Multiple Metabolism Pathways of Bentazone Potentially Regulated by Metabolic Enzymes in Rice.

Bentazone (BNTZ) is a selective and efficient herbicide used in crop production worldwide. However, the persistence of BNTZ residues in the environment has led to their increasing accumulation in farmland and crops, posing a high risk to human health. To evaluate its impact on crop growth and environmental safety, a comprehensive study was conducted on BNTZ toxicity, metabolic mechanism, and resultant pathways in rice. The rice growth was compromised to the treatment with BNTZ at 0.2-0.8 mg/L (529.95-1060.05 g a.i./ha), while the activities of enzymes including SOD, POD, CAT, GST, GT, and CYP450 were elevated under BNTZ stress. A genome-wide RNA-sequencing (RNA-Seq) was performed to dissect the variation of transcriptomes and metabolic mechanisms in rice exposed to BNTZ. The degradative pathways of BNTZ in rice are involved in glycosylation, hydrolysis, acetylation, and conjugation processes catalyzed by the enzymes. Our data provided evidence that helps understand the BNTZ metabolic and detoxic mechanisms.

Chlorinated benzothiadiazines inhibit angiogenesis through suppression of VEGFR2 phosphorylation.

Angiogenesis inhibitors are a critical pharmacological tool for the treatment of solid tumors. Suppressing vascular permeability leads to inhibition of tumor growth, invasion, and metastatic potential by blocking the supply of oxygen and nutrients. Disruption of the vascular endothelial growth factor (VEGF) signaling pathway is a validated target for the design of antiangiogenic agents. Several VEGFR2 inhibitors have been clinically approved over the past years. Structural analysis of these clinical VEGFR2 inhibitors highlighted key functional group overlap with the benzothiadiazine core contained in a library of in-house compounds. Herein we ascribe anti-angiogenic activity to a series of chlorinated benzothiadiazines. Selected compounds show significant activity to completely ameliorate VEGF-induced endothelial cell proliferation by suppression of VEGFR2 phosphorylation. The scaffold is devoid of activity to inhibit carbonic anhydrases and generally lacks cytotoxicity across a range of cancer and non-malignant cell lines. Assay of activity at 468 kinases shows remarkable selectivity with only four kinases inhibited > 65% at 10 µM concentration, and with significant activity to inhibit TNK2/ACK1 and PKRD2 by > 90%. All four identified kinase targets are known modulators of angiogenesis, thus highlighting compound 17b as a novel angiogenesis inhibitor for further development.

Comparative Molecular Field Analysis and Molecular Docking Studies on Quinolinone Derivatives Indicate Potential Hepatitis C Virus Inhibitors.

Presently, there are no effective vaccines and anti-virals for the prevention and treatment of Hepatitis C virus infections and hence there is an urgent need to develop potent HCV inhibitors. In this study, we have carried out molecular docking, molecular dynamics and 3D-QSAR on heteroaryl 3-(1,1-dioxo-2H-(1,2,4)-benzothiadizin-3-yl)-4-hydroxy-2(1H)-quinolinone series using NS5B protein. Total of 41 quinolinone derivatives is used for molecular modeling study. The binding conformation and hydrogen bond interaction of the docked complexes were analyzed to model the inhibitors. We identified the molecule XXXV that had a higher affinity with NS5B. The molecular dynamics study confirmed the stability of the compound XXXV-NS5B complex. The developed CoMFA descriptors parameters, which were calculated using a test set of 13 compounds, were statistically significant. Our results will provide useful insights and lead to design potent anti-Hepatitis C virus molecules.

Evidence of co-metabolic bentazone transformation by methanotrophic enrichment from a groundwater-fed rapid sand filter.

The herbicide bentazone is recalcitrant in aquifers and is therefore frequently detected in wells used for drinking water production. However, bentazone degradation has been observed in filter sand from a rapid sand filter at a waterworks with methane-rich groundwater. Here, the association between methane oxidation and removal of bentazone was investigated with a methanotrophic enrichment culture derived from methane-fed column reactors inoculated with that filter sand. Several independent lines of evidence obtained from microcosm experiments with the methanotrophic enrichment culture, tap water and bentazone at concentrations below 2 mg/L showed methanotrophic co-metabolic bentazone transformation: The culture removed 53% of the bentazone in 21 days in presence of 5 mg/L of methane, while only 31% was removed in absence of methane. Addition of acetylene inhibited methane oxidation and stopped bentazone removal. The presence of bentazone partly inhibited methane oxidation since the methane consumption rate was significantly lower at high (1 mg/L) than at low (1 µg/L) bentazone concentrations. The transformation yield of methane relative to bentazone normalized by their concentration ratio ranged from 58 to 158, well within the range for methanotrophic co-metabolic degradation of trace contaminants calculated from the literature, with normalized substrate preferences varying from 3 to 400. High-resolution mass spectrometry revealed formation of the transformation products (TPs) 6-OH, 8-OH, isopropyl-OH and di-OH-bentazone, with higher abundances of all TPs in the presence of methane. Overall, we found a suite of evidence all showing that bentazone was co-metabolically transformed to hydroxy-bentazone by a methanotrophic culture enriched from a rapid sand filter at a waterworks.

