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1.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299330

RESUMO

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer's disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-ß peptide (Aß). The morphological evaluation showed that Aß treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aß. Moreover, URB597 reduced both the increase of Rho protein activation in Aß-treated BV-2 cells and the Aß-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.


Assuntos
Amidoidrolases/antagonistas & inibidores , Benzamidas/farmacologia , Carbamatos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amidoidrolases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Citocinas/metabolismo , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Camundongos , Microglia/patologia , Alcamidas Poli-Insaturadas/metabolismo
2.
J Enzyme Inhib Med Chem ; 36(1): 1411-1423, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34256657

RESUMO

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations.


Assuntos
Amidoidrolases/antagonistas & inibidores , Aminas/farmacologia , Inibidores Enzimáticos/farmacologia , Amidoidrolases/metabolismo , Aminas/química , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Coelhos , Relação Estrutura-Atividade
3.
Int J Nanomedicine ; 16: 3661-3678, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093011

RESUMO

Introduction: Brain ischemia is a common neurological disorder worldwide that activates a cascade of pathophysiological events involving decreases in oxygen and glucose levels. Despite substantial efforts to explore its pathogenesis, the management of ischemic neuronal injury remains an enormous challenge. Accumulating evidence suggests that VEGF modified nanofiber (NF) materials and the fatty-acid amide hydrolase (FAAH) inhibitor URB597 exert an influence on alleviating ischemic brain damage. We aimed to further investigate their effects on primary hippocampal neurons, as well as the underlying mechanisms following oxygen-glucose deprivation (OGD). Methods: Different layers of VEGF-A loaded polycaprolactone (PCL) nanofibrous membranes were first synthesized by using layer-by-layer (LBL) self-assembly of electrospinning methods. The physicochemical and biological properties of VEGF-A NF membranes, and their morphology, hydrophilicity, and controlled-release of VEGF-A were then estimated. Furthermore, the effects of VEGF-A NF and URB597 on OGD-induced mitochondrial oxidative stress, inflammatory responses, neuronal apoptosis, and endocannabinoid signaling components were assessed. Results: The VEGF-A NF membrane and URB597 can not only promote hippocampal neuron adhesion and viability following OGD but also exhibited antioxidant/anti-inflammatory and mitochondrial membrane potential protection. The VEGF-A NF membrane and URB597 also inhibited OGD-induced cellular apoptosis through activating CB1R signaling. These results indicate that VEGF-A could be controlled-released by LBL self-assembled NF membranes. Discussion: The VEGF-A NF membrane and URB597 displayed positive synergistic neuroprotective effects through the inhibition of mitochondrial oxidative stress and activation of CB1R/PI3K/AKT/BDNF signaling, suggesting that a VEGF-A loaded NF membrane and the FAAH inhibitor URB597 could be of therapeutic value in ischemic cerebrovascular diseases.


Assuntos
Benzamidas/farmacologia , Carbamatos/farmacologia , Nanofibras/química , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Células Cultivadas , Endocanabinoides/metabolismo , Glucose/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Membranas Artificiais , Neurônios/metabolismo , Neurônios/patologia , Oxigênio/metabolismo , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/química
4.
J Biol Chem ; 296: 100257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33837735

RESUMO

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.


Assuntos
Mycobacterium tuberculosis/enzimologia , Fenilalanina-tRNA Ligase/ultraestrutura , Conformação Proteica , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Sequência de Aminoácidos/genética , Antibacterianos/química , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Cinética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/química , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/enzimologia , Tuberculose Resistente a Múltiplos Medicamentos/genética
5.
J Enzyme Inhib Med Chem ; 36(1): 940-953, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33896320

RESUMO

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.


