RESUMEN
Cellular signaling via binding of the cytokines IL-36α, ß, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.
Asunto(s)
Anticuerpos Monoclonales/química , Fragmentos Fab de Inmunoglobulinas/química , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/química , Cristalografía por Rayos X , Células HEK293 , Humanos , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.
Asunto(s)
Isoxazoles/química , Prolina/química , Ácido Pirrolidona Carboxílico/análogos & derivados , Receptor Cannabinoide CB2/agonistas , Animales , Neuropatías Diabéticas/inducido químicamente , Neuropatías Diabéticas/tratamiento farmacológico , Semivida , Humanos , Isoxazoles/farmacocinética , Isoxazoles/uso terapéutico , Ligandos , Masculino , Microsomas Hepáticos/metabolismo , Prolina/farmacocinética , Prolina/uso terapéutico , Unión Proteica , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/farmacocinética , Ácido Pirrolidona Carboxílico/uso terapéutico , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Solubilidad , Relación Estructura-ActividadRESUMEN
Computer-aided drug design scaffold hopping strategies were utilized to identify new classes of CB2 agonists when compounds of an established series with low nanomolar potency were challenging to optimize for good drug-like properties. Use of ligand-based design strategies through BI Builder (a tool for de novo design) and PharmShape (a virtual screening software package) approaches led to the discovery of new chemotypes. Specifically, compounds containing azetidine-, proline-, and piperidine-based cores were found to have low nanomolar and picomolar CB2 agonist activities with drug-like properties considered appropriate for early profiling.
Asunto(s)
Diseño de Fármacos , Receptor Cannabinoide CB2/agonistas , Diseño Asistido por Computadora , Humanos , Ligandos , Microsomas Hepáticos/metabolismo , Unión Proteica , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Solubilidad , Relación Estructura-ActividadRESUMEN
A novel class of potent cannabinoid receptor 2 (CB2) agonists based on a (S)-piperidine scaffold was identified using ligand-based pharmacophore models. Optimization of solubility and metabolic stability led to the identification of several potent CB2 agonists (e.g., 30) that displayed selectivity over cannabinoid receptor 1 (CB1) and acceptable drug like properties. In rats, compound 30 demonstrated a favorable pharmacokinetic profile and efficacy in a Streptozotocin-induced diabetic neuropathy model, with full reversal of mechanical hyperalgesia.
Asunto(s)
Ácidos Pipecólicos/química , Piperidinas/química , Receptor Cannabinoide CB2/agonistas , Tiazinas/química , Animales , Neuropatías Diabéticas/inducido químicamente , Neuropatías Diabéticas/tratamiento farmacológico , Semivida , Humanos , Ligandos , Masculino , Microsomas Hepáticos/metabolismo , Dolor/tratamiento farmacológico , Ácidos Pipecólicos/farmacocinética , Ácidos Pipecólicos/uso terapéutico , Piperidinas/farmacocinética , Piperidinas/uso terapéutico , Unión Proteica , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Solubilidad , Relación Estructura-Actividad , Tiazinas/farmacocinética , Tiazinas/uso terapéuticoRESUMEN
GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Análisis por Conglomerados , Cricetulus , AMP Cíclico , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Perfilación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Células Mieloides/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Bile acids (BAs) and BA receptors, including G protein-coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined. The results demonstrated that the infusion of three structurally diverse and selective GPBAR1 agonists produced marked reductions in vascular tone and blood pressure in dog, but not in rat, as well as reflex tachycardia and a positive inotropic response, effects that manifested in an enhanced cardiac output. Changes in cardiovascular function were unrelated to modulation of the levothyroxine/thyroxine axis and were nitric oxide independent. A direct effect on vascular tone was confirmed in dog isolated vascular rings, whereby concentration-dependent decreases in tension that were tightly correlated with reductions in vascular tone observed in vivo and were blocked by iberiotoxin. Compound concentrations in which cardiovascular effects occurred, both ex vivo and in vivo, could not be separated from those necessary for modulation of GPBAR1-mediated efficacy, resulting in project termination. These results are the first to clearly demonstrate direct and potent peripheral arterial vasodilation due to GPBAR1 stimulation in vivo through activation of large conductance Ca(2+) activated potassium channel K(Ca)1.1.
Asunto(s)
Arterias/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Vasodilatación/efectos de los fármacos , Animales , Arterias/fisiología , Factor Natriurético Atrial/sangre , Células CHO , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/patología , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Citocinas/sangre , Dinitrofluorobenceno/análogos & derivados , Perros , Endotelina-1/sangre , Humanos , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Óxido Nítrico/biosíntesis , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Tiroxina/sangre , Triazoles/farmacologíaRESUMEN
This paper details exploration of a class of triazole-based cathepsin S inhibitors originally reported by Ellman and co-workers. SAR studies involving modifications across the whole inhibitor provide a perspective on the strengths and weaknesses of this class of inhibitors. In addition, we put the unique characteristics of this class of compounds into perspective with other classes of cathepsin S inhibitors.
Asunto(s)
Amidas/química , Catepsinas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Tiofenos/química , Triazoles/química , Catepsinas/metabolismo , Semivida , Humanos , Microsomas Hepáticos/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Unión Proteica , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/farmacocinética , Triazoles/síntesis química , Triazoles/farmacocinéticaRESUMEN
Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.
