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1.
Oxid Med Cell Longev ; 2022: 2350857, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35509834

RESUMO

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cerebral ischemia injury via regulating the level of asymmetric dimethylarginine (ADMA). This study is aimed at exploring the effect of adiponectin resistance on ADMA-induced neuronal loss in ischemic stroke (IS) and the underlying mechanism. DDAH1 knockout (DDAH1-/-) and wild-type (DDAH1+/+) rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). Plasma and brain adiponectin levels and the expressions of adiponectin receptor 1 (APR1), adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK were determined after 24 h, 3 days, and 7 days. Neurological behavior, infarct volume, and adiponectin signaling were evaluated using adiponectin peptide or AdipoRon. The levels of reactive oxygen species (ROS) and Forkhead box O1 (FOXO1) (a transcription factor for APR1) were also assessed. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in primary neurons. DDAH1 was overexpressed in neurons, after which FOXO1 expression, ROS production, adiponectin resistance, and cell viability were detected. DDAH1-/- rats showed no significant difference in adiponectin level in either plasma or brain after MCAO/R in DDAH1+/+ rats, but downregulated APR1 expression and suppressed adiponectin signaling were observed. AdipoRon, but not adiponectin peptide, attenuated the neurological deficits and adiponectin resistance in DDAH1-/- rats. ROS accumulation and phosphorylated FOXO1 expression also increased with DDAH1 depletion. Following DDAH1 overexpression, decreased cell viability and inhibited adiponectin signaling induced by OGD/R were alleviated in primary neurons, accompanied by reduced ROS production and phosphorylated FOXO1 expression. Our study elucidated that in IS, DDAH1 protected against adiponectin resistance in IS via the ROS/FOXO1/APR1 pathway.


Assuntos
Amidoidrolases/metabolismo , Isquemia Encefálica , Traumatismo por Reperfusão , Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina , Animais , Arginina/análogos & derivados , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Proteínas do Tecido Nervoso , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo
2.
Bioorg Med Chem Lett ; 68: 128763, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35500728

RESUMO

A small library of FAAH and dual FAAH/MAGL inhibitors designed for peripheral selectivity were targeted. Of these compounds, three were identified to have desirable FAAH inhibition and reduced permeability in a PAMPA assay. Those three compounds were advanced into a MAGL inhibitor assay and one was found to be a relative selective FAAH inhibitor, FAAH to MAGL IC50 ratio of 1:27, and one was found to be more characteristic of a true dual enzyme inhibitor, FAAH to MAGL IC50 ratio of 1:4. Both compounds showed activity in an ABPP assay, blockage of TAMRA-FP labeling of FAAH and MAGL in rat eye homogenate.


Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Amidoidrolases , Animais , Inibidores Enzimáticos/farmacologia , Ratos
3.
Bioorg Med Chem ; 65: 116791, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35537325

RESUMO

Fourteen novel vanin-1 inhibitors coded OMP-# were designed from RR6 and successfully synthesized by a nucleophilic addition-elimination reaction of the pantetheinic acid-derived Weinreb amide as a key step under Barbier conditions. The synthesized OMP compounds exhibited inhibitory activity against human serum vanin-1 in vitro. Among the synthesized compounds, OMP-7, which possesses a trifluoromethoxy group at the para-position on the phenyl ring, exhibited the most potent activity, approximately 20 times that of the mother compound RR6. OMP-7 was further subjected to an in vivo assay using a normal hamster. More potent activity was observed than that of RR6 against both serum and renal vanin-1. The activity lasted for 4 h after injection against serum vanin-1 and 1 h after injection against renal vanin-1, whereas RR6 did not show the desired activity.


