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1.
Nutrients ; 13(9)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34578891

RESUMO

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.


Assuntos
Brassica/química , Microbioma Gastrointestinal , Glucosinolatos/metabolismo , Isotiocianatos/metabolismo , Nitrilas/metabolismo , Sulfóxidos/metabolismo , Clostridiaceae , Enterobacteriaceae , Feminino , Humanos , Masculino , Brotos de Planta/química , Tiocianatos/metabolismo
2.
Chem Commun (Camb) ; 57(72): 9096-9099, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34498651

RESUMO

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Simulação de Dinâmica Molecular , Nitrilas/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilas/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/metabolismo , Teoria Quântica , SARS-CoV-2/isolamento & purificação , Termodinâmica
3.
Bioorg Med Chem Lett ; 50: 128333, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34418570

RESUMO

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.


Assuntos
COVID-19/patologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilas/química , Nitrilas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Prolina/química , Prolina/metabolismo , Inibidores de Proteases/metabolismo , SARS-CoV-2/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Microb Cell Fact ; 20(1): 133, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256737

RESUMO

BACKGROUND: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinamide insecticide that regulates insect growth. Because of its wide application in agricultural production and high solubility in water, it poses potential risks to aquatic environments and food chain. RESULTS: In the present study, Ensifer adhaerens CGMCC 6315 was shown to efficiently transform FLO into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) via a hydration pathway mediated by two nitrile hydratases, PnhA and CnhA. In pure culture, resting cells of E. adhaerens CGMCC 6315 degraded 92% of 0.87 mmol/L FLO within 24 h at 30 °C (half-life 7.4 h). Both free and immobilized (by gel beads, using calcium alginate as a carrier) E. adhaerens CGMCC 6315 cells effectively degraded FLO in surface water. PnhA has, to our knowledge, the highest reported degradation activity toward FLO, Vmax = 88.7 U/mg (Km = 2.96 mmol/L). Addition of copper ions could increase the enzyme activity of CnhA toward FLO by 4.2-fold. Structural homology modeling indicated that residue ß-Glu56 may be important for the observed significant difference in enzyme activity between PnhA and CnhA. CONCLUSIONS: Application of E. adhaerens may be a good strategy for bioremediation of FLO in surface water. This work furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and provides effective transformation strategies for microbial remediation of FLO contamination.


Assuntos
Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Hidroliases/metabolismo , Inseticidas/metabolismo , Niacinamida/análogos & derivados , Rhizobiaceae/enzimologia , Rhizobiaceae/metabolismo , Niacinamida/metabolismo , Nitrilas/metabolismo
5.
Biochem J ; 478(14): 2811-2823, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34190988

RESUMO

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.


Assuntos
Domínio Catalítico , Conformação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Benzamidas/metabolismo , Benzamidas/farmacologia , Biocatálise/efeitos dos fármacos , Humanos , Modelos Moleculares , Nitrilas/metabolismo , Nitrilas/farmacologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
6.
Sci Rep ; 11(1): 13244, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168179

RESUMO

Two-component plant defenses such as cyanogenic glucosides are produced by many plant species, but phloem-feeding herbivores have long been thought not to activate these defenses due to their mode of feeding, which causes only minimal tissue damage. Here, however, we report that cyanogenic glycoside defenses from cassava (Manihot esculenta), a major staple crop in Africa, are activated during feeding by a pest insect, the whitefly Bemisia tabaci, and the resulting hydrogen cyanide is detoxified by conversion to beta-cyanoalanine. Additionally, B. tabaci was found to utilize two metabolic mechanisms to detoxify cyanogenic glucosides by conversion to non-activatable derivatives. First, the cyanogenic glycoside linamarin was glucosylated 1-4 times in succession in a reaction catalyzed by two B. tabaci glycoside hydrolase family 13 enzymes in vitro utilizing sucrose as a co-substrate. Second, both linamarin and the glucosylated linamarin derivatives were phosphorylated. Both phosphorylation and glucosidation of linamarin render this plant pro-toxin inert to the activating plant enzyme linamarase, and thus these metabolic transformations can be considered pre-emptive detoxification strategies to avoid cyanogenesis.


