RESUMO
The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high inâ vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.
Assuntos
Amidas/química , Benzoxazóis/química , Ceramidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologiaRESUMO
Inhibition of intracellular N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity is a promising approach to manage the inflammatory response under disabling conditions. In fact, NAAA inhibition preserves endogenous palmitoylethanolamide (PEA) from degradation, thus increasing and prolonging its anti-inflammatory and analgesic efficacy at the inflamed site. In the present work, we report the identification of a potent, systemically available, novel class of NAAA inhibitors, featuring a pyrazole azabicyclo[3.2.1]octane structural core. After an initial screening campaign, a careful structure-activity relationship study led to the discovery of endo-ethoxymethyl-pyrazinyloxy-8-azabicyclo[3.2.1]octane-pyrazole sulfonamide 50 (ARN19689), which was found to inhibit human NAAA in the low nanomolar range (IC50 = 0.042 µM) with a non-covalent mechanism of action. In light of its favorable biochemical, in vitro and in vivo drug-like profile, sulfonamide 50 could be regarded as a promising pharmacological tool to be further investigated in the field of inflammatory conditions.
Assuntos
Amidoidrolases/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Tropanos/farmacologia , Amidoidrolases/metabolismo , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Pirazóis/síntese química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tropanos/síntese química , Tropanos/metabolismo , Tropanos/farmacocinéticaRESUMO
Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.
Assuntos
Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tetrazóis/farmacologia , Animais , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Indazóis/síntese química , Indazóis/farmacocinética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/farmacocinéticaRESUMO
Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of ß-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzyme's catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.
Assuntos
Amidoidrolases/antagonistas & inibidores , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Ativação de Macrófagos/efeitos dos fármacos , beta-Lactamas/química , beta-Lactamas/farmacologia , Amidoidrolases/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , beta-Lactamas/uso terapêuticoRESUMO
N-(2-Oxo-3-oxetanyl)carbamic acid esters have recently been reported to be noncompetitive inhibitors of the N-acylethanolamine acid amidase (NAAA) potentially useful for the treatment of pain and inflammation. In the present study, we further explored the structure-activity relationships of the carbamic acid ester side chain of 2-methyl-4-oxo-3-oxetanylcarbamic acid ester derivatives. Additional favorable features in the design of potent NAAA inhibitors have been found together with the identification of a single digit nanomolar inhibitor. In addition, we devised a 3D QSAR using the atomic property field method. The model turned out to be able to account for the structural variability and was prospectively validated by designing, synthesizing, and testing novel inhibitors. The fairly good agreement between predictions and experimental potency values points to this 3D QSAR model as the first example of quantitative structure-activity relationships in the field of NAAA inhibitors.
Assuntos
Amidoidrolases/antagonistas & inibidores , Carbamatos/síntese química , Inibidores Enzimáticos/síntese química , Ésteres/síntese química , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Relação Estrutura-AtividadeRESUMO
Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/uso terapêutico , Carbamatos/uso terapêutico , Endocanabinoides/fisiologia , Inibidores Enzimáticos/uso terapêutico , Éteres Cíclicos/uso terapêutico , Hiperalgesia/tratamento farmacológico , Ácidos Oleicos/fisiologia , PPAR alfa/fisiologia , Percepção da Dor/efeitos dos fármacos , Amidas , Amidoidrolases/genética , Amidoidrolases/fisiologia , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Queimaduras/tratamento farmacológico , Queimaduras/etiologia , Carbamatos/administração & dosagem , Carbamatos/farmacologia , Carragenina/toxicidade , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Etanolaminas , Éteres Cíclicos/administração & dosagem , Éteres Cíclicos/farmacologia , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Lisossomos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR alfa/agonistas , PPAR alfa/deficiência , Percepção da Dor/fisiologia , Ácidos Palmíticos , Lesões por Radiação/tratamento farmacológico , Lesões por Radiação/etiologia , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Nervo Isquiático/lesões , Acetato de Tetradecanoilforbol/toxicidade , Raios Ultravioleta/efeitos adversosRESUMO
N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid agonists of peroxisome proliferator-activated receptor-α, which include oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). The ß-lactone derivatives (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide (2) and (S)-N-(2-oxo-3-oxetanyl)-biphenyl-4-carboxamide (3) inhibit NAAA, prevent FAE hydrolysis in activated inflammatory cells, and reduce tissue reactions to pro-inflammatory stimuli. Recently, our group disclosed ARN077 (4), a potent NAAA inhibitor that is active in vivo by topical administration in rodent models of hyperalgesia and allodynia. In the present study, we investigated the structure-activity relationship (SAR) of threonine-derived ß-lactone analogues of compound 4. The main results of this work were an enhancement of the inhibitory potency of ß-lactone carbamate derivatives for NAAA and the identification of (4-phenylphenyl)-methyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate (14q) as the first single-digit nanomolar inhibitor of intracellular NAAA activity (IC50 = 7 nM on both rat NAAA and human NAAA).
Assuntos
Amidoidrolases/antagonistas & inibidores , Carbamatos/química , Carbamatos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Sequência de Bases , Carbamatos/síntese química , Primers do DNA , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Espectrometria de Massas em TandemRESUMO
The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted ß-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography-tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA.