ABSTRACT
Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) has a high status in the development of new inhibitors. To develop novel and highly effective PPO inhibitors, active substructure linking and bioisosterism replacement strategies were used to design and synthesize novel tetrahydrophthalimide derivatives containing oxadiazole/thiadiazole moieties, and their inhibitory effects on Nicotiana tobacco PPO (NtPPO) and herbicidal activity were evaluated. Among them, compounds B11 (Ki = 9.05 nM) and B20 (Ki = 10.23 nM) showed significantly better inhibitory activity against NtPPO than that against flumiclorac-pentyl (Ki = 46.02 nM). Meanwhile, compounds A20 and B20 were 100% effective against three weeds (Abutilon theophrasti, Amaranthus retroflexus, and Portulaca oleracea) at 37.5 g a.i./ha. It was worth observing that compound B11 was more than 90% effective against three weeds (Abutilon theophrasti, Amaranthus retroflexus, and Portulaca oleracea) at 18.75 and 9.375 g a.i./ha. It was also safer to rice, maize, and wheat than flumiclorac-pentyl at 150 g a.i./ha. In addition, the molecular docking results showed that compound B11 could stably bind to NtPPO and it had a stronger hydrogen bond with Arg98 (2.9 Å) than that of flumiclorac-pentyl (3.2 Å). This research suggests that compound B11 could be used as a new PPO inhibitor, and it could help control weeds in agricultural production.
Subject(s)
Amaranthus , Drug Design , Enzyme Inhibitors , Herbicides , Molecular Docking Simulation , Oxadiazoles , Phthalimides , Plant Weeds , Protoporphyrinogen Oxidase , Thiadiazoles , Herbicides/chemistry , Herbicides/pharmacology , Herbicides/chemical synthesis , Thiadiazoles/chemistry , Thiadiazoles/pharmacology , Thiadiazoles/chemical synthesis , Plant Weeds/drug effects , Plant Weeds/enzymology , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Oxadiazoles/chemical synthesis , Structure-Activity Relationship , Phthalimides/chemistry , Phthalimides/pharmacology , Phthalimides/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Amaranthus/chemistry , Amaranthus/drug effects , Plant Proteins/chemistry , Plant Proteins/antagonists & inhibitors , Molecular Structure , Nicotiana/chemistryABSTRACT
Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.
Subject(s)
Drug Design , Enzyme Inhibitors , Herbicides , Nicotiana , Plant Proteins , Protoporphyrinogen Oxidase , Pyridazines , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/metabolism , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/genetics , Pyridazines/chemistry , Pyridazines/pharmacology , Herbicides/pharmacology , Herbicides/chemistry , Herbicides/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Structure-Activity Relationship , Nicotiana/metabolism , Nicotiana/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Molecular Docking Simulation , Molecular Structure , Plant Weeds/drug effects , Plant Weeds/enzymology , KineticsABSTRACT
Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) is one of the most important targets for the discovery of green herbicides. In order to find novel PPO inhibitors with a higher herbicidal activity, a series of novel N-phenyltriazinone derivatives containing oxime ether and oxime ester groups were designed and synthesized based on the strategy of pharmacophore and scaffold hopping. Bioassay results revealed that some compounds showed herbicidal activities; especially, compound B16 exhibited broad-spectrum and excellent 100% herbicidal effects to Echinochloa crusgalli, Digitaria sanguinalis, Setaria faberii, Abutilon juncea, Amaranthus retroflexus, and Portulaca oleracea at a concentration of 37.5 g a.i./ha, which were comparable to trifludimoxazin. Nicotiana tabacum PPO (NtPPO) enzyme inhibitory assay indicated that B16 showed an excellent enzyme inhibitory activity with a value of 32.14 nM, which was similar to that of trifludimoxazin (31.33 nM). Meanwhile, compound B16 revealed more safety for crops (rice, maize, wheat, peanut, soybean, and cotton) than trifludimoxazin at a dose of 150 g a.i./ha. Moreover, molecular docking and molecular dynamics simulation further showed that B16 has a very strong and stable binding to NtPPO. It indicated that B16 can be used as a potential PPO inhibitor and herbicide candidate for application in the field.