Assuntos
Glicosídeos/metabolismo , Hemípteros , Manihot/metabolismo , Animais , Glucose/metabolismo , Herbivoria , Nitrilas/metabolismo , Fosforilação
7.
Angew Chem Int Ed Engl ; 60(35): 19162-19168, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-33886145

RESUMO

In this contribution, the unique and unprecedented stereochemical phenomenon of an aldoxime dehydratase-catalyzed enantioselective dehydration of racemic E- and Z-aldoximes with selective formation of both enantiomeric forms of a chiral nitrile is rationalized by means of molecular modelling, comprising in silico mutations and docking studies. This theoretical investigation gave detailed insight into why with the same enzyme the use of racemic E- and Z-aldoximes leads to opposite forms of the chiral nitrile. The calculated mutants with a larger or smaller cavity in the active site were then prepared and used in biotransformations, showing the theoretically predicted decrease and increase of the enantioselectivities in these nitrile syntheses. This validated model also enabled the rational design of mutants with a smaller cavity, which gave superior enantioselectivities compared to the known wild-type enzyme, with excellent E-values of up to E>200 when the mutant OxdRE-Leu145Phe was utilized.


Assuntos
Hidroliases/metabolismo , Simulação de Acoplamento Molecular , Nitrilas/metabolismo , Hidroliases/química , Estrutura Molecular , Nitrilas/química , Estereoisomerismo
8.
Life Sci ; 270: 119123, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33548287

RESUMO

Chronic ulceration of the colon is associated with the activation of TLR4/NF-κB and P2X7R/NLRP3 signaling pathways. We investigated the effect of individual or combined administration of BBG, a P2X7R blocker, and OLT1177, a selective NLRP3 inhibitor, in the dextran sodium sulfate-induced ulcerative colitis (UC) rat model. The ulcerative rats were treated orally with brilliant blue G (BBG) (50 mg/kg/day) or OLT1177 (200 mg/kg/day) or a combination of both. Myd88 and NF-κB levels were measured by ELISA, qRT-PCR, and immunohistochemical staining. Cytokines known to be associated with TLR4/NF-κB or P2X7R/NLRP3 signaling were measured by ELISA. P2X7R and NLRP3 expression were measured by ELISA and qRT-PCR. The administration of BBG or OLT1177 ameliorated the toxic effects of DSS on the colon as they restored normal colonic macroscopic and microscopic morphology. BBG administration, but not OLT1177, reduced the expression of Myd88, NF-κB, IL-6, and TNF-α in addition to lowering P2X7R and oxidative stress levels. Individual BBG or OLT1177 administration decreased NLRP3 inflammasome recruitment and subsequent activation of caspase-1, IL-1ß, and IL-18. However, the combined administration of OLT1177 with BBG potentiated its inhibitory effect on the NLRP3, which was reflected by the additional suppressive effect on caspase-1, IL-1ß, IL-18 levels. In conclusion, BBG/OLT1177 exhibited complementary effects and effectively ameliorated UC. This novel approach provides a basis for the clinical application of this combination for the treatment of IBDs and might also be promising for the pharmacological intervention of other NLRP3 inflammasome-dependent inflammatory conditions.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Nitrilas/farmacologia , Corantes de Rosanilina/farmacologia , Animais , Caspase 1/metabolismo , Colite/induzido quimicamente , Colite Ulcerativa/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nitrilas/metabolismo , Ratos , Ratos Wistar , Receptores Purinérgicos P2X7/metabolismo , Corantes de Rosanilina/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Acc Chem Res ; 54(7): 1711-1722, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33576600

