4-hydroxyphenylpyruvate Dioxygenase Research Articles

Discovery of tetrazolium nicotinamide derivatives as novel 4-Hydroxyphenylpyruvate dioxygenase inhibiting-based herbicides

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is currently one of the popular targets of herbicide research, and herbicides targeting it have shown promising results in treating resistant weeds. However, the long-term application of HPPD inhibitors with in their classical scaffolds inevitably leads to the development of drug resistance. To further delay the underlying development of weed resistance to HPPD inhibitors, we successfully screened HPPD inhibitor pharmacophore with novel chelate structures using computer-aided drug molecular design and obtained 2-trifluoromethyltetrazolium nicotinamide derivatives through active fragment splicing. Among these derivatives, 6-(3,3-diethyl-1-methylureido)-N-(1-methyl-1H-tetrazol-5-yl)-2-(trifluoromethyl)nicotinamide (compound 31), exhibited the most promising in vitro enzyme inhibitory activity, with IC50 value of 0.072 μM, which was approximately five times better than that of the control agent mesotrione (IC50 = 0.363 μM). The crystal structure of the AtHPPD complexed with compound 31 (2.0 Å) and the binding energy calculations of the representative compounds revealed several important interactions of the ligand binding to the target protein, which explained the superior enzyme inhibitory activity of the compounds at the molecular level. In addition, compounds 7 and 31 possessed 100% inhibition against the five target weeds at the tested dosage and both were more effective against Setaria viridis than mesotrione. In general, it is promising to design novel HPPD inhibitors by developing novel chelating structures, and compounds 7 or 31 could be used as the leads for further development of valuable HPPD inhibitors.

Filago pyramidata Tolerant to ALS-Inhibiting Herbicides: A New Invasive Weed in Olive Groves of Southern Spain

Weeds that usually grow in non-agricultural areas have become increasingly common invading perennial crops. Species of the genus Filago, in addition to invading Spanish olive groves, have developed certain levels of natural tolerance to the acetolactate synthase (ALS) inhibiting herbicide flazasulfuron. The objective of this study was to determine the level and the mechanism involved in the tolerance to flazasulfuron in Filago pyramidata L., which occurs in olive groves of southern Spain, as well as to identify possible cross- or multiple-tolerances by evaluating alternative herbicides for its control. A population resistant (R) to flazasulfuron and a susceptible (S) one of Conyza canadensis were used as references. The accessions of F. pyramidata presented LD50 values (from 72 to 81 g active ingredient (ai) ha−1) higher than the field dose of flazasulfuron (50 g ai ha−1), being 11–12.5 times more tolerant than the S population of C. canadensis, but less than half the R population (170 g ai ha−1). Enzymatically, F. pyramidata was as sensitive to flazasulfuron (I50 = 17.3 μM) as the S population of C. canadensis. Filago pyramidata plants treated with flazasulfuron, combined with 4-chloro-7-nitro-2,1,3-benzoxadiazole, had a growth reduction of up to 85%, revealing the participation of glutathione-S-transferases in herbicide metabolism. Filago pyramidata presented cross-tolerance to the different chemical groups of ALS inhibitors, except triazolinones (florasulam). Synthetic auxins (2,4-D and fluroxypyr) presented good control, but some individuals survived (low multiple resistance). Cellulose synthesis, 5-enolpyruvylshikimate-3-phosphate, 4-hydroxyphenylpyruvate dioxygenase, protoporphyrinogen oxidase, photosystem I, and photosystem II inhibitor herbicides, applied in PRE or POST-emergence, presented excellent levels of control of F. pyramidata. These results confirmed the natural tolerance of F. pyramidata to flazasulfuron and cross-tolerance to most ALS-inhibiting herbicides. The mechanism involved was enhanced metabolism mediated by glutathione-S-transferases, which also conferred low multiple tolerance to synthetic auxins. Even so, herbicides with other mechanisms of action still offer excellent levels of control of F. pyramidata.

Design, Synthesis, and Bioactivity of Novel Ester-Substituted Cyclohexenone Derivatives as Safeners.