Subject(s)
Enzyme Inhibitors , Herbicides , Molecular Docking Simulation , Oximes , Plant Proteins , Plant Weeds , Protoporphyrinogen Oxidase , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Herbicides/pharmacology , Herbicides/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Oximes/chemistry , Oximes/pharmacology , Structure-Activity Relationship , Plant Weeds/drug effects , Plant Weeds/enzymology , Plant Proteins/chemistry , Plant Proteins/antagonists & inhibitors , Plant Proteins/metabolism , Triazines/chemistry , Triazines/pharmacology , Esters/chemistry , Esters/pharmacology , Molecular Structure , Ethers/chemistry , Ethers/pharmacology , Drug DiscoveryABSTRACT
In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.
Subject(s)
Drug Design , Enzyme Inhibitors , Herbicides , Molecular Docking Simulation , Plant Weeds , Protoporphyrinogen Oxidase , Pyrrolidinones , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Herbicides/pharmacology , Herbicides/chemistry , Herbicides/chemical synthesis , Plant Weeds/drug effects , Plant Weeds/enzymology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Structure-Activity Relationship , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Pyrrolidinones/chemical synthesis , Plant Proteins/chemistry , Plant Proteins/antagonists & inhibitors , Pyridines/chemistry , Pyridines/pharmacology , Pyridines/chemical synthesis , Amaranthus/drug effects , Amaranthus/chemistry , Echinochloa/drug effects , Echinochloa/enzymology , Digitaria/drug effects , Digitaria/enzymology , Digitaria/chemistry , Lolium/drug effects , Lolium/enzymology , Molecular StructureABSTRACT
BACKGROUND: Fluorine plays a significant role in agrochemical science because approximately 25% of herbicides licensed worldwide contain this element. In a pool of previously synthesized benzoxazinones, some compounds contained fluorine and demonstrated inhibitory activities against protoporphyrinogen IX oxidase (PPO). Therefore, three data sets of benzoxazinone derivatives with known inhibitory activity against PPO were employed to build a multivariate image analysis applied to a quantitative structure-activity relationships (MIA-QSAR) model to identify improved analogs with at least one fluorine substituent. RESULTS: The QSAR model was vigorously validated and demonstrated to be highly predictive (r2 = 0.85, q2 = 0.71, and r2 pred = 0.88); thus, the model can provide reliable estimations for the PPO inhibitory activity of unknown derivatives. From these compounds, a couple of N-substituted benzoxazinones that contained the -CH2CHF2 group were found with predicted pKi values larger than 8 (Ki in mol L-1) and higher lipophilicity than the most active data set compounds. In addition, we carried out a systematic investigation of the binding mode of PPO by performing computational docking followed by molecular dynamics simulations. The proposed binding mode was consistent with experimental studies, and several potential key residues were identified. CONCLUSION: Two new proposed benzoxazinones exhibited better performance than compounds of the data set, and fluorine substituents played pivotal roles in describing the biological activities. © 2024 Society of Chemical Industry.