RESUMO

Organonitrogen chemicals are essential in many aspects of modern life. Over 80% of the top 200 prescribed pharmaceutical products contain at least one nitrogen atom in the molecule, while all top 10 agrochemicals contain nitrogen, just to name a few. At present, the prevailing industrial processes for manufacturing organonitrogen chemicals start from nonrenewable fossil resources, but eventually we have to make these chemicals in a more sustainable manner. Biomass represents the largest renewable carbon resource on earth, which is inexpensive and widely available. Integrating biomass into the organonitrogen chemical supply chain will mitigate the carbon footprint, diversify the product stream, and enhance the economic competitiveness of biorefinery. Short-cut synthesis routes can be created for oxygen-containing organonitrogen compounds by exploiting the inherent oxygen functionalities in the biomass resources. Moreover, for nitrogen-containing biomass components such as chitin, a unique opportunity to make organonitrogen chemicals bypassing the energy-intensive Haber-Bosch ammonia synthesis process arises. Estimated at 100 billion tons of annual production in the world, chitin captures more nitrogen than the Haber-Bosch process in the form of amide functional groups in its polymer side chain.In this Account, we intend to summarize our efforts to establish new reaction routes to synthesize valuable organonitrogen chemicals from renewable resources. Enabled by tailor-designed catalytic systems, diverse nitrogen-containing products including amines, amino acids, nitriles, and N-heterocycles have been obtained from a range of biomass feedstock either directly or via intermediate platform compounds. Two strategies to produce organonitrogen chemicals are presented. For platform chemicals derived from cellulose, hemicellulose, lignin, and lipids, which are enriched with oxygen functionalities, in particular, hydroxyl groups, the key chemistry to be developed is the catalytic transformation of hydroxyl groups into nitrogen-containing groups using NH3 as the nitrogen source. Along this line, Ru- and Ni-based heterogeneous catalysts are developed to convert alcohols to amines and/or nitriles via a thermal catalytic pathway, while CdS nanomaterials are explored to promote -OH to -NH2 conversion under visible-light irradiation. Metal-zeolite multifunctional systems are further established to enable the synthesis of N-heterocycles from O-heterocycles. The second strategy involves the use of chitin and chitin derivatives as the starting materials. Under the concept of shell biorefinery, distinctive protocols have been established to chemically transform chitin as the sole feedstock to amino sugars, amino alcohols, furanic amides, and N-heterocycles. By combining mechanochemistry with biotransformation, an integrated process to convert shrimp shell waste to complex, high-value, chiral compounds including tyrosine and l-DOPA is also demonstrated.


Assuntos
Aminas/metabolismo , Aminoácidos/metabolismo , Quitina/metabolismo , Nitrilas/metabolismo , Aminas/química , Aminoácidos/química , Biomassa , Quitina/química , Estrutura Molecular , Nitrilas/química
10.
Angew Chem Int Ed Engl ; 60(13): 6965-6969, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33529432

RESUMO

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).


Assuntos
Acetofenonas/química , Álcool Desidrogenase/química , Derivados de Benzeno/química , Oxigenases de Função Mista/química , Nitrilas/química , Álcool Feniletílico/química , Acetofenonas/metabolismo , Agrocybe/enzimologia , Álcool Desidrogenase/metabolismo , Derivados de Benzeno/metabolismo , Catálise , Luz , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Nitrilas/metabolismo , Oxirredução , Álcool Feniletílico/metabolismo , Processos Fotoquímicos , Rhodococcus/enzimologia , Estereoisomerismo
11.
Theranostics ; 11(6): 2490-2504, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33456555

