RESUMO
Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.
Assuntos
Cistinil Aminopeptidase , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Humanos , Cristalografia por Raios X , Especificidade por Substrato , Pirimidinas/química , Pirimidinas/farmacologia , Modelos Moleculares , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Conformação ProteicaRESUMO
The discovery of a dual MAO-B/SSAO inhibitor PXS-5131 is reported. The compound offers a compact and rigid three-dimensional structure with superior selectivity over MAO-A. Potency and selectivity are linked to both the double bond geometry and stereochemistry of the allylamine moiety, highlighting the importance of optimal set up of these features in the class of amine oxidase inhibitors. PXS-5131 possesses an attractive preclinical pharmacokinetic profile and has anti-inflammatory properties in models of acute inflammation and neuroinflammation.
Assuntos
Amina Oxidase (contendo Cobre) , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Monoaminoxidase , Inibidores da Monoaminoxidase/farmacologiaRESUMO
Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.
Assuntos
Aminas/química , Aminas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Animais , Desenho de Fármacos , Humanos , Camundongos , Triazóis/químicaRESUMO
Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Colágeno/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrose/prevenção & controle , Infarto do Miocárdio/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Tetracloreto de Carbono/toxicidade , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Reagentes de Ligações Cruzadas/química , Elastina/antagonistas & inibidores , Elastina/efeitos dos fármacos , Elastina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibrose/induzido quimicamente , Fibrose/enzimologia , Fibrose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Ratos WistarRESUMO
The synthesis of the structure, 1, assigned to the anti-inflammatory natural product myrsinoic acid F is reported together with a means for preparing its Z-isomer 21. While neither of these compounds corresponds to the natural product, both of them are anti-inflammatory agents (as determined using a mouse ear edema assay) with congener 1 being notably more potent than the widely prescribed NSAID indometacin.
Assuntos
Alcenos/síntese química , Benzofuranos/síntese química , Animais , Anti-Inflamatórios não Esteroides , Produtos Biológicos , Estrutura MolecularRESUMO
Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Inibidores Enzimáticos/farmacologia , Aminoácido Oxirredutases/genética , Aminopropionitrilo/farmacologia , Animais , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Camundongos , Metástase Neoplásica , Neovascularização Patológica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A. METHODS: Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A. RESULTS: Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.
Assuntos
Alilamina/análogos & derivados , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Benzamidas/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Infecções por Klebsiella/tratamento farmacológico , Pulmão/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Infecções por Picornaviridae/tratamento farmacológico , Pneumonia/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Alilamina/farmacocinética , Alilamina/farmacologia , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Anti-Inflamatórios/farmacocinética , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Asma/virologia , Benzamidas/farmacocinética , Broncoconstrição/efeitos dos fármacos , Ceco/microbiologia , Ceco/cirurgia , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Inibidores Enzimáticos/farmacocinética , Infecções por Klebsiella/enzimologia , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/patogenicidade , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Ligadura , Lipopolissacarídeos , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Picornaviridae/enzimologia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/fisiopatologia , Infecções por Picornaviridae/virologia , Pneumonia/enzimologia , Pneumonia/etiologia , Pneumonia/imunologia , Punções , Ratos Wistar , Infecções Respiratórias/enzimologia , Infecções Respiratórias/etiologia , Infecções Respiratórias/imunologia , Rhinovirus/patogenicidadeRESUMO
Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.
Assuntos
Compostos Alílicos/farmacologia , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/enzimologia , Compostos Alílicos/química , Compostos Alílicos/farmacocinética , Compostos Alílicos/uso terapêutico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Dermatite/tratamento farmacológico , Dermatite/enzimologia , Dermatite/imunologia , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Técnicas In Vitro , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Modelos Biológicos , Estrutura Molecular , Pneumonia/tratamento farmacológico , Pneumonia/enzimologia , Pneumonia/imunologia , Coelhos , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Especificidade da Espécie , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêuticoRESUMO
A new class of 3-fluoroallyl amine-based SSAO/VAP-1 inhibitors is reported. These compounds have excellent selectivity over diamine oxidase, MAO-A and MAO-B. Synthesis and SAR studies leading to compound 28 (PXS-4159A) are reported. The pharmacokinetic profile of 28 in the rat, together with activity in a murine model of lung inflammation are also disclosed.