Screening of hepatitis C NS5B polymerase inhibitors containing benzothiadiazine core: a steered molecular dynamics.

Hepatic C virus (HCV) is a global health problem, resulting in liver cirrhosis and inflammation that can develop to hepatocellular carcinoma and fatality. The NS5B polymerase of HCV plays an important role in viral RNA replication process, making it an attractive therapeutic target for design and development of anti-HCV drugs. To search new potent compounds against the HCV NS5B polymerase, the molecular docking and the steered molecular dynamics (SMD) simulation techniques were performed. The potential potent inhibitors of the NS5B polymerase were screened out from the ZINC database using structural similarity search and molecular docking technique. Five top-hit compounds (the ZINC compounds 49888724, 49054741, 49777239, 49793673, and 49780355) were then studied by the SMD simulations based on the hypothesis that a high rupture force relates to a high binding efficiency. The results demonstrated that the ZINC compound 49888724 had a greater maximum rupture force, reflecting a good binding strength and inhibitory potency than known inhibitors and the rest four ZINC compounds. Therefore, our finding indicated that the ZINC compound 49888724 is a potential candidate to be a novel NS5B inhibitor for further design. Besides, the van der Waals interaction could be considered as the main contribution for stabilizing the NS5B-ligand complex.

Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties.

AMPA receptor (AMPAR) function is modulated by auxiliary subunits. Here, we report on three AMPAR interacting proteins-namely CKAMP39, CKAMP52 and CKAMP59-that, together with the previously characterized CKAMP44, constitute a novel family of auxiliary subunits distinct from other families of AMPAR interacting proteins. The new members of the CKAMP family display distinct regional and developmental expression profiles in the mouse brain. Notably, despite their structural similarities they exert diverse modulation on AMPAR gating by influencing deactivation, desensitization and recovery from desensitization, as well as glutamate and cyclothiazide potency to AMPARs. This study indicates that AMPAR function is very precisely controlled by the cell-type specific expression of the CKAMP family members.

Leaf proteome analysis provides insights into the molecular mechanisms of bentazon detoxification in rice.

Bentazon is a widely used herbicide that selectively removes broad-leaf weeds by competing with plastoquinone for the binding site in the D1 protein and interrupting the PET (photosynthetic electron transfer) chain. However, monocotyledonous plants, such as rice, show strong resistance to bentazon due to CYP81A6 induction, which results in herbicide detoxification. Here, we confirmed that rice was sensitive to bentazon treatment during the initial exposure period, in which bentazon rapidly inhibited photosynthesis efficiency and electron transfer, based on results of chlorophyll fluorescence analysis. In order to gain a comprehensive, pathway-oriented, mechanistic understanding of the effects directly induced by bentazon, we employed 2D-DIGE (two-dimensional difference gel electrophoresis) to analyze the leaf proteome after 8h of bentazon treatment coupled with individual protein identification by MALDI-TOF (Matrix assisted laser desorption/ionization-time of flight) MS/MS. Proteomic analyses revealed that bentazon induced the relative upregulation or downregulation of 30 and 71 proteins (by 1.5-fold or more, p<0.05), respectively. The pathways involved include photosynthesis processes, carbohydrate metabolism, antioxidant systems, and DNA stabilization and protein folding. Protein analysis data revealed that bentazon primarily suppressed photosynthesis processes, and showed inhibitory effects on carbohydrate metabolism and ATP synthesis, whereas several stress response proteins were induced in response to bentazon. Importantly, we identified a 519kD protein containing two histidine kinase-like ATPase domains and a C3HC4 RING type zinc finger domain which may function as a transcript factor to drive expression of detoxification genes such as CYP81A6, leading to bentazon tolerance. This study identifies, for the first time, a candidate transcription factor that could up-regulate CYP81A6 expression, and provides a foundation for further research to advance our knowledge of mechanisms of bentazon resistance in rice.