RESUMO

Background: Magnetic resonance imaging (MRI) is indispensable for diagnosing neurological conditions such as multiple sclerosis (MS). MRI also supports decisions regarding the choice of disease-modifying drugs (DMDs). Determining in vivo tissue concentrations of DMDs has the potential to become an essential clinical tool for therapeutic drug monitoring (TDM). The aim here was to examine the feasibility of fluorine-19 (19F) MR methods to detect the fluorinated DMD teriflunomide (TF) during normal and pathological conditions. Methods: We used 19F MR spectroscopy to detect TF in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) in vivo. Prior to the in vivo investigations we characterized the MR properties of TF in vitro. We studied the impact of pH and protein binding as well as MR contrast agents. Results: We could detect TF in vivo and could follow the 19F MR signal over different time points of disease. We quantified TF concentrations in different tissues using HPLC/MS and showed a significant correlation between ex vivo TF levels in serum and the ex vivo 19F MR signal. Conclusion: This study demonstrates the feasibility of 19F MR methods to detect TF during neuroinflammation in vivo. It also highlights the need for further technological developments in this field. The ultimate goal is to add 19F MR protocols to conventional 1H MRI protocols in clinical practice to guide therapy decisions.


Assuntos
Crotonatos/metabolismo , Radioisótopos de Flúor/metabolismo , Flúor/metabolismo , Hidroxibutiratos/metabolismo , Inflamação/diagnóstico , Nitrilas/metabolismo , Toluidinas/metabolismo , Animais , Meios de Contraste/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Imagem por Ressonância Magnética de Flúor-19/métodos , Inflamação/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/metabolismo , Ratos
12.
Proteins ; 89(3): 336-347, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33118210

RESUMO

Predicting the range of substrates accepted by an enzyme from its amino acid sequence is challenging. Although sequence- and structure-based annotation approaches are often accurate for predicting broad categories of substrate specificity, they generally cannot predict which specific molecules will be accepted as substrates for a given enzyme, particularly within a class of closely related molecules. Combining targeted experimental activity data with structural modeling, ligand docking, and physicochemical properties of proteins and ligands with various machine learning models provides complementary information that can lead to accurate predictions of substrate scope for related enzymes. Here we describe such an approach that can predict the substrate scope of bacterial nitrilases, which catalyze the hydrolysis of nitrile compounds to the corresponding carboxylic acids and ammonia. Each of the four machine learning models (logistic regression, random forest, gradient-boosted decision trees, and support vector machines) performed similarly (average ROC = 0.9, average accuracy = ~82%) for predicting substrate scope for this dataset, although random forest offers some advantages. This approach is intended to be highly modular with respect to physicochemical property calculations and software used for structural modeling and docking.


Assuntos
Aminoidrolases , Proteínas de Bactérias , Aprendizado de Máquina , Simulação de Acoplamento Molecular/métodos , Aminoidrolases/química , Aminoidrolases/genética , Aminoidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Fenômenos Químicos , Ligantes , Nitrilas/química , Nitrilas/metabolismo , Ligação Proteica
13.
Angew Chem Int Ed Engl ; 60(7): 3679-3684, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33141478

RESUMO

A mirror-image strategy, that is, symmetry analysis of the substrate-binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3-isobutyl glutaronitrile (1 a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90 % ee to R, 47 % ee). By further reshaping the substrate-binding pocket via routine site-saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99 % ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)-3-isobutyl-4-cyanobutanoic acid ((R)-2 a) and showed similar stereopreference inversion towards a series of 3-substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.


Assuntos
Aminoidrolases/metabolismo , Nitrilas/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Biocatálise , Hidrólise , Estrutura Molecular , Nitrilas/química , Estereoisomerismo , Synechocystis/enzimologia
14.
Br J Anaesth ; 126(1): 245-255, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32859366

RESUMO

BACKGROUND: The mechanisms underlying the role of T-type calcium channels (T-channels) in thalamocortical excitability and oscillations in vivo during neurosteroid-induced hypnosis are largely unknown. METHODS: We used patch-clamp electrophysiological recordings from acute brain slices ex vivo, recordings of local field potentials (LFPs) from the central medial thalamic nucleus in vivo, and wild-type (WT) and Cav3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH). RESULTS: Patch-clamp recordings showed that 3ß-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3ß-OH inhibited the spike firing mode more profoundly in WT than in Cav3.1 knockout mice. Furthermore, 3ß-OH significantly hyperpolarised neurones, reduced the amplitudes of low threshold spikes, and diminished rebound burst firing only in WT mice. We found that 80 mg kg-1 i.p. injections of 3ß-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3ß-OH (20 mg kg-1 i.p.) accelerated induction of hypnosis by isoflurane only in WT mice, but had similar effects on the maintenance of isoflurane-induced hypnosis in both WT and Cav3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3ß-OH increased δ, θ, α, and ß oscillations in WT mice in comparison with Cav3.1 knock-out mice. CONCLUSIONS: The Cav3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.