Subject(s)
Benzoxazines , Enzyme Inhibitors , Molecular Docking Simulation , Molecular Dynamics Simulation , Protoporphyrinogen Oxidase , Quantitative Structure-Activity Relationship , Protoporphyrinogen Oxidase/antagonists & inhibitors , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Benzoxazines/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Herbicides/chemistry , Herbicides/pharmacology , Halogenation , Molecular Structure , Drug DesignABSTRACT
To find novel herbicidal compounds with high activity and broad spectrum, a series of phenylpyridine moiety-containing α-trifluoroanisole derivatives were designed, synthesized, and identified via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Greenhouse-based herbicidal activity assays revealed that compound 7a exhibited > 80% inhibitory activity against Abutilon theophrasti, Amaranthus retroflexus, Eclipta prostrate, Digitaria sanguinalis, and Setaria viridis at a dose of 37.5 g a.i./hm2, which was better than fomesafen. Compound 7a further exhibited excellent herbicidal activity against Abutilon theophrasti and Amaranthus retroflexus in this greenhouse setting, with respective median effective dose (ED50) values of 13.32 and 5.48 g a.i./hm2, both of which were slightly superior to fomesafen (ED50 = 36.39, 10.09 g a.i./hm2). The respective half-maximal inhibitory concentration (IC50) for compound 7a and fomesafen when used to inhibit the Nicotiana tabacum protoporphyrinogen oxidase (NtPPO) enzyme, were 9.4 and 110.5 nM. The docking result of compound 7a indicated that the introduction of 3-chloro-5-trifluoromethylpyridine and the trifluoromethoxy group was beneficial to the formation of stable interactions between these compounds and NtPPO. This work demonstrated that compound 7a could be further optimized as a PPO herbicide candidate to control various weeds.
Subject(s)
Amaranthus , Herbicides , Benzamides/pharmacology , Herbicides/chemistry , Herbicides/pharmacology , Plant Weeds , Protoporphyrinogen Oxidase/chemistry , Structure-Activity Relationship , NicotianaABSTRACT
Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.
Subject(s)
Flavoproteins/genetics , Mitochondrial Proteins/genetics , Mutation/genetics , Protoporphyrinogen Oxidase/genetics , Amino Acid Sequence , Biophysical Phenomena , Cell Line , Enzyme Stability , Flavoproteins/chemistry , Flavoproteins/isolation & purification , Humans , Kinetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/isolation & purification , Models, Molecular , Protein Multimerization , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/isolation & purification , Subcellular Fractions/metabolism , TemperatureABSTRACT
Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.
Subject(s)
Arginine/chemistry , Arginine/metabolism , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Amino Acid Sequence , Arginine/genetics , Enzyme Assays/methods , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Mutagenesis, Site-Directed/methods , Protein Structural Elements , Protoporphyrinogen Oxidase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structure-Activity RelationshipABSTRACT
Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204-210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the ß-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction.
Subject(s)
Protoporphyrinogen Oxidase/metabolism , Catalysis , Computer Simulation , Protoporphyrinogen Oxidase/chemistry , Protoporphyrins/chemistry , Tetrapyrroles/chemistryABSTRACT
Protoporphyrinogen oxidase (PPO) has been identified as one of the most promising targets for herbicide discovery. A series of novel phthalimide derivatives were designed by molecular docking studies targeting the crystal structure of mitochondrial PPO from tobacco (mtPPO, PDB: 1SEZ) by using Flumioxazin as a lead, after which the derivatives were synthesized and characterized, and their herbicidal activities were subsequently evaluated. The herbicidal bioassay results showed that compounds such as 3a (2-(4-bromo-2,6-difluorophenyl) isoindoline-1,3-dione), 3d (methyl 2-(4-chloro-1,3-dioxoisoindolin-2-yl)-5-fluorobenzoate), 3g (4-chloro-2-(5-methylisoxazol-3-yl) isoindoline-1,3-dione), 3j (4-chloro-2-(thiophen-2-ylmethyl) isoindoline-1,3-dione) and 3r (2-(4-bromo-2,6-difluorophenyl)-4-fluoroisoindoline-1,3-dione) had good herbicidal activities; among them, 3a showed excellent herbicidal efficacy against A. retroflexus and B. campestris via the small cup method and via pre-emergence and post-emergence spray treatments. The efficacy was comparable to that of the commercial herbicides Flumioxazin, Atrazine, and Chlortoluron. Further, the enzyme activity assay results suggest that the mode of action of compound 3a involves the inhibition of the PPO enzyme, and 3a showed better inhibitory activity against PPO than did Flumioxazin. These results indicate that our molecular design strategy contributes to the development of novel promising PPO inhibitors.
