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1.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555144

RESUMO

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.


Assuntos
Endocanabinoides , Neuralgia , Humanos , Endocanabinoides/metabolismo , Neuralgia/tratamento farmacológico , Amidoidrolases/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Inflamação/tratamento farmacológico , Simulação de Dinâmica Molecular
2.
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
3.
Molecules ; 22(7)2017 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-28698463

RESUMO

Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3'UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3'UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/uso terapêutico , Regiões 3' não Traduzidas/genética , Células A549 , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Humanos , MicroRNAs/antagonistas & inibidores , Mutação , Ácidos Nucleicos Peptídicos/genética , RNA Mensageiro/genética , Transfecção
4.
Eur J Med Chem ; 136: 523-542, 2017 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-28535469

RESUMO

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes.


Assuntos
Amidoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ibuprofeno/farmacologia , Amidoidrolases/metabolismo , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ibuprofeno/síntese química , Ibuprofeno/química , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Relação Estrutura-Atividade , Termodinâmica
5.
PLoS One ; 9(11): e112082, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25375166

RESUMO

UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1-E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.


Assuntos
Modelos Moleculares , Complexos Multienzimáticos/química , Enzimas Ativadoras de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/metabolismo , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Estrutura Quaternária de Proteína , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
6.
Biomed Res Int ; 2014: 610718, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24829907

RESUMO

Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3'UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.


Assuntos
Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Ácidos Nucleicos Peptídicos/farmacologia , Ácidos Nucleicos Peptídicos/uso terapêutico , Linhagem Celular Tumoral , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos da radiação , Ácidos Nucleicos Peptídicos/síntese química , Espectrofotometria Ultravioleta , Raios Ultravioleta
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