Assuntos
Androstanóis/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Canais de Cálcio Tipo T/metabolismo , Hipnóticos e Sedativos/farmacologia , Nitrilas/farmacologia , Androstanóis/metabolismo , Animais , Fenômenos Eletrofisiológicos , Hipnóticos e Sedativos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Neuroesteroides/metabolismo , Neuroesteroides/farmacologia , Nitrilas/metabolismo
15.
Crit Rev Biotechnol ; 41(1): 72-93, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33045860

RESUMO

Nitrilases are widely distributed in nature and are able to hydrolyze nitriles into their corresponding carboxylic acids and ammonia. In industry, nitrilases have been used as green biocatalysts for the production of high value-added products. To date, biocatalysts are considered to be important alternatives to chemical catalysts due to increasing environmental problems and resource scarcity. This review provides an overview of recent advances of nitrilases in aspects of distribution, enzyme screening, molecular structure and catalytic mechanism, protein engineering, and their potential applications in industry.


Assuntos
Aminoidrolases , Química Verde , Engenharia de Proteínas , Aminoidrolases/genética , Aminoidrolases/metabolismo , Ácidos Carboxílicos/metabolismo , Química Verde/tendências , Nitrilas/metabolismo
16.
Ecotoxicol Environ Saf ; 209: 111861, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33383338

RESUMO

Soybean pod borer (Leguminivora glycinivorella) is an important pest in soybean production, and chemical pesticides was major way for prevention. However, it is difficult to balance the efficiency and safety of pesticide application. In this paper, we evaluated safety and effectiveness of common insecticides (chlorpyrifos and lambda-cyhalothrin) on soybean from three aspects, including distribution, dissipation and control effect, around three major soybean production area (Anhui, Jilin and Shandong) in China. For chlorpyrifos, the initial deposition of each position (upper leaf, lower leaf, upper stem, lower stem, soybean and root) was determinated for 0.23 mg/kg to 70.7 mg/kg, and the half-lifes ranged from 1.96 days to 5.36 days. For lambda-cyhalothrin, the initial deposition of the position was determinated for 0.10 mg/kg to 2.54 mg/kg, and the half-lifes ranged from 2.45 days to 7.26 days. We found that the target insecticides were major deposition and faster degradation in upper stem and leaf. Through comparing the relationship between field control effect and residue, it can be suggested that 40% chlorpyrifos EC and 2.5% lambda-cyhalothrin WE should be sprayed at 600 g a.i./ha and 5.63 g a.i./ha for SPB prevention. This study enhanced our understanding of distribution, dissipation and relationship between residue and control effect. The results provided data support for guiding the precise and scientific application of chemical insecticides on soybean.


Assuntos
Clorpirifos/metabolismo , Inseticidas/metabolismo , Mariposas , Nitrilas/metabolismo , Piretrinas/metabolismo , Soja/metabolismo , Animais , China , Inseticidas/análise , Praguicidas/metabolismo , Folhas de Planta/química
17.
Pharm Dev Technol ; 26(1): 48-59, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33121318

RESUMO

The pharmaceutical industry has to tackle the explosion of high amounts of poorly soluble APIs. This phenomenon leads to numerous sophisticated solutions. These include the use of multifactorial data analysis identifying correlations between the components and dosage form properties, laboratory and production process parameters with respect to the API liberation Example of such API is bicalutamide. Improved liberation is achieved by particle size reduction. Laboratory batches, with different PSD of API, were filled into gelatinous capsules and consequently granulated for tablet compression. Comparative dissolution profiles with Casodex 150 mg (Astra Zeneca) were performed. The component analysis was used for the statistical evaluation of f1 and f2 factors and D(v,0.9) and D[4,3] parameters of PSD to identify optimal PSD values. Suitable PSD limits for API were statistically confirmed in laboratory and in commercial scale with respect to optimized tablet properties. The tablets were bioequivalent with originator (n = 20; 90% CI for ln AUC0-120: 99.8-111.9%; 90% CI for ln cmax: 101.1-112.9%). In conclusion, the micronisation of the API is still an efficient and inexpensive method improving the bioavailability, although there are more complicated and expensive methods available. Statistical multifactorial methods improved the safety and reproducibility of production.


Assuntos
Anilidas/síntese química , Anilidas/metabolismo , Química Farmacêutica/métodos , Nitrilas/síntese química , Nitrilas/metabolismo , Compostos de Tosil/síntese química , Compostos de Tosil/metabolismo , Disponibilidade Biológica , Análise Multivariada , Comprimidos , Equivalência Terapêutica
18.
J Biol Chem ; 296: 100231, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33361191

RESUMO

The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.


Assuntos
Proteínas de Bactérias/química , Dioxigenases/química , Glicina/química , Ferro/química , Ácidos Cetoglutáricos/química , Nitrilas/metabolismo , Streptomyces/enzimologia , Aminobutiratos/química , Aminobutiratos/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicina/metabolismo , Histidina/química , Histidina/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Nitrilas/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Streptomyces/química , Streptomyces/genética , Especificidade por Substrato , Vanadatos/química , Vanadatos/metabolismo
19.
Xenobiotica ; 51(1): 40-50, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32757971

RESUMO

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 µM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.


Assuntos
Citosol/metabolismo , Fígado/metabolismo , Permetrina/metabolismo , Animais , Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Humanos , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Nitrilas/metabolismo , Piretrinas/metabolismo , Ratos , Ratos Sprague-Dawley
20.
J Agric Food Chem ; 68(50): 14988-14995, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33287534

RESUMO

The aim of this study was to monitor the degradation of three insecticides licensed for the control of cabbage moths during the 14-day fermentation period of sauerkraut samples. The hypothesis of this study is that the different sauerkraut fermentation processes could affect the degradation of applied insecticides. For this purpose, the fresh cabbage leaves contaminated with (λ-cyhalothrin, malathion, and chlorpyrifos-methyl) were left for fermentation with and without (natural) starter addition (Lactobacillus plantarum 112), and vacuum-packed as a control under laboratory conditions. The pH values and microbial growth were periodically monitored in sauerkraut samples during the fermentation period. During this time, the insecticide residues were determined in control and treatment samples using LC-MS-MS. In control samples, the degradation of chlorpyrifos-methyl and malathion was higher with rates of 69 and 98%, respectively, compared with the sauerkraut samples (12 and 59%; 31 and 34%, respectively) 14 days after the insecticide application. At the end of fermentation (14 d), no significant reduction in λ-cyhalothrin was detected in both treatments and control (13-19% reduction). The current study demonstrated that the presence of the lactic acid bacteria in the sauerkraut fermentation accelerated pH decline (below 4.0), and these fermentation conditions probably decelerated the degradation of malathion and chlorpyrifos-methyl. The results showed that the stability of different insecticides varied during the same fermentation process.


Assuntos
Brassica/microbiologia , Inseticidas/metabolismo , Lactobacillus plantarum/metabolismo , Biodegradação Ambiental , Brassica/química , Brassica/metabolismo , Contagem de Colônia Microbiana , Fermentação , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Inseticidas/química , Lactobacillus plantarum/crescimento & desenvolvimento , Malation/química , Malation/metabolismo , Nitrilas/química , Nitrilas/metabolismo , Piretrinas/química , Piretrinas/metabolismo
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