Tembotrione, a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor, has been widely used in many types of plants. Tembotrione has been reported for its likelihood of causing injury and plant death to certain corn hybrids. Safeners are co-applied with herbicides to protect certain crops without compromising weed control efficacy. Alternatively, herbicide safeners may effectively improve herbicide selectivity. To address tembotrione-induced Zea mays injury, a series of novel ester-substituted cyclohexenone derivatives were designed using the fragment splicing method. In total, 35 title compounds were synthesized via acylation reactions. All the compounds were characterized using infrared spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry. The configuration of compound II-15 was confirmed using single-crystal X-ray diffraction. The bioactivity assay proved that tembotrione phytotoxicity to maize could be reduced by most title compounds. In particular, compound II-14 exhibited the highest activity against tembotrione. The molecular structure comparisons as well as absorption, distribution, metabolism, excretion, and toxicity predictions demonstrated that compound II-14 exhibited pharmacokinetic properties similar to those of the commercial safener isoxadifen-ethyl. The molecular docking model indicated that compound II-14 could prevent tembotrione from reaching or acting with Z. mays HPPD (PDB: 1SP8). Molecular dynamics simulations showed that compound II-14 maintained satisfactory stability with Z. mays HPPD. This research revealed that ester-substituted cyclohexenone derivatives can be developed as potential candidates for discovering novel herbicide safeners in the future.

Optimization of annealing temperature for amplification of the exon one region of the HPPD gene in HA1 accession sunflowers

The HPPD gene is one of the genes that is actively involved in the biosynthesis of tocopherol, which is the main component of Vitamin E. Sunflower is raw materials for natural medicine since their oil contains vitamin E. To obtain complete information regarding the whole sequences of the 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) gene in sunflower accession HA1, the exon one region needs to be identified using a PCR-based method. One factor affecting the success rate of PCR is the annealing temperature. The optimum annealing temperature will produce specific target gene products. This study aimed to determine the optimal temperature for amplifying the exon 1 region of the HPPD gene using the PCR method. This research began with primer design activities, followed by in vitro amplification (PCR), then optimization of annealing temperature using gradient PCR. The results showed that the optimum temperature for amplifying fragment one was an annealing temperature of 53 °C, fragment two was at an annealing temperature of 45 °C, and fragment three had an annealing temperature of 57 °C with the expected size of PCR products are 306 bp, 506 bp, and 534 bp respectively.

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.

Identification of Potential Inhibitors for the Treatment of Alkaptonuria Using an Integrated In Silico Computational Strategy.

Alkaptonuria (AKU) is a rare genetic autosomal recessive disorder characterized by elevated serum levels of homogentisic acid (HGA). In this disease, tyrosine metabolism is interrupted because of the alterations in homogentisate dioxygenase (HGD) gene. The patient suffers from ochronosis, fractures, and tendon ruptures. To date, no medicine has been approved for the treatment of AKU. However, physiotherapy and strong painkillers are administered to help mitigate the condition. Recently, nitisinone, an FDA-approved drug for type 1 tyrosinemia, has been given to AKU patients in some countries and has shown encouraging results in reducing the disease progression. However, this drug is not the targeted treatment for AKU, and causes keratopathy. Therefore, the foremost aim of this study is the identification of potent and druggable inhibitors of AKU with no or minimal side effects by targeting 4-hydroxyphenylpyruvate dioxygenase. To achieve our goal, we have performed computational modelling using BioSolveIT suit. The library of ligands for molecular docking was acquired by fragment replacement of reference molecules by ReCore. Subsequently, the hits were screened on the basis of estimated affinities, and their pharmacokinetic properties were evaluated using SwissADME. Afterward, the interactions between target and ligands were investigated using Discovery Studio. Ultimately, compounds c and f were identified as potent inhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Chemical Evaluation, Phytotoxic Potential, and In Silico Study of Essential Oils from Leaves of Guatteria schomburgkiana Mart. and Xylopia frutescens Aubl. (Annonaceae) from the Brazilian Amazon.

The essential oils (EOs) of Guatteria schomburgkiana (Gsch) and Xylopia frutescens (Xfru) (Annonaceae) were obtained by hydrodistillation, and their chemical composition was evaluated by gas chromatography-mass spectrometry (GC/MS). Herbicide activity was measured by analyzing the seed germination percentage and root and hypocotyl elongation of two invasive species: Mimosa pudica and Senna obtusifolia. The highest yield was obtained for the EO of Xfru (1.06%). The chemical composition of Gsch was characterized by the presence of the oxygenated sesquiterpenes spathulenol (22.40%) and caryophyllene oxide (14.70%). Regarding the EO of Xfru, the hydrocarbon monoterpenes α-pinene (35.73%) and β-pinene (18.90%) were the components identified with the highest concentrations. The germination of seeds of S. obtusifolia (13.33 ± 5.77%) showed higher resistance than that of seeds of M. pudica (86.67 ± 5.77%). S. obtusifolia was also more sensitive to the EO of Xfru in terms of radicle (55.22 ± 2.72%) and hypocotyl (71.12 ± 3.80%) elongation, while M. pudica showed greater sensitivity to the EO of Gsch. To screen the herbicidal activity, the molecular docking study of the major and potent compounds was performed against 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein. Results showed good binding affinities and attributed the strongest inhibitory activity to δ-cadinene for the target protein. This work contributes to the study of the herbicidal properties of the EOs of species of Annonaceae from the Amazon region.