Assuntos
Amidas/farmacologia , Amidoidrolases/antagonistas & inibidores , Analgésicos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Flurbiprofeno/farmacologia , Amidas/síntese química , Amidas/química , Amidoidrolases/metabolismo , Analgésicos/síntese química , Analgésicos/química , Animais , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Flurbiprofeno/síntese química , Flurbiprofeno/química , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Eletricidade Estática , Relação Estrutura-Atividade
6.
Biomolecules ; 11(4)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810505

RESUMO

Growing evidence shows that the immune system is critically involved in Alzheimer's disease (AD) pathogenesis and progression. The modulation and targeting of peripheral immune mechanisms are thus promising therapeutic or preventive strategies for AD. Given the critical involvement of the endocannabinoid (eCB) system in modulating immune functions, we investigated the potential role of the main elements of such a system, namely type-1 and type-2 cannabinoid receptors (CB1 and CB2), and fatty acid amide hydrolase (FAAH), in distinct immune cell populations of the peripheral blood of AD patients. We found that, compared to healthy controls, CB1 and CB2 expression was significantly lower in the B-lymphocytes of AD patients. Moreover, we found that CB2 was significantly lower and FAAH was significantly higher in monocytes of the same subjects. In contrast, T-lymphocytes and NK cells did not show any variation in any of these proteins. Of note, monocytic CB2 and FAAH levels significantly correlated with clinical scores. Furthermore, the pharmacological inactivation of FAAH in monocytes and monocyte-derived macrophages obtained from AD patients was able to modulate their immune responses, by reducing production of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12, and enhancing that of the anti-inflammatory cytokine IL-10. Furthermore, FAAH blockade skewed AD monocyte-derived macrophages towards a more anti-inflammatory and pro-resolving phenotype. Collectively, our findings highlight a central role of FAAH in regulating AD monocytes/macrophages that could be of value in developing novel monocyte-centered therapeutic approaches aimed at promoting a neuroprotective environment.


Assuntos
Doença de Alzheimer/patologia , Amidoidrolases/metabolismo , Macrófagos/metabolismo , Idoso , Amidoidrolases/antagonistas & inibidores , Benzamidas/farmacologia , Carbamatos/farmacologia , Feminino , Humanos , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
J Med Chem ; 64(9): 5956-5972, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33900772

RESUMO

N-Acylethanolamines are signaling lipid molecules implicated in pathophysiological conditions associated with inflammation and pain. N-Acylethanolamine acid amidase (NAAA) favorably hydrolyzes lipid palmitoylethanolamide, which plays a key role in the regulation of inflammatory and pain processes. The synthesis and structure-activity relationship studies encompassing the isothiocyanate pharmacophore have produced potent low nanomolar inhibitors for hNAAA, while exhibiting high selectivity (>100-fold) against other serine hydrolases and cysteine peptidases. We have followed a target-based structure-activity relationship approach, supported by computational methods and known cocrystals of hNAAA. We have identified systemically active inhibitors with good plasma stability (t1/2 > 2 h) and microsomal stability (t1/2 ∼ 15-30 min) as pharmacological tools to investigate the role of NAAA in inflammation, pain, and drug addiction.


Assuntos
Amidoidrolases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isotiocianatos/química , Isotiocianatos/farmacologia , Amidoidrolases/metabolismo , Estabilidade de Medicamentos , Humanos , Hidrólise , Relação Estrutura-Atividade
8.
Molecules ; 26(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916405

RESUMO

The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.


Assuntos
Albuminas 2S de Plantas/química , Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Moringa oleifera/química , Mostardeira/química , Albuminas 2S de Plantas/isolamento & purificação , Albuminas 2S de Plantas/farmacologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Sítios de Ligação , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Folhas de Planta/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
9.
Biochemistry ; 60(12): 908-917, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33721990

RESUMO

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Sulfatos/farmacologia , Amidoidrolases/metabolismo , Cristalografia por Raios X , Neisseria meningitidis/enzimologia
10.
Acc Chem Res ; 54(7): 1623-1634, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33720682