Thermodynamic characterization of new positive allosteric modulators binding to the glutamate receptor A2 ligand-binding domain: combining experimental and computational methods unravels differences in driving forces.

Positive allosteric modulation of the ionotropic glutamate receptor GluA2 presents a potential treatment of cognitive disorders, for example, Alzheimer's disease. In the present study, we describe the synthesis, pharmacology, and thermodynamic studies of a series of monofluoro-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven, and 5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI, and combined with the shorter total simulation time, we found the OSP method to be more effective for this setup. Furthermore, from the molecular dynamics simulations, we extracted the enthalpies and entropies, and along with the ITC data, this suggested that the differences in binding free energies are largely explained by the direct ligand-surrounding enthalpies. Furthermore, we used the OSP setup to predict binding affinities for a series of polysubstituted fluorine compounds and monosubstituted methyl compounds and used these predictions to characterize the modulator binding pocket for this scaffold of positive allosteric modulators.

1,2,4-Benzothiadiazine-1,1-dioxide derivatives as ionotropic glutamate receptor ligands: synthesis and structure-activity relationships.

Ionotropic glutamate receptor (iGluR) modulators, specially AMPA receptor antagonists, are potential tools for numerous therapeutic applications in neurological disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, epilepsy, chronic pain, and neuropathology ensuing from cerebral ischemia or cardiac arrest. In this work, the synthesis and binding affinities at the Gly/NMDA, AMPA, and kainic acid (KA) receptors of a new series of 1,2,4-benzothiadiazine-1,1-dioxide derivatives are reported. The results show that 1,2,4-benzothiadiazine-1,1-dioxide is a new scaffold for obtaining iGluR ligands. Moreover, this work has led us to the 7-(3-formylpyrrol-1-yl)-6-trifluoromethyl substituted compound 7, which displays the highest AMPA receptor affinity and high selectivity versus the Gly/NMDA (90-fold) and KA (46-fold) receptors.

Stimulation of aerobic degradation of bentazone, mecoprop and dichlorprop by oxygen addition to aquifer sediment.

In order to investigate aerobic degradation potential for the herbicides bentazone, mecoprop and dichlorprop, anaerobic groundwater samples from two monitoring and three drinking water wells near a drinking water abstraction field in Nybølle, Denmark, were screened for their degradation potential for the herbicides. In the presence of oxygen (14)C-labelled bentazone and mecoprop were removed significantly from the two monitoring wells' groundwater samples. Oxygen was added to microcosms in order to investigate whether different oxygen concentrations stimulate the biodegradation of the three herbicides in microcosms using groundwater and sandy aquifer materials. To maintain a certain oxygen concentration this level was measured from the outside of the bottles with a fibre oxygen meter using oxygen-sensitive luminescent sensor foil mounted inside the microcosm, to which supplementary oxygen was added. The highest oxygen concentrations (corresponding to 4-11 mg L(-1)) stimulated degradation (a 14-27% increase for mecoprop, 3-9% for dichlorprop and 15-20% for bentazone) over an experimental period of 200 days. Oxygen was required to biodegrade the herbicides, since no degradation was observed under anaerobic conditions. This is the first time bentazone degradation has been observed in aquifer material at low oxygen concentrations (2 mg L(-1)). The sediment had substantial oxygen consumption (0.92-1.45O2 g(-1)dw over 200 days) and oxygen was depleted rapidly in most incubations soon after its addition, which might be due to the oxidation of organic matter and other reduced species such as Fe(2+), S(2-) and Mn in sediment before the biodegradation of herbicides takes place. This study suggests that oxygen enhancement around a drinking water abstraction field could stimulate the bioremediation of diffuse source contamination.

Molecular modeling and residue interaction network studies on the mechanism of binding and resistance of the HCV NS5B polymerase mutants to VX-222 and ANA598.

Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about the mechanisms of drug resistance conferred by the studied mutations and will be helpful to design more potent inhibitors which could effectively overcome drug resistance of antivirus agents.

Effect of C7 modifications on benzothiadiazine-1,1-dioxide derivatives on their inhibitory activity and selectivity toward aldose reductase.