Assuntos
Amidoidrolases , Rim , Proteínas Ligadas por GPI , Humanos
4.
J Mol Graph Model ; 114: 108200, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35453045

RESUMO

UDP-3-O-acyl-N acetylglucosamine deacetylase (LpxC), Zn metalloenzyme for Gram-negative bacteria is an attractive target for developing novel therapeutic agents. Since LpxC has the similar binding pocket as the human matrix metalloproteinases (MMPs), LpxC inhibitors might also inhibit MMP functions producing side effects in human bodies. Here, we investigated specific interactions between LpxC/MMP and their inhibitors using ab initio molecular simulations to elucidate the reason of selective inhibition for LpxC by non-hydroxamate compounds. The evaluated binding properties between LpxC and the compounds are comparable to the trend of their observed inhibitory affinities. It was also elucidated that compound 22 binds most strongly to LpxC due to its specific interactions with Zn ion and Asp241 side chain of LpxC. In contrast, the interactions between the compounds and MMP are significantly weakened due to the water molecules, which are tightly coordinated with the Zn ion in MMP and interrupt the binding of the compounds to the Zn ion. Accordingly, the present molecular simulations revealed that these water molecules around the Zn ion in MMP are causally related to the selective inhibition of these compounds for LpxC rather than MMP.


Assuntos
Água , Zinco , Amidoidrolases/química , Antibacterianos/farmacologia , Inibidores Enzimáticos , Humanos , Metaloproteinases da Matriz , Zinco/química , Zinco/farmacologia
5.
Int J Mol Sci ; 23(8)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35457225

RESUMO

Migraine is a disabling neurovascular disorder characterized by severe pain with still limited efficient treatments. Endocannabinoids, the endogenous painkillers, emerged, alternative to plant cannabis, as promising analgesics against migraine pain. In this thematic review, we discuss how inhibition of the main endocannabinoid-degrading enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), could raise the level of endocannabinoids (endoCBs) such as 2-AG and anandamide in order to alleviate migraine pain. We describe here: (i) migraine pain signaling pathways, which could serve as specific targets for antinociception; (ii) a divergent distribution of MAGL and FAAH activities in the key regions of the PNS and CNS implicated in migraine pain signaling; (iii) a complexity of anti-nociceptive effects of endoCBs mediated by cannabinoid receptors and through a direct modulation of ion channels in nociceptive neurons; and (iv) the spectrum of emerging potent MAGL and FAAH inhibitors which efficiently increase endoCBs levels. The specific distribution and homeostasis of endoCBs in the main regions of the nociceptive system and their generation 'on demand', along with recent availability of MAGL and FAAH inhibitors suggest new perspectives for endoCBs-mediated analgesia in migraine pain.


Assuntos
Endocanabinoides , Transtornos de Enxaqueca , Amidoidrolases/metabolismo , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Carbamatos/farmacologia , Endocanabinoides/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Canais Iônicos , Transtornos de Enxaqueca/tratamento farmacológico , Monoacilglicerol Lipases/metabolismo , Dor
6.
J Exp Biol ; 225(9)2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35438163

RESUMO

The endocannabinoid system (eCS) plays a critical role in a variety of homeostatic and developmental processes. Although the eCS is known to be involved in motor and sensory function, the role of endocannabinoid (eCB) signaling in sensorimotor development remains to be fully understood. In this study, the catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) were inhibited either simultaneously or individually during the first ∼24 h of zebrafish embryogenesis, and the properties of contractile events and escape responses were studied in animals ranging in age from 1 day post-fertilization (dpf) to 10 weeks. This perturbation of the eCS resulted in alterations to contractile activity at 1 dpf. Inhibition of MAGL using JZL 184 and dual inhibition of FAAH/MAGL using JZL 195 decreased escape swimming activity at 2 dpf. Treatment with JZL 195 also produced alterations in the properties of the 2 dpf short latency C-start escape response. Animals treated with JZL 195 exhibited deficits in escape responses elicited by auditory/vibrational stimuli at 5 and 6 dpf. These deficits were also present during the juvenile developmental stage (8- to 10-week-old fish), demonstrating a prolonged impact to sensory systems. These findings demonstrate that eCS perturbation affects sensorimotor function, and underscores the importance of eCB signaling in the development of motor and sensory processes.


Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Amidoidrolases/metabolismo , Animais , Desenvolvimento Embrionário , Endocanabinoides/metabolismo , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/metabolismo , Peixe-Zebra/metabolismo
8.
Bioorg Med Chem ; 60: 116698, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35296453

RESUMO

Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are promising targets for neuropathic pain and other CNS disorders. Based on our previous lead compound SIH 3, we designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones and evaluated for FAAH and MAGL inhibition properties. Most of the compounds showed potency towards both enzymes with leading FAAH selectivity. Compound (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC50 = 11 nM) and MAGL (IC50 = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Molecular docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π-π stacking via phenyl ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by molecular simulation studies. PAMPA assay revealed that the lead compound is suitable for blood-brain penetration. The lead compound showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo experiments unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compound exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compound can be a potential template for the development of agents for neuropathic pain.


Assuntos
Neuralgia , Semicarbazonas , Amidoidrolases , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Monoacilglicerol Lipases , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico
9.
Methods Enzymol ; 664: 85-102, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35331380

RESUMO

Bile acids are important molecules that participate in digestion and regulate many host physiological processes, including metabolism and inflammation. Primary bile acids are biosynthesized from cholesterol in the liver, where they are conjugated to glycine and taurine before secretion into the intestines. A small fraction of these molecules remain in the gut, where they are modified by a microbial enzyme, bile salt hydrolase (BSH), which deconjugates the glycine and taurine groups. This deconjugation precedes all subsequent biotransformation in the intestines, including regioselective dehydroxylation and epimerization reactions, to produce numerous secondary bile acids. Thus, BSH is considered the gatekeeper enzyme of secondary bile acid metabolism, and, as a result, it controls the overall bile acid composition in the host. Despite the critical role that BSH plays in bile acid metabolism, there exist few tools to probe its activity in complex biological mixtures. In this chapter, we describe a chemoproteomic approach termed BSH-TRAP (bile salt hydrolase tagging and retrieval with activity-based probes) that enables visualization and identification of BSH activity in bacteria. Here, we describe application of BSH-TRAP to cultured bacterial strains and the gut microbes derived from mice. We envision that BSH-TRAP could be used to profile changes in BSH activity and identify novel BSH enzymes in complex biological samples, such as the gut microbiome.


Assuntos
Amidoidrolases , Ácidos e Sais Biliares , Amidoidrolases/metabolismo , Animais , Bactérias/metabolismo , Glicina , Camundongos , Taurina
10.
Cells ; 11(5)2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35269478

RESUMO

(1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB1R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV.


Assuntos
Canabinoides , Infecções por HIV , Amidoidrolases , Animais , Canabinoides/farmacologia , Endocanabinoides/metabolismo , Inibidores Enzimáticos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Piperidinas , Piridinas , Receptores de Canabinoides , Transmissão Sináptica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
11.
Int J Mol Sci ; 23(5)2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35269926

RESUMO

Acute lung injury (ALI) is a group of lung illnesses characterized by severe inflammation, with no treatment. The fatty acid amide hydrolase (FAAH) enzyme is an integral membrane protein responsible for the hydrolysis of the main endocannabinoids, such as anandamide (AEA). In pre-clinical pain and inflammation models, increasing the endogenous levels of AEA and other bioactive fatty acid amides (FAAs) via genetic deletion or the pharmacological inhibition of FAAH produces many analgesic benefits in several different experimental models. To date, nobody has investigated the role of FAAH inhibition on an ALI mouse model. Mice were subjected to a carrageenan injection and treated orally 1 h after with the FAAH inhibitor URB878 dissolved in a vehicle consisting of 10% PEG-400, 10% Tween-80 and 80% saline at different doses: The inhibition of FAAH activity was able to counteract not only the CAR-induced histological alteration, but also the cascade of related inflammatory events. URB878 clears the way for further studies based on FAAH inhibition in acute lung pathologies.


Assuntos
Lesão Pulmonar Aguda , Amidoidrolases , Lesão Pulmonar Aguda/tratamento farmacológico , Amidoidrolases/metabolismo , Animais , Endocanabinoides/metabolismo , Inflamação/tratamento farmacológico , Camundongos , Dor/patologia , Alcamidas Poli-Insaturadas/metabolismo
12.
Methods Enzymol ; 664: 243-265, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35331377