β-triketone herbicide exposure cause tyrosine and fat accumulation in Caenorhabditis elegans

β-triketone herbicides have been efficiently employed as an alternate to atrazine. Triketones are 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme inhibitors and exposure is reported to cause significant increase in plasma tyrosine levels. In this study, we have employed a non-target organism Caenorhabditis elegans to determine the impact of β-triketone exposures at recommended field doses (RfD). Our results indicate sulcotrione and mesotrione, negatively influence the survival, behavior, and reproduction of the organism at RfD. Additionally, we have traced the parallels regarding the impact of triketones on the tyrosine metabolism pathway, in C. elegans to those in mammalian models, wherein the expression of the tyrosine metabolism pathway genes are altered, directly influencing tyrosine catabolism leading to significant tyrosine accumulation in exposed organism. Further, we investigated the impact of sulcotrione and mesotrione exposure on fat deposition (triglyceride levels, Oil-Red-O staining and lipidomics) and the fatty acid metabolism pathway. In the exposed worms, the expression of enlongases and fatty acid desaturases were up-regulated along with an increase in the levels of triglycerides. Thus, the data indicates a positive association of β-triketone exposure to mis-regulation of the fatty acid metabolism pathway genes leading to fat accumulation in worms. Therefore, β-triketone might be a potential obesogen.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Discovery of novel HPPD inhibitors: Virtual screening, molecular design, structure modification and biological evaluation

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD, a Fe(II)/α-ketoglutarate dependent oxygenases), is a popular herbicide target. In this work, two pharmacophore models based on common molecular characteristics (HipHop) and receptor-ligand complex (CBP) were generated for virtual screening for HPPD inhibitors. About 1,000,000 molecules containing diketone structure from PubChem were filtered by Lipinski's rules to build a 3D database. Then the database was screened through combining HipHop model, CBP model, ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. Subsequently, based on the specific binding mode and affinity of HPPD inhibitors, 4 molecules with high -CDOCKER energy, good aqueous solubility and human safety predicative properties values were screened. From the screening results and combined with previous work, three novel HPPD inhibitors were designed and synthesized through fragment splicing and bioisosterism strategies. Compound IV-a exhibited similar inhibition of Arabidopsis thaliana HPPD (AtHPPD) and herbicidal activity as mesotrione. Crop selectivity showed that compound IV-a had better crop safety than mesotrione. Comparing the molecular properties, ADMET and molecular docking studies indicated that compounds IV-a exhibited better properties than mesotrione, which could be further modified as novel HPPD inhibitor herbicides.

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes.

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Photodegradation of the Novel Herbicide Pyraquinate in Aqueous Solution: Kinetics, Photoproducts, Mechanisms, and Toxicity Assessment.

Pyraquinate, a newly developed 4-hydroxyphenylpyruvate dioxygenase class herbicide, has shown excellent control of resistant weeds in paddy fields. However, its environmental degradation products and corresponding ecotoxicological risks after field application remain ambiguous. In this study, we systematically investigate the photolytic behaviors of pyraquinate in aqueous solutions and in response to xenon lamp irradiation. The degradation follows first-order kinetics, and its rate depends on pH and the amount of organic matter. No vulnerability to light radiation is indicated. Ultrahigh-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry and UNIFI software analysis reveals six photoproducts generated by methyl oxidation, demethylation, oxidative dechlorination, and ester hydrolysis. Gaussian calculation suggests that activities due to hydroxyl radicals or aquatic oxygen atoms caused these reactions on the premise of obeying thermodynamic criteria. Practical toxicity test results show that the toxicity of pyraquinate to zebrafish embryos is low but increases when the compound is combined with its photoproducts.

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

ABSTRACT 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 r 2, q 2, and r 2 pred 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.

Discovery of Novel Pyrazole Derivatives with Improved Crop Safety as 4-Hydroxyphenylpyruvate Dioxygenase-Targeted Herbicides.