The development and progression of chronic complications in diabetic patients, such as retinopathy, nephropathy, neuropathy, cataracts, and stroke, are related to the activation and/or overexpression of aldose reductase (ALR2), which is a member of the aldo-keto reductase superfamily. A structure-activity relationship study focused on the C7 position of 1,2,4-benzothiadiazine-1,1-dioxide derivatives was pursued in an attempt to discover ALR2 inhibitors with enhanced potency and selectivity. These studies led to a series of new C7-substituted compounds, which were evaluated for their inhibitory activity against ALR2; they exhibited IC(50) values in the range of 2.80-45.13 nM. Two compounds with a C7-dimethylcarbamoyl and a C7-diethylcarbamoyl substituent, respectively, were found to be the most active and presented excellent selectivity for ALR2 over aldehyde reductase (ALR1). The structure-activity relationship analyses and molecular modeling studies presented herein highlight the importance of hydrophobic and bulky groups at the C7 position for inhibitory activity and selectivity toward ALR2.

3D-QSAR and docking studies on a series of benzothiadiazine derivatives as genotype 1 HCV polymerase inhibitors.

The Benzothiadiazine derivatives have been regarded as a novel class of HCV genotype 1 polymerase inhibitors. To explore the relationship between the structures of substituted Benzothiadiazine derivatives and their inhibitory activities against HCV, 3D-QSAR and molecular docking studies were performed on a dataset of ninty-eight compounds. The 3D-QSAR models resulted from seventy-eight molecules in the training set gave q(2) value of 0.81 and a test set of twenty compounds, gave predictive r(2) value of 0.94. 3D-QSAR model generated from kNN-MFA along with the docking binding structures provided enough information about the structural requirements for better activity. The results can serve as a useful guideline to design novel HCV genotype 1 inhibitors with better potencies.

Insight into the structural requirements of benzothiadiazine scaffold-based derivatives as hepatitis C virus NS5B polymerase inhibitors using 3D-QSAR, molecular docking and molecular dynamics.

Hepatitis C virus (HCV) infection is a significant world health threat with frequently ineffective problem existed in the present treatment, thus representing a major unmet medical need. The nonstructural viral protein 5B (NS5B), one of the best-studied polymerase, has emerged as an attractive target for the development of novel therapeutics against hepatitis C virus. In this work, both ligand- and receptor- based three-dimensional quantitative structure activity relationship (3D-QSAR) studies were carried out using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques on 360 benzothiadiazine scaffold-based derivatives as HCV GT-1b NS5B polymerase allosteric inhibitors. The resultant optimum 3D-QSAR model exhibited R(2)(ev) of 0.54, R(2)(nev) of 0.72 and the predictive ability was validated by using an independent test set of 90 compounds which gave R(2)(pred) value of 0.64. In addition, docking analysis and molecular dynamics simulation (MD) were also applied to elucidate the probable binding modes of these inhibitors at the allosteric site of the enzyme. Interpretation of the 3D contour maps in context of the topology of the allosteric binding site of NS5B provided insight into NS5B-inhibitor interactions. The information obtained from this work can be utilized to accurately predict the binding affinity of related analogues and also facilitate the future rational design of novel inhibitors with improved activity.

Thiourea isosteres as anion receptors and transmembrane transporters.

Compounds containing cyanoguanidine and 3-amino-1,2,4-benzothiadiazine-1,1-dioxide have been studied as anion receptors and transporters. Significant affinity for oxo-anions was observed in organic solution and the receptors were found to function as transmembrane chloride/nitrate antiporters with transport rates enhanced in the presence of valinomycin-K(+) complex.

Measurements and modeling of pesticide persistence in soil at the catchment scale.

Degradation of pesticides in soils is both spatially variable and also one of the most sensitive factors determining losses to surface water and groundwater. To date, no general guidance is available on suitable approaches for dealing with spatial variation in pesticide degradation in catchment or regional scale modeling applications. The purpose of the study was therefore to study the influence of various soil physical, chemical and microbiological characteristics on pesticide persistence in the contrasting cultivated soils found in a small (13 km(2)) agricultural catchment in Sweden and to develop and test a simple model approach that could support catchment scale modeling. Persistence of bentazone, glyphosate and isoproturon was investigated in laboratory incubation experiments. Degradation rate constants were highly variable with coefficients of variation ranging between 42 and 64% for the three herbicides. Multiple linear regression analysis and Mallows Cp statistic were employed to select the best set of independent parameters accounting for the variation in degradation. Soil pH and the proportion of active microorganisms (r) together explained 69% of the variation in the bentazone degradation rate constant; the Freundlich sorption co-efficient (K(f)) and soil laccase activity together explained 88% of the variation in degradation rate of glyphosate, while soil pH was a significant predictor (p<0.05) for isoproturon persistence. However, correlations between many potential predictor variables made clear interpretations of the statistical analysis difficult. Multiplicative models based on two predictors chosen 'a priori', one accounting for microbial activity (e.g. microbial respiration, laccase activity or the surrogate variable soil organic carbon, SOC) and one accounting for the effects of sorption on bioavailability, showed promise to support predictions of degradation for large-scale modeling applications, explaining up to 50% of the variation in herbicide persistence.

Transport and degradation of pesticides in a biopurification system under variable flux Part II: A macrocosm study.

Transport of bentazone, isoproturon, linuron, metamitron and metalaxyl were studied under three different flows in macrocosms. The aim was to verify the observations from Part I of the accompanying paper, with an increase in column volume and decrease in chemical and hydraulic load. Very limited breakthrough occurred in the macrocosms for all pesticides, except bentazone, at all flows. From batch degradation experiments, it was observed that the lag time of metamitron and linuron decreased drastically in time for all flows, indicating a growth in the pesticide degrading population. This in contrast to isoproturon and metalaxyl, where an increase in lag time could be observed in time for all flows. From the batch degradation experiments, it could be concluded that the influence of flow on the lag time was minimal and that the inoculation of the pesticide-primed soil had a little surplus value on degradation.

Transport and degradation of pesticides in a biopurification system under variable flux, Part I: a microcosm study.

The efficiency of a biopurification system, developed to treat pesticide contaminated water, is to a large extent determined by the chemical and hydraulic load. Insight into the behaviour of pesticides under different fluxes is necessary. The behaviour of metalaxyl, bentazone, linuron, isoproturon and metamitron was studied under three different fluxes with or without the presence of pesticide-primed soil in column experiments. Due to the time-dependent sorption process, retention of the pesticides with intermediate mobility was significantly influenced by the flux. The higher the flux, the slower pesticides will be sorbed, which resulted in a lower retention. Degradation of the intermediate mobile pesticides was also submissive to variations in flux. An increase in flux, led to a decrease in retention, which in turn decreased the opportunity time for biodegradation. Finally, the presence of pesticide-primed soil was only beneficial for the degradation of metalaxyl.

Comparative removal of bentazon by Ganoderma lucidum in liquid and solid state cultures.

Bentazon removal by Ganoderma lucidum cultured in liquid and solid state conditions was compared in this work. In solid state cultures, the fungus produced both ligninolytic enzymes, namely laccase and Mn peroxidase. In liquid cultures, the main ligninolytic enzyme produced was laccase. In both types of cultures bentazon improved the production of laccase without significant alteration in the production of Mn peroxidase. In solid state cultures, where high levels of both laccase and Mn peroxidase activities were found, the fungus was more resistant to the action of the herbicide (50 mM in solid state cultures against 20 mM in liquid cultures) and more efficient in removing bentazon (90% removal against 55% in liquid cultures after 10 days of cultivation). Furthermore, the solid state culture filtrates were more efficient in the in vitro degradation of bentazon than the liquid culture filtrates. These observations suggest that both enzymes, laccase and Mn peroxidase, are involved in bentazon degradation. The results further suggest that solid state cultures of Ganoderma lucidum could be useful in strategies designed to reduce environmental contamination by bentazon.

Coupling of liquid chromatography/tandem mass spectrometry and liquid chromatography/solid-phase extraction/NMR techniques for the structural identification of metabolites following in vitro biotransformation of SUR1-selective ATP-sensitive potassium channel openers.

SUR1-selective ATP-sensitive potassium channel openers (PCOs) have been shown to be of clinical value for the treatment of several metabolic disorders, including type I and type II diabetes, obesity, and hyperinsulinemia. Taking into account these promising therapeutic benefits, different series of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide were developed. In view of the lead optimization process of the series, knowledge of absorption, distribution, metabolism, excretion, and toxicity parameters, and more particularly the metabolic fate of these compounds, is a fundamental requirement. For such a purpose, two selected promising compounds [7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) and 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 157)] were incubated in the presence of phenobarbital-induced rat liver microsomes to produce expected mammal in vivo phase I metabolites. The resulting major metabolites were then analyzed by both mass spectrometry (MS) and NMR to completely elucidate their chemical structures. The two compounds were also further incubated in the presence of nontreated rats and human microsomes to compare the metabolic profiles. In the present study, the combined use of an exact mass liquid chromatography (LC)/tandem MS platform and an LC/solid-phase extraction/NMR system allowed the clarification of some unresolved structural assessments in the accurate chemical structure elucidation process of the selected PCO drugs. These results greatly help the optimization of the lead compounds.