RESUMO

Bacterial infections caused by multi-drug-resistant Gram-negative pathogens pose a serious threat to public health. Gram-negative bacteria are characterized by the enrichment of lipid A-anchored lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in the outer leaflet of their outer membrane. Constitutive biosynthesis of lipid A via the Raetz pathway is essential for bacterial viability and fitness in the human host. The inhibition of early-stage lipid A enzymes such as LpxC not only suppresses the growth of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter spp., and other clinically important Gram-negative pathogens but also sensitizes these bacteria to other antibiotics. The inhibition of late-stage lipid A enzymes such as LpxH is uniquely advantageous because it has an extra mechanism of bacterial killing through the accumulation of toxic lipid A intermediates, rendering LpxH inhibition additionally lethal to Acinetobacter baumannii. Because essential enzymes of the Raetz pathway have never been exploited by commercial antibiotics, they are excellent targets for the development of novel antibiotics against multi-drug-resistant Gram-negative infections.This Account describes the ongoing research on characterizing the structure and inhibition of LpxC and LpxH, the second and fourth enzymes of the Raetz pathway of lipid A biosynthesis, in the laboratories of Dr. Pei Zhou and Dr. Jiyong Hong at Duke University. Our studies have elucidated the molecular basis of LpxC inhibition by the first broad-spectrum inhibitor, CHIR-090, as well as the mechanism underlying its spectrum of activity. Such an analysis has provided a molecular explanation for the broad-spectrum antibiotic activity of diacetylene-based LpxC inhibitors. Through the structural and biochemical investigation of LpxC inhibition by diacetylene LpxC inhibitors and the first nanomolar LpxC inhibitor, L-161,240, we have elucidated the intrinsic conformational and dynamics difference in individual LpxC enzymes near the active site. A similar approach has been taken to investigate LpxH inhibition, leading to the establishment of the pharmacophore model of LpxH inhibitors and subsequent structural elucidation of LpxH in complex with its first reported small-molecule inhibitor based on a sulfonyl piperazine scaffold.Intriguingly, although our crystallographic analysis of LpxC- and LpxH-inhibitor complexes detected only a single inhibitor conformation in the crystal lattice, solution NMR studies revealed the existence of multiple ligand conformations that together delineate a cryptic ligand envelope expanding the ligand-binding footprint beyond that observed in the crystal structure. By harnessing the ligand dynamics information and structural insights, we demonstrate the feasibility to design potent LpxC and LpxH inhibitors by merging multiple ligand conformations. Such an approach has enabled us to rationally design compounds with significantly enhanced potency in enzymatic assays and outstanding antibiotic activities in vitro and in animal models of bacterial infection. We anticipate that continued efforts with structure and ligand dynamics-based lead optimization will ultimately lead to the discovery of LpxC- and LpxH-targeting clinical antibiotics against a broad range of Gram-negative pathogens.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Simulação de Dinâmica Molecular , Pirofosfatases/antagonistas & inibidores , Amidoidrolases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/enzimologia , Humanos , Ligantes , Estrutura Molecular , Pirofosfatases/metabolismo
11.
Exp Neurol ; 341: 113699, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33736974

RESUMO

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.


Assuntos
Amidoidrolases/antagonistas & inibidores , Doenças Neurodegenerativas/prevenção & controle , Neuroproteção/fisiologia , Fármacos Neuroprotetores/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Amidoidrolases/deficiência , Amidoidrolases/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/metabolismo , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo
12.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33671948

RESUMO

Alveolar bone loss, the major feature of periodontitis, results from the activation of osteoclasts, which can consequently cause teeth to become loose and fall out; the development of drugs capable of suppressing excessive osteoclast differentiation and function is beneficial for periodontal disease patients. Given the difficulties associated with drug discovery, drug repurposing is an efficient approach for identifying alternative uses of commercially available compounds. Here, we examined the effects of PF-3845, a selective fatty acid amide hydrolase (FAAH) inhibitor, on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis, its function, and the therapeutic potential for the treatment of alveolar bone destruction in experimental periodontitis. PF-3845 significantly suppressed osteoclast differentiation and decreased the induction of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific markers. Actin ring formation and osteoclastic bone resorption were also reduced by PF-3845, and the anti-osteoclastogenic and anti-resorptive activities were mediated by the suppression of phosphorylation of rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase, (ERK) and nuclear factor κB (NF-κB) inhibitor (IκBα). Furthermore, the administration of PF-3845 decreased the number of osteoclasts and the amount of alveolar bone destruction caused by ligature placement in experimental periodontitis in vivo. The present study provides evidence that PF-3845 is able to suppress osteoclastogenesis and prevent alveolar bone loss, and may give new insights into its role as a treatment for osteoclast-related diseases.


Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Amidoidrolases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Osteogênese/efeitos dos fármacos , Periodontite/tratamento farmacológico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Animais , Reabsorção Óssea/tratamento farmacológico , Células Cultivadas , Modelos Animais de Doenças , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Resultado do Tratamento
13.
Crit Rev Toxicol ; 51(1): 65-75, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33528291

RESUMO

In 2016, one subject died and four were hospitalized with neurological symptoms during a clinical trial with the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. The present paper reviews the regulatory toxicology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with national and international standards including European regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). The CNS effects seen in the rat and mouse appear to be common in rodents in such studies and do not in principle seem to be of the type to generate a signal. In the same way in non-human primates, insignificant alterations in the mesencephalon, and especially of the autonomic nervous system (Meissner's plexus in the bowel) in rodents and monkeys were observed in some animals treated with a high dose. Overall, these data, as well as the extensive additional data generated since the accident, support the conclusion that the tragic fatality that occurred during the clinical trial with BIA 10-2474 was unpredictable and that the mechanism responsible remains unknown, from a non-clinical toxicological perspective.


Assuntos
Óxidos N-Cíclicos/toxicidade , Inibidores Enzimáticos/toxicidade , Piridinas/toxicidade , Administração Oral , Amidoidrolases/antagonistas & inibidores , Animais , Humanos , Camundongos , Ratos
14.
Bioorg Chem ; 109: 104684, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33607363

RESUMO

As anandamide (N-arachidonoylethanolamine, AEA) shows neuroprotective effects, the inhibition of its degradative enzyme, fatty acid amide hydrolase (FAAH) has been considered as a hopeful avenue for the treatment of neurodegenerative diseases, like Alzheimer's disease (AD). Memory loss, cognitive impairment and diminution of the cholinergic tone, due to the dying cholinergic neurons in the basal forebrain, are common hallmarks in patients with AD. By taking advantage of cholinesterase inhibitors (ChEIs), the degradation of acetylcholine (ACh) is decreased leading to enhanced cholinergic neurotransmission in the aforementioned region and ultimately improves the clinical condition of AD patients. In this work, new carbamates were designed as inhibitors of FAAH and cholinestrases (ChEs) (acetylcholinestrase (AChE), butyrylcholinestrase (BuChE)) inspired by the structure of the native substrates, structure of active sites and the SARs of the well-known inhibitors of these enzymes. All the designed compounds were synthesized using different reactions. All the target compounds were tested for their inhibitory activity against FAAH and ChEs by employing the Cayman assay kit and Elman method respectively. Generally, compounds possessing aminomethyl phenyl linker was more potent compared to their corresponding compounds possessing piperazinyl ethyl linker. The inhibitory potential of the compounds 3a-q extended from 0.83 ± 0.03 µM (3i) to ˃100 µM (3a) for FAAH, 0.39 ± 0.02 µM (3i) to 24% inhibition in 113 ± 4.8 µM (3b) for AChE, and 1.8 ± 3.2 µM (3i) to 23.2 ± 0.2 µM (3b) for BuChE. Compound 3i a heptyl carbamate analog possessing 2-oxo-1,2-dihydroquinolin ring and aminomethyl phenyl linker showed the most inhibitory activity against three enzymes. Also, compound 3i was investigated for memory improvement using the Morris water maze test in which the compound showed better memory improvement at 10 mg/kg compared to reference drug rivastigmine at 2.5 mg/kg. Molecular docking and molecular dynamic studies of compound 3i into the enzymes displayed the possible interactions of key residues of the active sites with compound 3i. Finally, kinetic study indicated that 3i inhibits AChE through the mixed- mode mechanism and non-competitive inhibition mechanism was revealed for BuChE.