Subject(s)
Enzyme Inhibitors , Herbicides , Molecular Docking Simulation , Plant Proteins/antagonists & inhibitors , Plant Weeds/enzymology , Protoporphyrinogen Oxidase/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Herbicides/chemical synthesis , Herbicides/chemistry , Herbicides/pharmacology , Phthalimides/chemical synthesis , Phthalimides/chemistry , Phthalimides/pharmacology , Plant Proteins/chemistry , Plant Proteins/metabolism , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolismABSTRACT
Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the discovery and systematic SAR-based structure optimization of novel N-isoxazolinylphenyltriazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the new synthetic compounds displayed strong PPO inhibitory activity with Ki values in the nanomolar range. Some compounds exhibited excellent and broad-spectrum weed control at the rate of 9.375-37.5 g ai/ha by postemergence application and showed improved monocotyledonous weed control compared to saflufenacil. Most promisingly, ethyl 3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylate, 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold higher potency than saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 5a showed excellent and broad-spectrum weed control against 32 kinds of weeds at 37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by postemergence application, indicating that 5a could be a potential herbicide candidate for weed control in paddy fields.
Subject(s)
Enzyme Inhibitors/pharmacology , Herbicides/chemistry , Herbicides/pharmacology , Plant Proteins/antagonists & inhibitors , Protoporphyrinogen Oxidase/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/chemistry , Kinetics , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Weeds/chemistry , Plant Weeds/drug effects , Plant Weeds/enzymology , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Quantitative Structure-Activity Relationship , Nicotiana/chemistry , Nicotiana/drug effects , Nicotiana/enzymology , Weed ControlABSTRACT
The herbicide fomesafen has the advantages of low toxicity and high selectivity, and the target of this compound is protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4). However, this herbicide has a long residual period and can have phytotoxic effects on succeeding crops. To protect maize from fomesafen, a series of thiazole phenoxypyridines were designed based on structure-activity relationships, active substructure combinations, and bioisosterism. Bioassays showed that thiazole phenoxypyridines could improve maize tolerance under fomesafen toxicity stress to varying degrees at a dose of 10 mg·kg-1. Compound 4i exhibited the best effects. After being treated by compound 4i, average recovery rates of growth index exceeded 72%, glutathione content markedly increased by 167% and glutathione S-transferase activity was almost 163% of fomesafen-treated group. More importantly, after being treated by compound 4i, the activity of PPO, the main target enzyme of fomesafen, recovered to 93% of the control level. The molecular docking result exhibited that the compound 4i could compete with fomesafen to bind with the herbicide target enzyme, which consequently attained the herbicide detoxification. The present work suggests that compound 4i could be developed as a potential safener to protect maize from fomesafen.
Subject(s)
Protoporphyrinogen Oxidase/metabolism , Pyridines/chemical synthesis , Thiazoles/chemical synthesis , Zea mays/growth & development , Benzamides/adverse effects , Benzamides/pharmacology , Drug Design , Gene Expression Regulation, Plant/drug effects , Glutathione/metabolism , Glutathione Transferase/metabolism , Models, Molecular , Molecular Docking Simulation , Plant Proteins/chemistry , Plant Proteins/metabolism , Protoporphyrinogen Oxidase/chemistry , Pyridines/chemistry , Pyridines/pharmacology , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Zea mays/drug effects , Zea mays/metabolismABSTRACT
In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO (NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80% control of all three tested broadleaf weeds at 37.5-150 g ai/ha by postemergent application, and a majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha. Promisingly, 17i (Ki = 6.7 nM) was 6 and 4 times more potent than flumioxazin (Ki = 46 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent, broad-spectrum herbicidal activity, even at levels as low as 37.5 g ai/ha, and it was determined to be safe for wheat at 150 g ai/ha in postemergent application, indicating the great potential for 17i development as a herbicide for weed control in wheat fields.
Subject(s)
Benzoxazines/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Herbicides/chemistry , Herbicides/pharmacology , Plant Proteins/antagonists & inhibitors , Protoporphyrinogen Oxidase/antagonists & inhibitors , Quinazolines/chemistry , Benzoxazines/pharmacology , Drug Design , Kinetics , Plant Proteins/chemistry , Plant Weeds/drug effects , Plant Weeds/enzymology , Protoporphyrinogen Oxidase/chemistry , Quantitative Structure-Activity Relationship , Quinazolines/pharmacology , Nicotiana/drug effects , Nicotiana/enzymology , Weed ControlABSTRACT
Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.
Subject(s)
Coproporphyrinogen Oxidase/metabolism , Heme/metabolism , Protoporphyrinogen Oxidase/metabolism , Synechocystis/enzymology , Tetrapyrroles/metabolism , Coproporphyrinogen Oxidase/chemistry , Heme/chemistry , Models, Molecular , Oxidation-Reduction , Protoporphyrinogen Oxidase/chemistry , Tetrapyrroles/chemistryABSTRACT
Tyrosinases and catechol oxidases belong to the polyphenol oxidase (PPO) enzyme family, which is mainly responsible for the browning of fruits. Three cDNAs encoding PPO pro-enzymes have been cloned from leaves of Malus domestica (apple, MdPPO). The three pro-enzymes MdPPO1-3 were heterologously expressed in E. coli yielding substantial amounts of protein and have been characterized with regard to their optimum of activity resulting from SDS, acidic and proteolytic activation. Significant differences were found in the kinetic characterization of MdPPO1-3 when applying different mono- and diphenolic substrates. All three enzymes have been classified as tyrosinases, where MdPPO1 exhibits the highest activity with tyramine (kcat = 9.5 s-1) while MdPPO2 and MdPPO3 are also clearly active on this monophenolic substrate (kcat = 0.92 s-1 and kcat = 1.0 s-1, respectively). Based on the activity, sequence data and homology modelling it is proposed that the monophenolase and diphenolase activity of PPOs can be manipulated by the appropriate combination of two amino acids, which are located within the active site cleft and were therefore named "activity controllers".
Subject(s)
Amino Acids/chemistry , Catechol Oxidase/chemistry , Catechol Oxidase/metabolism , Malus/enzymology , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/metabolism , Recombinant Proteins , Amino Acid Sequence , Catechol Oxidase/genetics , Gene Expression , Malus/genetics , Models, Molecular , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/isolation & purification , Protein Conformation , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
To search for new protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors with improved bioactivity, a series of novel pyrido[2,3-d]pyrimidine-2,4-dione-benzoxazinone hybrids, 9-13, were designed and synthesized. Several compounds with improved tobacco PPO (mtPPO)-inhibiting and promising herbicidal activities were found. Among them, the most potent compound, 3-(7-fluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[b][1,4] oxazin-6-yl)-1-methylpyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione, 11q, with a Ki value of 0.0074 µM, showed six times more activity than flumioxazin (Ki = 0.046 µM) against mtPPO. Compound 11q displayed a strong and broad spectrum of weed control at 37.5-150 g of active ingredient (ai)/ha by both post- and pre-emergence application, which was comparable to that of flumioxazin. 11q was safe to maize, soybean, peanut, and cotton at 150 g ai/ha, and selective to rice and wheat at 75 g ai/ha by pre-emergence application, indicating potential applicability in these fields.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Herbicides/chemical synthesis , Herbicides/pharmacology , Plant Proteins/antagonists & inhibitors , Protoporphyrinogen Oxidase/antagonists & inhibitors , Pyrimidines/chemistry , Benzoxazines/chemistry , Enzyme Inhibitors/chemistry , Herbicides/chemistry , Kinetics , Plant Proteins/chemistry , Plant Weeds/drug effects , Plant Weeds/enzymology , Protoporphyrinogen Oxidase/chemistry , Structure-Activity Relationship , Nicotiana/enzymologyABSTRACT
Tuning the binding selectivity through appropriate ways is a primary goal in the design and optimization of a lead toward agrochemical discovery. However, how to achieve rational design of selectivity is still a big challenge. Herein, we developed a novel computational fragment generation and coupling (CFGC) strategy that led to a series of highly potent and bioselective inhibitors targeting protoporphyrinogen IX oxidase. This enzyme plays a vital role in heme and chlorophyll biosynthesis, which has been proven to be associated with many drugs and agrochemicals. However, existing agrochemicals are nonbioselective, resulting in a great threat to nontargeted organisms. To the best of our knowledge, this is the first bioselective inhibitor targeting the tetrapyrrole biosynthesis pathway. In addition, the candidate showed excellent in vivo bioactivity and much better safety toward humans.