As one of the essential herbicide targets, 4-hydroxyphenylpyruvate dioxygenase (HPPD) has recently been typically used to produce potent new herbicides. In continuation with the previous work, several pyrazole derivatives comprising a benzoyl scaffold were designed and synthesized, and their inhibitory effects on Arabidopsis thaliana hydroxyphenylpyruvate dioxygenase (AtHPPD) and herbicidal activities were comprehensively evaluated in this study. Compound Z9 showed top-rank inhibitory activity to AtHPPD with an half-maximal inhibitory concentration (IC50) value of 0.05 μM, which was superior to topramezone (1.33 μM) and mesotrione (1.76 μM). Compound Z21 exhibited superior preemergence inhibitory activity against Echinochloa crusgalli, with stem and root inhibition rates of 44.3 and 69.6%, respectively, compared to topramezone (16.0 and 53.0%) and mesotrione (12.8 and 41.7%). Compounds Z5, Z15, Z20, and Z21 showed excellent postemergence herbicidal activities at a dosage of 150 g ai/ha, along with distinct bleaching symptoms and higher crop safety than topramezone and mesotrione, and they all were safe for maize, cotton, and wheat with injury rates of 0 or 10%. In addition, the molecular docking analysis also revealed that these compounds formed hydrophobic π-π interactions with Phe360 and Phe403 to AtHPPD. This study suggests that pyrazole derivatives containing a benzoyl scaffold could be used as new HPPD inhibitors to develop pre- and postemergence herbicides and be applied to additional crop fields.

Adjuvant and nozzle effects on weed control using mesotrione and rimsulfuron plus thifensulfuron-methyl

Limitations on the available herbicide portfolio for weed control have led to weed control failures. Application techniques such as proper selection of nozzle types, adjuvants, and herbicide selection have been suggested as one of the ways to maximize efficacy. The launch of HPPD (hydroxyphenylpyruvate dioxygenase)-tolerant crops will increase HPPD herbicide applications, while effective herbicide tank mixtures might prolong herbicide resistance evolution. Therefore, a greenhouse experiment was conducted to evaluate the effects of mesotrione (MES) and rimsulfuron plus thifensulfuron-methyl (RIMTHIF) in tank mixtures with five adjuvants sprayed with three different nozzle types, XR (Extended Range), AIXR (Air Induction Extended Range), and TTI (Turbo TeeJet Induction), on weed control. Five weed species were tested: common lambsquarters (Chenopodium album L.), velvetleaf (Abutilon theophrasti Medic.), common waterhemp (Amaranthus tuberculatus (Moq.) J.D. Sauer), Palmer amaranth (Amaranthus palmeri S. Wats.), barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.), and a grain sorghum (Sorghum bicolor (L.) Moench) crop. A dose‒response study using MES and RIMTHIF was conducted to estimate the effective dose to reduce the aboveground biomass by 50% from the nontreated control (ED50). Another study was conducted to evaluate the efficacy of MES and RIMTHIF in tank mixtures with five adjuvants (ammonium sulfate – AMS, crop oil concentrate – COC, drift reducing adjuvant – DRA, methylated seed oil – MSO, and nonionic surfactant – NIS) sprayed with three nozzle types (XR, AIXR, and TTI). The selected nozzles influenced velvetleaf, common lambsquarters, and sorghum, indicating the possibility of applying herbicides with Coarser sprays, avoiding off-target movement. Our findings suggest adjuvants are an essential factor for improving weed control over the active ingredient, up to 18.7% and 38.3% for MES and RIMTHIF, respectively. Barnyardgrass biomass reduction was greater than 98% with RIMTHIF in tank mixtures with COC and MSO compared to 65% when applied alone. Common lambsquarters had 52.1% biomass reduction when RIMTHIF was sprayed alone, while adding adjuvants to the tank-mixture resulted in biomass reduction ranging from 60.1% (COC) to 94.1% (AMS). Velvetleaf biomass reduction was 98% when the MES and RIMTHIF mixture was applied with the addition of MSO, while 87% without the adjuvant added. These results indicate that adding adjuvants in herbicide solution can improve herbicide efficacy (COC and MSO for barnyardgrass; NIS, AMS, and MSO for velvetleaf; and NIS, COC, and MSO for common lambsquaters).