Assuntos
Amidoidrolases/antagonistas & inibidores , Carbamatos/química , Carbamatos/farmacologia , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Desenho de Fármacos , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
15.
J Pharmacol Exp Ther ; 377(2): 242-253, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33622769

RESUMO

Enhanced signaling of the endocannabinoid (eCB) system through inhibition of the catabolic enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) has received increasing interest for development of candidate analgesics. This study compared effects of MAGL and FAAH inhibitors with effects of ∆9-tetrahydrocannabinol (THC) using a battery of pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to stimulate two behaviors (stretching, facial grimace) and depress two behaviors (rearing, nesting). Nesting and locomotion were also assessed in the absence of IP acid as pain-independent behaviors. THC and a spectrum of six eCB catabolic enzyme inhibitors ranging from MAGL- to FAAH-selective were assessed for effectiveness to alleviate pain-related behaviors at doses that did not alter pain-independent behaviors. The MAGL-selective inhibitor MJN110 produced the most effective antinociceptive profile, with 1.0 mg/kg alleviating IP acid effects on stretching, grimace, and nesting without altering pain-independent behaviors. MJN110 effects on IP acid-depressed nesting had a slow onset and long duration (40 minutes to 6 hours), were blocked by rimonabant, and tended to be greater in females. As inhibitors increased in FAAH selectivity, antinociceptive effectiveness decreased. PF3845, the most FAAH-selective inhibitor, produced no antinociception up to doses that disrupted locomotion. THC decreased IP acid-stimulated stretching and grimace at doses that did not alter pain-independent behaviors; however, it did not alleviate IP acid-induced depression of rearing or nesting. These results support further consideration of MAGL-selective inhibitors as candidate analgesics for acute inflammatory pain. SIGNIFICANCE STATEMENT: This study characterized a spectrum of endocannabinoid catabolic enzyme inhibitors ranging in selectivity from monoacylglycerol lipase-selective to fatty acid amide hydrolase-selective in a battery of pain-stimulated, pain-depressed, and pain-independent behaviors previously pharmacologically characterized in a companion paper. This battery provides a method for prioritizing candidate analgesics by effectiveness to alleviate pain-related behaviors at doses that do not alter pain-independent behaviors, with inclusion of pain-depressed behaviors increasing translational validity and decreasing susceptibility to motor-depressant false positives.


Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/farmacologia , Comportamento Animal/efeitos dos fármacos , Dronabinol/farmacologia , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Analgésicos/efeitos adversos , Animais , Dronabinol/efeitos adversos , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Inibidores Enzimáticos/efeitos adversos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nociceptividade/efeitos dos fármacos
16.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530477

RESUMO

In migraine pain, cannabis has a promising analgesic action, which, however, is associated with side psychotropic effects. To overcome these adverse effects of exogenous cannabinoids, we propose migraine pain relief via activation of the endogenous cannabinoid system (ECS) by inhibiting enzymes degrading endocannabinoids. To provide a functional platform for such purpose in the peripheral and central parts of the rat nociceptive system relevant to migraine, we measured by activity-based protein profiling (ABPP) the activity of the main endocannabinoid-hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). We found that in trigeminal ganglia, the MAGL activity was nine-fold higher than that of FAAH. MAGL activity exceeded FAAH activity also in DRG, spinal cord and brainstem. However, activities of MAGL and FAAH were comparably high in the cerebellum and cerebral cortex implicated in migraine aura. MAGL and FAAH activities were identified and blocked by the selective and potent inhibitors JJKK-048/KML29 and JZP327A, respectively. The high MAGL activity in trigeminal ganglia implicated in the generation of nociceptive signals suggests this part of ECS as a priority target for blocking peripheral mechanisms of migraine pain. In the CNS, both MAGL and FAAH represent potential targets for attenuation of migraine-related enhanced cortical excitability and pain transmission.


