular interaction model.

Model-Based Ligand Design and Molecular Docking of Patulin Derivatives
On the basis of our previous studies of the patulin-D1 protein binding model, it was indicated that the O2 carbonyl oxygen atom is a core scaffold for patulin's biological activity, which forms a major hydrogen bond between the residue D1-His252 [23].Consequently, the substituents at 1-, C3, C4, 5-, C6, or C7 positions of patulin were modified to find possible novel derivatives with high potency based on the molecular interaction modeling of patulin-A.thaliana D1 protein.The 81 patulin's derivatives designed and energetically minimized through ChemDraw 18.0 and Chem3D Pro 14.0, respectively, were divided into six types on the basis of the substituents modifying in 1-, C3, C4, 5-, C6, or C7 positions.Then, the molecular properties of each ligand were evaluated by Discovery Studio.Subsequently, we hypothesized that these designed derivatives show a similar binding manner with patulin at the Q B side of A. thaliana D1 protein.Notably, the binding site, which forms by the residues including D1-Met214, D1-His252, D1-Phe255, D1-Ser264, D1-Phe265, and D1-Leu271, was delineated for these derivatives.The method of the molecular docking of these 81 derivatives is similar to that described in the "3.4 Interaction Model of Patulin Binding to D1 Protein of A. thaliana" section.The interaction energy of each ligand at the Q B binding site of A. thaliana D1 protein was calculated and is presented in Table 1.

Prediction of Toxicity of Patulin Derivatives by Computational Analysis
Due to the current requests for environmental protection and safe food production, the most important pesticide properties used in pesticide registration refer to physical and chemical characteristics, toxicology and metabolism, environmental fate and behavior, and ecotoxicology (https://www.fao.org/pesticide-registration-toolkit/information-sources/

Figure 1 .
Figure 1.Flow chart illustrating the structure-based ligand design and discovery of novel p derivatives with high herbicidal activity.

Figure 1 .
Figure 1.Flow chart illustrating the structure-based ligand design and discovery of novel patulin derivatives with high herbicidal activity.

Figure 2 .
Figure 2. Simulated modeling of patulin binding to the D1 protein of Arabidopsis.(A) The chemical structure of patulin.(B) Hydrogen bonding interactions of patulin binding to the D1 protein.(C) The stereo view of the patulin binding environment of the D1 protein, in which carbon, oxygen, nitrogen, and hydrogen atoms are displayed in gray, red, blue, and white, respectively.The green dashed lines represent the possible hydrogen bonds.(D) The surface representation of the QB binding site with bound patulin.

Figure 2 .
Figure 2. Simulated modeling of patulin binding to the D1 protein of Arabidopsis.(A) The chemical structure of patulin.(B) Hydrogen bonding interactions of patulin binding to the D1 protein.(C) The stereo view of the patulin binding environment of the D1 protein, in which carbon, oxygen, nitrogen, and hydrogen atoms are displayed in gray, red, blue, and white, respectively.The green dashed lines represent the possible hydrogen bonds.(D) The surface representation of the Q B binding site with bound patulin.

Figure 2 .
Figure 2. Simulated modeling of patulin binding to the D1 protein of Arabidopsis.(A) The chemical structure of patulin.(B) Hydrogen bonding interactions of patulin binding to the D1 protein.(C) The stereo view of the patulin binding environment of the D1 protein, in which carbon, oxygen, nitrogen, and hydrogen atoms are displayed in gray, red, blue, and white, respectively.The green dashed lines represent the possible hydrogen bonds.(D) The surface representation of the QB binding site with bound patulin.

Figure 3 .
Figure 3. Binding interactions of patulin derivatives at the QB binding site of D1 protein of Arabidopsis.An illustration of the binding mode of compounds D3 (A), D6 (D), D34 (G), and D67 (J) binding to the D1 protein, respectively.Key interaction types are represented in the color code.The stereo view of compound D3 (B), D6 (E), D34 (H), and D67 (K) binding environments at the QB binding site.The surface representation of the QB binding site with compounds D3 (C), D6 (F), D34 (I), and D67 (L), respectively.

Figure 3 .
Figure 3. Binding interactions of patulin derivatives at the Q B binding site of D1 protein of Arabidopsis.An illustration of the binding mode of compounds D3 (A), D6 (D), D34 (G), and D67 (J) binding to the D1 protein, respectively.Key interaction types are represented in the color code.The stereo view of compound D3 (B), D6 (E), D34 (H), and D67 (K) binding environments at the Q B binding site.The surface representation of the Q B binding site with compounds D3 (C), D6 (F), D34 (I), and D67 (L), respectively.

Table 2 .
Structures and QED of new patulin's derivatives, which have different R group at C3 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).Five derivatives (D10, D12, D16, D18, and D19) show higher QED and binding affinity than patulin.

Table 2 .
Structures and QED of new patulin's derivatives, which have different R group at C3 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).Five derivatives (D10, D12, D16, D18, and D19) show higher QED and binding affinity than patulin.

Table 2 .
Structures and QED of new patulin's derivatives, which have different R group at C3 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).Five derivatives (D10, D12, D16, D18, and D19) show higher QED and binding affinity than patulin.

Table 4 .
Structures and QED of new patulin's derivatives, which have different group at 5-position, and the binding affinity between each compound and D1 protein of A. thaliana (5MDX).Five derivatives (D41, D43, D44, D45, and D46) show higher QED and binding affinity than patulin.

Table 4 .
Structures and QED of new patulin's derivatives, which have different group at 5-position, and the binding affinity between each compound and D1 protein of A. thaliana (5MDX).Five derivatives (D41, D43, D44, D45, and D46) show higher QED and binding affinity than patulin.

Table 6 .
Structures and QED of new patulin's derivatives, which have different R group at the C7 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).

Table 4 .
Structures and QED of new patulin's derivatives, which have different group at 5-position, and the binding affinity between each compound and D1 protein of A. thaliana (5MDX).Five derivatives (D41, D43, D44, D45, and D46) show higher QED and binding affinity than patulin.

Table 6 .
Structures and QED of new patulin's derivatives, which have different R group at the C7 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).

Table 6 .
Structures and QED of new patulin's derivatives, which have different R group at the C7 position, and the binding affinity between each compound and Arabidopsis D1 protein (5MDX).Seven derivatives (D69, D70, D71, D73, D75, D77, and D79) show higher QED and binding affinity than patulin.