RESUMO

Bile is a digestive fluid produced in the liver and stored in the gallbladder. It participates in absorption of fatty nutrients and vitamins, and aids in elimination of metabolic waste and toxins. The major chemical components of bile are bile salts that, apart from their function in digestion, are also known to participate in cell signaling by binding host farnesoid X (FXR), vitamin D (VDR), and G-protein coupled bile acid (TGR5) receptors. Microbial bile salt hydrolases (BSHs) catalyze bile salt deconjugation, a gatekeeper reaction that is a prerequisite for all subsequent microbial transformations of bile acids. As a result, BSH determines the composition of the bile salt and acid pools, which in turn affects its nutrient absorption and signaling capabilities. BSH profiling remains a challenge due to a paucity of tools that enable scientists to study its function. In this chapter, we discuss current BSH profiling approaches and demonstrate a novel fluorogenic probe-based assay that circumvents laborious and resource intensive BSH quantification methods. Alongside our assay protocol, we provide the reader with a detailed method for microbial cell extraction from fecal matter. We also cover probe validation protocols that can be adapted for Michaelis-Menten analysis with any BSH expressing strain.


Assuntos
Microbioma Gastrointestinal , Amidoidrolases/metabolismo , Ácidos e Sais Biliares , Fezes , Humanos
13.
ACS Chem Neurosci ; 13(7): 920-932, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35316021

RESUMO

Alzheimer's disease is identified by pathological hallmarks such as intracellular neurofibrillary tangles (NFTs) and extracellular amyloid ß plaques. Several hypotheses exist to define the neurodegeneration including microglial activation associated with neuroinflammatory processes. Recently, pharmacological inhibition of endocannabinoid (eCB)-degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), is being investigated to modulate the pathology of Alzheimer's disease. While MAGL inhibitors upregulate 2-acyl glycerol (2-AG) levels and reduce neuroinflammation, FAAH inhibitors elevate anandamide (AEA) levels and prevent the degradation of HSP-70, thereby preventing the phosphorylation of tau protein and formation of NFTs in neural cells. We investigated the possible neuroprotective potential of the dual MAGL/FAAH inhibitor JZL-195 (20 mg/kg) against ICV-STZ-induced sporadic Alzheimer's disease (SAD) in Swiss albino mice using donepezil (5 mg/kg) as the standard. The protective effects of JZL-195 were observed by the reversal of altered levels of Aß1-42, HSP-70, neuroinflammatory cytokines, and oxidative stress markers. However, JZL-195 expressed no cognitive improvement when assessed by spontaneous alternation behavior and Morris water maze tests and no effects on the AChE enzyme level in the hippocampal tissues of mice. Therefore, the findings of the present study indicate that although JZL-195 exhibited no improvement in cognitive deficits associated with sporadic Alzheimer's disease, it displayed significant reversal of the biochemical anomalies, thereby suggesting its therapeutic potential against the sporadic Alzheimer's disease model.


Assuntos
Doença de Alzheimer , Monoacilglicerol Lipases , Doença de Alzheimer/tratamento farmacológico , Amidoidrolases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Endocanabinoides/metabolismo , Camundongos , Monoacilglicerol Lipases/metabolismo , Monoglicerídeos , Estresse Oxidativo , Estreptozocina/toxicidade
14.
Food Funct ; 13(6): 3318-3328, 2022 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-35257124

RESUMO

Bile salt hydrolases (BSHs), a group of cysteine-hydrolases produced by gut microbes, play a crucial role in the hydrolysis of glycine- or taurine-conjugated bile acids and have been validated as key targets to modulate bile acid metabolism. This study aims to discover one or more efficacious inhibitors against a BSH produced by Lactobacillus salivarius (lsBSH) from natural products and to characterize the mechanism of the newly identified BSH inhibitor(s). Following screening of the inhibition potentials of more than 100 natural compounds against lsBSH, amentoflavone (AMF), a naturally occurring biflavone isolated from various medicinal plants, was discovered to be an efficacious BSH inhibitor (IC50 = 0.34 µM). Further investigation showed that AMF could strongly inhibit the lsBSH-catalyzed hydrolytic reaction in living gut microbes. Inhibition kinetic analyses demonstrated that AMF reversibly inhibited the lsBSH-catalyzed hydrolytic reaction in a mixed-inhibition manner, with an apparent Ki value of 0.65 µM. Fluorescence quenching assays suggested that AMF could quench the fluorescence of lsBSH via a static quenching procedure. Docking simulations suggested that AMF could be fitted into lsBSH at two distinct ligand-binding sites, mainly via hydrophobic interactions and hydrogen bonding, which explained well the mixed inhibition mode of this agent. Animal tests showed that the hydrolytic activities of BSHs in mice feces could be significantly blocked by AMF. In summary, this study reports that AMF is a strong, naturally occurring inhibitor of lsBSH, which offers a promising lead compound to develop novel agents for modulating bile acid metabolism in the host via targeting BSHs.