Assuntos
Amidoidrolases/metabolismo , Endocanabinoides/metabolismo , Transtornos de Enxaqueca/etiologia , Transtornos de Enxaqueca/metabolismo , Monoacilglicerol Lipases/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hidrólise , Masculino , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/genética , Nervos Periféricos/metabolismo , Nervos Periféricos/fisiopatologia , Ratos
17.
Bioorg Chem ; 107: 104603, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33429229

RESUMO

LpxC inhibitors represent a promising class of novel antibiotics selectively combating Gram-negative bacteria. In chiral pool syntheses starting from D- and L-xylose, a series of four 2r,3c,4t-configured C-furanosidic LpxC inhibitors was obtained. The synthesized hydroxamic acids were tested for antibacterial and LpxC inhibitory activity, the acquired biological data were compared with those of previously synthesized C-furanosides, and molecular docking studies were performed to rationalize the observed structure-activity relationships. Additionally, bacterial uptake and susceptibility to efflux pump systems were investigated for the most promising stereoisomers.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Xilose/farmacologia , Amidoidrolases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Relação Estrutura-Atividade , Xilose/síntese química , Xilose/química
18.
Int J Mol Sci ; 22(3)2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494322

RESUMO

Altered activity of fatty acid amide hydrolase (FAAH), an enzyme of the endocannabinoid system, has been implicated in several neuropsychiatric disorders, including major depressive disorder (MDD). It is speculated that increased brain FAAH expression is correlated with increased depressive symptoms. The aim of this scoping review was to establish the role of FAAH expression in animal models of depression to determine the translational potential of targeting FAAH in clinical studies. A literature search employing multiple databases was performed; all original articles that assessed FAAH expression in animal models of depression were considered. Of the 216 articles that were screened for eligibility, 24 articles met inclusion criteria and were included in this review. Three key findings emerged: (1) FAAH expression is significantly increased in depressive-like phenotypes; (2) genetic knockout or pharmacological inhibition of FAAH effectively reduces depressive-like behavior, with a dose-dependent effect; and (3) differences in FAAH expression in depressive-like phenotypes were largely localized to animal prefrontal cortex, hippocampus and striatum. We conclude, based on the animal literature, that a positive relationship can be established between brain FAAH level and expression of depressive symptoms. In summary, we suggest that FAAH is a tractable target for developing novel pharmacotherapies for MDD.


Assuntos
Amidoidrolases/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Depressão/etiologia , Depressão/metabolismo , Suscetibilidade a Doenças , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Antidepressivos/farmacologia , Biomarcadores , Encéfalo/efeitos dos fármacos , Depressão/tratamento farmacológico , Depressão/psicologia , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Predisposição Genética para Doença , Humanos , Neurônios/metabolismo , Roedores
19.
ACS Chem Biol ; 16(2): 264-269, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33492128

RESUMO

Electrophilic heterocycles offer attractive features as covalent fragments for inhibitor and probe development. A focused library of heterocycles for which protonation can enhance reactivity (called "switchable electrophiles") is screened for inhibition of the proposed drug target dimethylarginine dimethylaminohydrolase (DDAH). Several novel covalent fragments are identified: 4-chloroquinoline, 4-bromopyridazine, and 4,4-dipyridylsulfide. Mechanistic studies of DDAH inactivation by 4,4-dipyridylsulfide reveal selective covalent S-pyridinylation of the active-site Cys through catalysis by a neighboring Asp residue. Inactivation (kinact/KI = 0.33 M-1 s-1) proceeds with release of 4-thiopyridone (0.78 equiv), and structure-activity relationships reveal that the leaving group pKa can be modulated to tune reactivity. The use of a "switchable electrophile" strategy helps impart selectivity, even to fragment-sized modifiers. Identification of 4,4-dipyridylsulfide analogs as inactivators offers an easily tunable covalent fragment with multiple derivatization sites on both the leaving and staying groups.


Assuntos
Amidoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/química , Piridinas/química , Sulfetos/química , Amidoidrolases/química , Amidoidrolases/metabolismo , Domínio Catalítico , Ensaios Enzimáticos , Inibidores Enzimáticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Piridinas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Sulfetos/metabolismo
20.
Bioorg Med Chem ; 30: 115964, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385955

RESUMO

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Imidazóis/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Amidoidrolases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Imidazóis/síntese química , Imidazóis/química , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
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