RESUMO
Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1ß to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.
Assuntos
Cinurenina , Pancreatite , Camundongos , Animais , Estado Terminal , Insuficiência de Múltiplos Órgãos , Doença Aguda , Camundongos Knockout , Inflamação , Quinurenina 3-Mono-Oxigenase/genéticaRESUMO
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
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
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
Endothelial barrier dysfunction leads to edema and vascular leak, causing high morbidity and mortality. Previously, Abl kinase inhibition has been shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes. We found that the inhibitor bosutinib most potently protected against inflammation-induced endothelial barrier disruption. In vivo, bosutinib prevented lipopolysaccharide (LPS)-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, mitogen-activated protein 4 kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signaled via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Abl-related gene (Arg, also known as ABL2) by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation. We conclude that MAP4K4 is an important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Because it inhibits both Arg and MAP4K4, use of the clinically available drug bosutinib might form a viable strategy against vascular leakage syndromes.
Assuntos
Adesões Focais , Preparações Farmacêuticas , Junções Aderentes , Compostos de Anilina , Animais , Permeabilidade Capilar , Camundongos , Nitrilas , QuinolinasRESUMO
ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.
Assuntos
Aminopeptidases/antagonistas & inibidores , Apresentação de Antígeno/efeitos dos fármacos , Diterpenos Clerodânicos/farmacologia , Epitopos/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Sítio Alostérico , Aminopeptidases/química , Aminopeptidases/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Epitopos/química , Células HeLa , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Peptídeos/química , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacosRESUMO
Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.
RESUMO
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
The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.
Assuntos
Benzamidinas/química , Calicreínas/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Descoberta de Drogas , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/metabolismo , Cinética , Modelos Moleculares , Mutação , Síndrome de Netherton/tratamento farmacológico , Síndrome de Netherton/enzimologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática , Especificidade por SubstratoRESUMO
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.
Assuntos
Desenho de Fármacos , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , HumanosRESUMO
Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.
Assuntos
Benzamidinas/química , Calicreínas/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Animais , Benzamidinas/síntese química , Benzamidinas/metabolismo , Domínio Catalítico , Desenho de Fármacos , Calicreínas/metabolismo , Síndrome de Netherton/tratamento farmacológico , Ligação Proteica , Salicilamidas/síntese química , Salicilamidas/química , Salicilamidas/metabolismo , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/metabolismo , Spodoptera/genéticaRESUMO
The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pele/efeitos dos fármacos , Quinase Syk/antagonistas & inibidores , Administração Tópica , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Quinase Syk/químicaRESUMO
Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington's and Alzheimer's. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.
Assuntos
Inibidores Enzimáticos/farmacologia , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Insuficiência de Múltiplos Órgãos/metabolismo , Pancreatite/metabolismo , Animais , Inibidores Enzimáticos/química , Escherichia coli/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Quinurenina 3-Mono-Oxigenase/química , Quinurenina 3-Mono-Oxigenase/metabolismo , Modelos Moleculares , Domínios Proteicos , Células Sf9RESUMO
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
We report a multi-component asymmetric Brønsted acid-catalyzed aza-Darzens reaction which is not limited to specific aromatic or heterocyclic aldehydes. Incorporating alkyl diazoacetates and, important for high ee's, ortho-tert-butoxyaniline our optimized reaction (i.e. solvent, temperature and catalyst study) affords excellent yields (61-98 %) and mostly >90 % optically active cis-aziridines. (+)-Chloramphenicol was generated in 4 steps from commercial starting materials. A tentative mechanism is outlined.
RESUMO
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
Inhibition of kynurenine 3-monooxygenase (KMO) protects against multiple organ dysfunction (MODS) in experimental acute pancreatitis (AP). We aimed to precisely define the kynurenine pathway activation in relation to AP and AP-MODS in humans, by carrying out a prospective observational study of all persons presenting with a potential diagnosis of AP for 90 days. We sampled peripheral venous blood at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. We measured tryptophan metabolite concentrations and analysed these in the context of clinical data and disease severity indices, cytokine profiles and C-reactive protein (CRP) concentrations. 79 individuals were recruited (median age: 59.6 years; 47 males, 59.5%). 57 met the revised Atlanta definition of AP: 25 had mild, 23 moderate, and 9 severe AP. Plasma 3-hydroxykynurenine concentrations correlated with contemporaneous APACHE II scores (R2 = 0.273; Spearman rho = 0.581; P < 0.001) and CRP (R2 = 0.132; Spearman rho = 0.455, P < 0.001). Temporal profiling showed early tryptophan depletion and contemporaneous 3-hydroxykynurenine elevation. Furthermore, plasma concentrations of 3-hydroxykynurenine paralleled systemic inflammation and AP severity. These findings support the rationale for investigating early intervention with a KMO inhibitor, with the aim of reducing the incidence and severity of AP-associated organ dysfunction.
RESUMO
The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.
Assuntos
Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Humanos , Testes de Mutagenicidade , Naftiridinas/administração & dosagem , Naftiridinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
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.