RESUMO
α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
Assuntos
Desenho de Fármacos , Dobramento de Proteína , alfa 1-Antitripsina/metabolismo , Cristalização , Desenvolvimento de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/metabolismo , Biblioteca Gênica , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , alfa 1-Antitripsina/genéticaRESUMO
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Assuntos
Benzamidinas/química , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Benzamidinas/farmacologia , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Humanos , Isomerismo , Modelos Moleculares , Estrutura Molecular , Mutação , Ligação Proteica , Inibidor de Serinopeptidase do Tipo Kazal 5/genética , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1ß (IL-1ß) injections. We initiated GSK247246 treatment (i.p at 7â¯mg/kg or 20â¯mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7â¯mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.
Assuntos
Antagonistas dos Receptores Histamínicos H3/farmacologia , Substância Branca/lesões , Substância Branca/patologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encefalopatias/tratamento farmacológico , Lesões Encefálicas/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos , Microglia/metabolismo , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Neurogênese , Neuroimunomodulação/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Oligodendroglia , Gravidez , Nascimento Prematuro/tratamento farmacológico , Receptores Histamínicos/metabolismo , Substância Branca/metabolismoRESUMO
A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Animais , Benzoxazóis/química , Benzoxazóis/farmacocinética , Benzoxazóis/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Indazóis/química , Indazóis/farmacocinética , Indazóis/farmacologia , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase/metabolismo , Pancreatite/enzimologia , Pancreatite/metabolismo , Ratos , Triptofano/metabolismoRESUMO
The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.
Assuntos
Apolipoproteína A-I/metabolismo , Descoberta de Drogas , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Quinolinas/química , Regulação para Cima/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células Hep G2 , Histona Acetiltransferases , Chaperonas de Histonas , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso , Proteínas Nucleares/metabolismo , Quinolinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency.
Assuntos
Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina , Animais , Retículo Endoplasmático , Hepatócitos , Camundongos , alfa 1-Antitripsina/genéticaRESUMO
Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.
Assuntos
Anti-Inflamatórios/uso terapêutico , Compostos de Boro/uso terapêutico , Inflamação/tratamento farmacológico , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , Pele/patologia , Administração Tópica , Animais , Modelos Animais de Doenças , Humanos , Calicreínas/genética , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Pele/efeitos dos fármacos , Creme para a PeleRESUMO
Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.
Assuntos
Inibidores Enzimáticos/farmacologia , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Doença Aguda , Animais , Inibidores Enzimáticos/uso terapêutico , Humanos , RatosRESUMO
Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.
Assuntos
Benzoxazóis/farmacologia , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Insuficiência de Múltiplos Órgãos/genética , Oxazolidinonas/farmacologia , Pancreatite/genética , Propionatos/farmacologia , RNA Mensageiro/metabolismo , Doença Aguda , Animais , Cromatografia Líquida , Cristalografia por Raios X , Modelos Animais de Doenças , Células HEK293 , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Rim/metabolismo , Rim/patologia , Quinurenina 3-Mono-Oxigenase/genética , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/complicações , Pancreatite/patologia , Ratos , Espectrometria de Massas em Tandem , Triptofano/metabolismoRESUMO
Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.