Asunto(s)
Benzamidas/síntesis química , Guanidinas/síntesis química , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Animales , Benzamidas/química , Benzamidas/farmacología , Disponibilidad Biológica , Plaquetas/citología , Plaquetas/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular , Tamaño de la Célula , Inhibidores Enzimáticos del Citocromo P-450 , Perros , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Guanidinas/química , Guanidinas/farmacología , Humanos , Masculino , Membranas Artificiales , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Permeabilidad , Isoformas de Proteínas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Intercambiador 1 de Sodio-Hidrógeno , Relación Estructura-ActividadRESUMEN
The discovery and SAR of a series of beta-aryl substituted pyrrolidine 2H-isoquinolin-1-one inhibitors of Rho-kinase (ROCK) derived from 2 is herein described. SAR studies have shown that aryl groups in the beta-position are optimal for potency. Our efforts focused on improving the ROCK potency of this isoquinolone class of inhibitors which led to the identification of pyrrolidine 32 which demonstrated a 10-fold improvement in aortic ring (AR) potency over 2.
Asunto(s)
Isoquinolinas/química , Inhibidores de Proteínas Quinasas/química , Pirrolidinas/síntesis química , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Aorta/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Hipertensión/tratamiento farmacológico , Concentración 50 Inhibidora , Isoquinolinas/farmacología , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/química , Pirrolidinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
In this paper, we describe an in silico first principal approach to predict the mutagenic potential of primary aromatic amines. This approach is based on the so-called "nitrenium hypothesis", which was developed by Ford et al. in the early 1990s. This hypothesis asserts that the mutagenic effect for this class of molecules is mediated through the transient formation of a nitrenium ion and that the stability of this cation is correlated with the mutagenic potential. Here we use quantum mechanical calculations at different levels of theory (semiempirical AM1, ab initio HF/3-21G, HF/6-311G(d,p), and DFT/B3LYP/6-311G(d,p)) to compute the stability of nitrenium ions. When applied to a test set of 257 primary aromatic amines, we show that this method can correctly differentiate between Ames active and inactive compounds, and furthermore that it is able to rationalize and predict SAR trends within structurally related chemical series. For this test set, the AM1 nitrenium stability calculations are found to provide a good balance between speed and accuracy, resulting in an overall accuracy of 85%, and sensitivity and specificity of 91% and 72%, respectively. The nitrenium-based predictions are also compared to the commercial software packages DEREK, MULTICASE, and the MOE-Toxicophore descriptor. One advantage of the approach presented here is that the calculation of relative stabilities results in a continuous spectrum of activities and not a simple yes/no answer. This allows us to observe and rationalize subtle trends due to the different electrostatic properties of the organic molecules. Our results strongly indicate that nitrenium ion stability calculations should be used as a complementary approach to assist the medicinal chemist in prioritizing and selecting nonmutagenic primary aromatic amines during preclinical drug discovery programs.
Asunto(s)
Aminas/química , Aminas/toxicidad , Biología Computacional , Fenómenos Químicos , Bases de Datos Factuales , Modelos Moleculares , Conformación Molecular , Pruebas de Mutagenicidad , Programas Informáticos , Relación Estructura-Actividad , TermodinámicaRESUMEN
An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.
Asunto(s)
2-Naftilamina/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacología , Urea/análogos & derivados , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , 2-Naftilamina/química , Animales , Química Orgánica/métodos , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Concentración 50 Inhibidora , Lipopolisacáridos/metabolismo , Ratones , Modelos Químicos , Estructura Molecular , Factor de Necrosis Tumoral alfa/metabolismo , Urea/químicaRESUMEN
Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.
Asunto(s)
Adenina/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Sitios de Unión , Humanos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity, we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds, seven hits were identified. Of those, one inhibitor, BIX-01294 (diazepin-quinazolin-amine derivative), does not compete with the cofactor S-adenosyl-methionine, and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays, transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly, chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin.
Asunto(s)
Azepinas/farmacología , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Histonas/metabolismo , Quinazolinas/farmacología , Animales , Línea Celular , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/efectos de los fármacos , Humanos , Lisina/química , Lisina/metabolismo , Metilación , Ratones , Regiones Promotoras Genéticas , Proteína MetiltransferasasRESUMEN
Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.
Asunto(s)
Adenilil Imidodifosfato/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Adenilil Imidodifosfato/química , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Proto-Oncogénicas c-pim-1 , Relación Estructura-ActividadRESUMEN
BIRB 796, a member of the N-pyrazole-N'-naphthly urea class of p38MAPK inhibitors, binds to the kinase with both slow association and dissociation rates. Prior to binding, the kinase undergoes a reorganization of the activation loop exposing a critical binding domain. We demonstrate that, independent of the loop movement, association rates are governed by low energy conformations of the inhibitor and polar functionality on the tolyl ring. As anticipated, the dissociation rates of the inhibitors from the kinase are slowed by lipophilic and hydrogen bond interactions. The value of structure-kinetic relationships (SKR) in drug design is discussed.
Asunto(s)
Inhibidores Enzimáticos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Naftalenos/química , Pirazoles/química , Enfermedades Autoinmunes/tratamiento farmacológico , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Cinética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/química , Naftalenos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Pirazoles/farmacología , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/química , Sitio Alostérico , Secuencias de Aminoácidos , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Moleculares , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacología , Unión Proteica , Conformación Proteica , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Especificidad por Sustrato , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Despite its preferred equatorial conformation in the solid state and in solution, the cis,cis-trispiro ether 1 binds readily and strongly to lithium ions. The 2:1 sandwich complex 2 is initially formed and transformed progressively into the 1:1 species 3 upon the addition of more LiClO4 .