Subject(s)
Enzyme Inhibitors/chemistry , Protoporphyrinogen Oxidase/antagonists & inhibitors , Chlorophyll/metabolism , Computational Biology , Heme/metabolism , Humans , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Nicotiana/chemistry , Nicotiana/enzymologyABSTRACT
Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX) in the haem/chlorophyll biosynthetic pathway. Although extensive studies of PPO have already afforded many insights into its biological function and its significance to agriculture and medicine, details of the enzymatic mechanism as well as the nature of the specific amino acids involved in substrate binding still remain largely unknown due to the lack of structural information about protogen IX binding to PPO. In this study, we carried out a detailed and systematic investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis. The proposed binding mode was consistent with experimental studies, and several potential key residues that have not been investigated in previous studies, such as Thr70, Arg233, Ser235, Ser474 and Lys477, were identified. In addition, we compared the binding modes of protogen IX in mtPPO and Homo sapiens PPO, and found their differences. Considering the low sequence identity and the differences of biochemical properties among the PPOs from various species, the investigation could provide a valuable basis for the design of PPO inhibitors with high potency and species-selectivity.
Subject(s)
Molecular Docking Simulation , Molecular Dynamics Simulation , Protoporphyrinogen Oxidase/chemistry , Protoporphyrins/chemistry , Molecular Conformation , Molecular Structure , Protoporphyrinogen Oxidase/metabolism , Protoporphyrins/metabolismABSTRACT
Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is known as a key action target for several structurally diverse herbicides. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel 3-(2'-halo-5'-substituted-benzothiazol-1'-yl)-1-methyl-6-(trifluoromethyl)pyrimidine-2,4-diones 9 were designed and synthesized. The bioassay results indicated that a number of the newly synthesized compounds exhibited higher inhibition activity against tobacco PPO (mtPPO) than the controls, saflufenacil and sulfentrazone. Compound 9F-5 was identified as the most potent inhibitor with a Ki value of 0.0072 µM against mtPPO, showing about 4.2-fold and 1.4-fold higher potency than sulfentrazone (Ki = 0.03 µM) and saflufenacil (Ki = 0.01 µM), respectively. An additional green house assay demonstrated that compound 9F-6 (Ki = 0.012 µM) displayed the most promising postemergence herbicidal activity with a broad spectrum even at a concentration as low as 37.5 g of active ingredient (ai)/ha. Maize exhibits relative tolerance against compound 9F-6 at the dosage of 150 g ai/ha, but it is susceptible to saflufenacil even at 75 g ai/ha. Thus, compound 9F-6 exhibits the potential to be a new herbicide for weed control in maize fields.
Subject(s)
Enzyme Inhibitors/pharmacology , Herbicides/pharmacology , Nicotiana/enzymology , Plant Proteins/chemistry , Protoporphyrinogen Oxidase/chemistry , Enzyme Inhibitors/chemistry , Herbicides/chemical synthesis , Herbicides/chemistry , Kinetics , Plant Proteins/metabolism , Protoporphyrinogen Oxidase/metabolism , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Quantitative Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Nicotiana/drug effects , Triazoles/chemistry , Triazoles/pharmacologyABSTRACT
Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.