Assuntos
Inibidores Enzimáticos/farmacologia , Lactamas/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lactamas/administração & dosagem , Lactamas/síntese química , Lactamas/química , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Tiazóis/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
The aim of this study was to test the hypothesis that the most appropriate model for studying the diffusional release of an active from a topical formulation is one in which the membrane offers minimal resistance to release and involves a receptor phase that presents the least possible interfacial discontinuity. Using ketoprofen as the active, a series of simple gels were prepared consisting of PEG400 thickened with Cabosil M5. Using Franz-type diffusion cells, three different types of membrane (two porous and one non-porous) were compared, as were receptor phases of PEG400 (component of formulation) and PBS. Of the membranes tested only 0.2 microm nylon provided consistent first order kinetics for a range of gel consistencies, indicating negligible influence of the membrane. The non-porous silicone membrane did not show first order kinetic profile confirming the diffusional nature of such a membrane. From the non-thickened formulations, diffusional release into a receptor phase of PEG400 was some 3x that into PBS, whereas from the formulation thickened with 5% Cabosil M5, diffusional release into a receptor phase of PEG400 was 6x lower than that into PBS. Diffusional release into PBS did not follow first order kinetics while diffusion into PEG400 did, suggesting that the existence of a discontinuity affected the release process. Although the importance of zero-resistance membranes is of perhaps obvious importance, it is often not stated in the literature. The existence of phase/hydrodynamic boundaries in release studies can be a source of significant inaccuracy.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/química , Cetoprofeno/administração & dosagem , Cetoprofeno/química , Administração Tópica , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Difusão , Géis , Membranas Artificiais , Nylons , Veículos Farmacêuticos , Polietilenoglicóis , Polipropilenos , Elastômeros de SiliconeRESUMO
The migration of ketoprofen through a series of simple gels that varied in solvent composition to simulate snapshots of a dynamically drying topical formulation was studied. Firstly, the release rate of ketoprofen was determined from formulations based on Cabosil and PEG 400, the proportion of which was varied to mimic progressively dryer states. Secondly, the apparent permeability of ketoprofen across the corresponding blank Cabosil gels was determined. Thirdly, the effect of macro viscosity on these data was probed by comparing permeation of ketoprofen across Cabosil and hydroxypropyl cellulose (HPC) gels of equal viscosity. Linear release profiles were produced for all formulations suggesting first-order release and the rate of ketoprofen liberated was inversely to the proportion of Cabosil, suggesting that the drier the film, the slower the rate of release. At the lowest level of thickener used (5%) the release rate was reduced to 45% of the control. At 25% the release rate was reduced to 24% of the control. The presence of the Cabosil had an even more dramatic effect on the apparent permeability of ketoprofen across the gels. At 5% Cabosil the apparent steady state flux was reduced to 4% of the control. At 25% the apparent steady state flux was reduced to < 1% of the control. Although the 0.5% HPC gel and the 1% Cabosil gel possessed identical macro viscosities, the permeation of ketoprofen through the HPC gel was almost double that of the Cabosil gel. The data from these experiments demonstrated that migration of active molecules through a gel is significantly affected by the amount of solvent present in, or lost from, the system. It is proposed that increased adsorption of active to the thickener plays a more important role than increased macro viscosity for reduced active release as the formulation becomes increasingly dry. Furthermore, such affects are profoundly influenced by the chemical nature of the thickener.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Cetoprofeno/administração & dosagem , Administração Tópica , Química Farmacêutica , Géis , PolietilenoglicóisRESUMO
Bromodomains (BRDs) are small protein domains found in a variety of proteins that recognize and bind to acetylated histone tails. This binding affects chromatin structure and facilitates the localisation of transcriptional complexes to specific genes, thereby regulating epigenetically controlled processes including gene transcription and mRNA elongation. Inhibitors of the bromodomain and extra-terminal (BET) proteins BRD2-4 and T, which prevent bromodomain binding to acetyl-modified histone tails, have shown therapeutic promise in several diseases. We report here the discovery of 1,5-naphthyridine derivatives as potent inhibitors of the BET bromodomain family with good cell activity and oral pharmacokinetic parameters. X-ray crystal structures of naphthyridine isomers have been solved and quantum mechanical calculations have been used to explain the higher affinity of the 1,5-isomer over the others. The best compounds were progressed in a mouse model of inflammation and exhibited dose-dependent anti-inflammatory pharmacology.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Proteínas Cromossômicas não Histona , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Histonas/química , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Naftiridinas/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.
Assuntos
Aminoquinolinas/síntese química , Anti-Inflamatórios/síntese química , Apolipoproteína A-I/metabolismo , Benzoatos/síntese química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinolinas/síntese química , Fatores de Transcrição/antagonistas & inibidores , Aminoquinolinas/farmacocinética , Aminoquinolinas/farmacologia , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Benzoatos/farmacocinética , Benzoatos/farmacologia , Proteínas de Ciclo Celular , Descoberta de Drogas , Humanos , Camundongos , Quinolinas/farmacocinética , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
AMP-activated protein kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme that is activated in shortage of energy and suppressed in its surfeit. AMPK activation stimulates fatty acid oxidation, enhances insulin sensitivity, alleviates hyperglycemia and hyperlipidemia, and inhibits proinflammatory changes. Thus, AMPK is a well-received therapeutic target for type 2 diabetes and other metabolic disorders. Here, we will report the discovery of pyrrolopyridone derivatives as AMPK direct activators. We will illustrate the synthesis and structure-activity relationships of the series as well as some pharmacokinetic results. Some compounds exhibited encouraging oral exposure and were evaluated in a mouse diabetic model. Compound 17 showed oral activity at 30 mg/kg on blood glucose.