Hydroxyphenylpyruvate Dioxygenase Is a Metabolic Immune Checkpoint for UTX-deficient Colorectal Cancer

Aberrant epigenetic events mediated by histone methyltransferases and demethylases contribute to malignant progression of colorectal cancer (CRC). However, the role of the histone demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in CRC remains poorly understood. UTX conditional knockout mice and UTX-silenced MC38 cells were used to investigate UTX function in tumorigenesis and development of CRC. We performed time of flight mass cytometry to clarify the functional role of UTX in remodeling immune microenvironment of CRC. To investigate metabolic interaction between myeloid-derived suppressor cells (MDSCs) and CRC, we analyzed metabolomics data to identify metabolites secreted by UTX-deficient cancer cells and taken up by MDSCs. We unraveled a tyrosine-mediated metabolic symbiosis between MDSC and UTX-deficient CRC. Loss of UTX in CRC resulted in methylation of phenylalanine hydroxylase, preventing its degradation and subsequently increasing tyrosine synthesis and secretion. Tyrosine taken up by MDSCs was metabolized to homogentisic acid by hydroxyphenylpyruvate dioxygenase. Homogentisic acid modified protein inhibitor of activated STAT3 via carbonylation of Cys 176, and relieved the inhibitory effect of protein inhibitor of activated STAT3 on signal transducer and activator of transcription 5 transcriptional activity. This in turn, promoted MDSC survival and accumulation, enabling CRC cells to acquire invasive and metastatic traits. Collectively, these findings highlight hydroxyphenylpyruvate dioxygenase as a metabolic checkpoint to restrict immunosuppressive MDSCs and to counteract malignant progression of UTX-deficient CRC.

Species differences and human relevance of the toxicity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and a new approach method in vitro for investigation

The mode of action (MoA) of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides in mammals is well described and is generally accepted to be due to a build-up of excess systemic tyrosine which is associated with the range of adverse effects reported in laboratory animals. What is less well accepted is the basis for the marked difference in the effects of HPPD inhibitors that has been observed across experimental species and humans, where some species show significant toxicities whereas in other species exposure causes few effects. The activity of the catabolic enzyme tyrosine aminotransferase (TAT) varies across species including humans and it is hypothesized that this primarily accounts for the different levels of tyrosinemia observed between species and leads to the subsequent differences in toxicity. The previously reported activities of TAT in different species showed large variation, were inconsistent, have methodological uncertainties and could lead to a reasonable challenge to the scientific basis for the species difference in response. To provide clarity, a new method was developed for the simultaneous and systematic measurement of TAT in vitro using robust methodologies in a range of mammalian species including human. The results obtained showed general correlation between high TAT activity and low in vivo toxicity when using a model based on hepatic cytosol and a very convincing correlation when using a primary hepatocyte model. These data fully support the role of TAT in explaining the species differences in toxicity. Moreover, this information should give greater confidence in selecting the most appropriate animal model (the mouse) for human health risk assessment and for key classification and labeling decision-making.

Role of metabolization and conjugation of herbicides in tolerance of white oats to 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) enzyme inhibitors

The success of chemical control is dependent on crop tolerance to herbicides and their efficiency in controlling weeds. Thus, knowledge of the tolerance level of cultivated plants and the tolerance mechanism involved is crucial for safe herbicide use. The objective of this study was to evaluate the effect of metabolization (malathion) and conjugation (NBD-Cl) inhibitors on the tolerance of white oats to the herbicides mesotrione and tembotrione. The experiment was carried out under field conditions in the 2021 crop, in an RBD, with nine treatments, namely: T0- control (no application); T1- malathion; T2- NBD-Cl; T3- mesotrione; T4- mesotrione+malathion; T5- mesotrione+NBD-Cl; T6- tembotrione; T7- tembotrione+malathion and T8- tembotrione+NBD-Cl. The plant characteristics were evaluated for plant intoxication, SPAD index, levels of chlorophyll a, b and total carotenoids, yield components, yield and industrial grain quality. Symptoms of intoxication in oat plants started at 7 DAA and increased up to 14 DAA in treatments containing mesotrione or tembotrione. Plant recovery started at 21 DAA, and it was intensified at 28 DAA. Intoxication levels were not affected by the addition of the metabolization inhibitors malathion (43.1%) and NBD-Cl (36.2%). There was no change in other variables tested under the control treatment. Based on these findings, it can be concluded that there was no change in the level of tolerance of white oats to the herbicides mesotrione or tembotrione after application of malathion and NBD-Cl. This outcome suggests that metabolization or conjugation is not the main mechanisms that make white oat tolerant to HPPD enzyme inhibitors.

Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.