Assuntos
Compostos Alílicos/síntese química , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Aminas/síntese química , Anti-Inflamatórios/síntese química , Inibidores da Monoaminoxidase/síntese química , Pneumonia/tratamento farmacológico , Compostos Alílicos/farmacocinética , Compostos Alílicos/farmacologia , Amina Oxidase (contendo Cobre)/metabolismo , Aminas/farmacocinética , Aminas/farmacologia , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Descoberta de Drogas , Halogenação , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Inibidores da Monoaminoxidase/farmacocinética , Inibidores da Monoaminoxidase/farmacologia , Pneumonia/enzimologia , Ratos , Relação Estrutura-AtividadeRESUMO
The need for an alternative to red cells for oxygen transport in transfusions has led to the creation of hemoglobin-based oxygen carriers, materials produced by chemical modification or genetic engineering of human or bovine hemoglobin. Modifications of the native proteins are necessitated by the spontaneous dissociation of the functional hemoglobin tetramers (alpha(2)beta(2)) into non-functional alphabeta dimers. Based on clinical observations of hypertension resulting from some of these materials, it was proposed that the stabilized tetramers are sufficiently small to extravasate through blood vessels and scavenge nitric oxide, depleting the endothelium of the signal for smooth muscle relaxation. In order to increase size and minimize extravasation while maintaining structure and function, methods for producing larger entities through protein-protein conjugation were developed. Approaches have included the use of nonspecific reagents that polymerize proteins (e.g., polyglutaraldehyde), conjugation to polyethylene glycol, expression of naturally occurring multimers and the use of selective reagents, which is the focus of this article.
Assuntos
Substitutos Sanguíneos/química , Substitutos Sanguíneos/uso terapêutico , Animais , Substitutos Sanguíneos/metabolismo , Ensaios Clínicos como Assunto , Reagentes de Ligações Cruzadas/química , Humanos , Nitrito Redutases/metabolismo , Oxigênio/metabolismo , Polietilenoglicóis/química , Engenharia de Proteínas , Multimerização Proteica , Proteínas/químicaRESUMO
Cross-linked hemoglobin-azides react with a bis-alkyne to form a bis-tetramer through sequential "click" reactions where the second step is promoted by the first.
Assuntos
Alcinos/química , Azidas/química , Reagentes de Ligações Cruzadas/química , Hemoglobinas/química , Multimerização Proteica , Hemoglobinas/metabolismo , Humanos , Estrutura Molecular , Estabilidade Proteica , SolubilidadeRESUMO
With certain substituent patterns, benzyl benzyl sulfone systems have been found to give unexpectedly high Z-stereoselectivity (up to E:Z = 1:16) in the Meyers variant of the Ramberg-Bäcklund reaction. A range of sulfones, bearing various aryl substituents, were explored to rationalize this unprecedented selectivity for Z-stilbene systems. This high level of double bond stereocontrol has also been utilized in the synthesis of integrastatin nucleus, the core of two highly bioactive anti-HIV compounds.
Assuntos
Inibidores de Integrase de HIV/síntese química , Estilbenos/síntese química , Sulfonas/química , Alcenos/síntese química , Alcenos/química , Inibidores de Integrase de HIV/química , Isomerismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Estilbenos/químicaRESUMO
The effect of the variation of the experimental parameters on the conversion of precursor to products in a typical flash vacuum pyrolysis (FVP) experiment was investigated empirically. Temperature-conversion plots can be used to optimise FVP conditions and their mechanistic significance is exemplified. At a given temperature, the conversion can be increased by an increase in the background pressure, or by packing a section of the furnace tube with inert material (particularly when placed at the trap end of the furnace tube) or by employing a catalyst. Despite the prevailing view that only intramolecular reactions take place by FVP, it has been shown by a 'dual-FVP' cross-over experiment that the dimerisation of benzyl radicals occurs in the gas-phase, before the cold trap, under standard conditions. However, reduction in through-put rate, increase in furnace temperature and reduction in background pressure all reduce the amount of gas-phase coupling.
RESUMO
[reaction: see text] The first synthesis of the tetracyclic nucleus of the Integrastatins, natural products that have been shown to selectively inhibit HIV-1 integrase, is reported. Key steps of this synthesis involve a novel cis-selective Ramberg-Bäcklund reaction and an unusual Lewis acid-promoted cyclization step.