Assuntos
Amidoidrolases/antagonistas & inibidores , Biflavonoides/farmacologia , Inibidores Enzimáticos/farmacologia , Lactobacillus salivarius/enzimologia , Amidoidrolases/química , Amidoidrolases/metabolismo , Animais , Biflavonoides/química , Biflavonoides/metabolismo , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fezes/enzimologia , Cinética , Camundongos , Simulação de Acoplamento Molecular
15.
Oxid Med Cell Longev ; 2022: 8151917, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35355865

RESUMO

Nitric oxide synthase- (NOS-) dependent endothelial dysfunction induced by oxidative stress (OS) is assumed to play a pivotal role in the pathogenesis and progression of diabetes mellitus-related erectile dysfunction (DMED). Cysteine-rich whey protein isolate (CR-WPI) is a widely used protein supplement and has been confirmed to reduce reactive oxygen species (ROS) by increasing cellular antioxidant glutathione (GSH). However, it is currently unknown whether CR-WPI elicits therapeutic effects in DMED. Here, we provide diabetic rats with CR-WPI to determine its effect on DMED and the underlying mechanisms. The results suggest that CR-WPI supplementation increased GSH biosynthesis and reduced ROS content and simultaneously upregulated the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/nitric oxide synthase (NOS) metabolic pathway. Evaluation of intracavernous pressure (ICP) also showed an improvement of penile erectile function in CR-WPI-treated rats. The results of the vitro cell culture showed that glutathione pretreatment protected corpus cavernosum smooth muscle cells (CCSMC) from H2O2-induced apoptosis by decreasing Caspase 9 and Caspase 3 expressions. These results augur well for the potential therapeutic application of dietary CR-WPI supplementation for treating diabetic erectile dysfunction.


Assuntos
Diabetes Mellitus Experimental , Disfunção Erétil , Amidoidrolases , Animais , Arginina/metabolismo , Cisteína/metabolismo , Cisteína/farmacologia , Cisteína/uso terapêutico , Disfunção Erétil/tratamento farmacológico , Humanos , Peróxido de Hidrogênio , Masculino , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Proteínas do Soro do Leite/farmacologia , Proteínas do Soro do Leite/uso terapêutico
16.
Artigo em Inglês | MEDLINE | ID: mdl-35162636

RESUMO

Tuberculosis (TB), the most frequent bacterium-mediated infectious disease caused by Mycobacterium tuberculosis, has been known to infect humans since ancient times. Although TB is common worldwide, the most recent report by the WHO (World Health Organization) listed the three countries of India, China, and Russia with 27%, 14%, and 8% of the global burden of TB, respectively. It has been reported that resistance to TB drugs, particularly by the pncA gene to the pyrazinamide drug due to mutations, significantly affects the effective treatment of TB. Understanding the mechanism of drug resistance using computational methods is of great interest to design effective TB treatment, exploring the structural features with these tools. Thus, keeping in view the importance of these methods, we employed state-of-the-art computational methods to study the mechanism of resistance caused by the W68L, L85P, and T87A mutations recently reported in 2021. We employed a molecular docking approach to predict the binding conformation and studied the dynamic properties of each complex using molecular dynamics simulation approaches. Our analysis revealed that compared to the wildtype, these three mutations altered the binding pattern and reduced the binding affinity. Moreover, the structural dynamic features also showed that these mutations significantly reduced the structural stability and packing, particularly by the W68L and L85P mutations. Moreover, principal component analysis, free energy landscape, and the binding free energy results revealed variation in the protein's motion and the binding energy. The total binding free energy was for the wildtype -9.61 kcal/mol, W68L -7.57 kcal/mol, L85P -6.99 kcal/mol, and T87A -7.77 kcal/mol. Our findings can help to design a structure-based drug against the MDR (multiple drug-resistant) TB.


Assuntos
Antituberculosos , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis , Pirazinamida , Amidoidrolases/química , Amidoidrolases/genética , Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mutação , Mycobacterium tuberculosis/genética , Pirazinamida/química , Pirazinamida/farmacologia
17.
Fitoterapia ; 158: 105161, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35217118

RESUMO

Seventeen triterpenoids including four new lanostane triterpenoids (1-3 and 5) were isolated from the fruiting bodies of Ganoderma lucidum by various chromatographic techniques. Their chemical structures were determined by extensive spectroscopic data, including 1D-NMR, 2D-NMR, and HRESIMS. In addition, the spectral data of compound 4 was reported for the first time. In an in vitro bioassay, most isolated triterpenoids could inhibit the hydrolysis activity of fatty acid amide hydrolase (FAAH). Furthermore, there is no cytotoxicity observed for these isolated triterpenoids. Therefore, G. lucidum showed the potential application for anti-neuroinflammation and more FAAH inhibitors may be explored from G. lucidum.


Assuntos
Ganoderma , Reishi , Triterpenos , Amidoidrolases , Carpóforos/química , Ganoderma/química , Estrutura Molecular , Reishi/química , Triterpenos/química , Triterpenos/farmacologia
18.
J Microbiol Biotechnol ; 32(4): 504-513, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35131956

RESUMO

Chitin deacetylase (CDA) inhibitors were developed as novel antifungal agents because CDA participates in critical fungal physiological and metabolic processes and increases virulence in soilborne fungal pathogens. However, few CDA inhibitors have been reported. In this study, 150 candidate CDA inhibitors were selected from the commercial Chemdiv compound library through structure-based virtual screening. The top-ranked 25 compounds were further evaluated for biological activity. The compound J075-4187 had an IC50 of 4.24 ± 0.16 µM for AnCDA. Molecular docking calculations predicted that compound J075-4187 binds to the amino acid residues, including active sites (H101, D48). Furthermore, compound J075-4187 inhibited food spoilage fungi and plant pathogenic fungi, with minimum inhibitory concentration (MIC) at 260 µg/ml and minimum fungicidal concentration (MFC) at 520 µg/ml. Therefore, compound J075-4187 is a good candidate for use in developing antifungal agents for fungi control.


Assuntos
Amidoidrolases , Antifúngicos , Amidoidrolases/metabolismo , Antifúngicos/química , Antifúngicos/farmacologia , Bioensaio , Quitina/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular
19.
Microbes Infect ; 24(4): 104951, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35151875

RESUMO

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Acil-Butirolactonas , Amidoidrolases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Humanos , Percepção de Quorum
20.
J Hazard Mater ; 430: 128444, 2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35183828

RESUMO

Triclocarban (TCC) is an emerging and intractable environmental contaminant due to its hydrophobicity and chemical stability. However, the antibacterial property of TCC limits its biodegradation, and only the functional enzyme TccA involved in TCC degradation has been characterized to date. In this study, we report a highly efficient TCC-degrading bacterium, Rhodococcus rhodochrous BX2, that could degrade and mineralize TCC (10 mg/L) by 76.8% and 56.5%, respectively, within 5 days. Subsequently, the TCC biodegradation pathway was predicted based on the detection of metabolites using modern mass spectrometry techniques. Furthermore, an amidase (TccS) and a novel phenol hydroxylase (PHIND) encoded by the tccS and PHIND genes, respectively, were identified by genomic and transcriptomic analyses of strain BX2, and these enzymes were further unequivocally proven to be the key enzymes responsible for the metabolism of TCC and its intermediate 4-chloroaniline (4-CA) by using a combination of heterologous expression and gene knockout. Our results shed new light on the mechanism of TCC biodegradation and better utilization of microbes to remediate TCC contamination.


Assuntos
Antibacterianos , Rhodococcus , Amidoidrolases , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Biodegradação Ambiental , Carbanilidas , Oxigenases de Função Mista , Rhodococcus/genética , Rhodococcus/metabolismo
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