RESUMEN
Two novel series of oxazepine and diazepine based HSP90 inhibitors are reported. This effort relied on structure based design and isothermal calorimetry to identify small drug like macrocycles. Computational modelling was used to build into a solvent exposed pocket near the opening of the ATP binding site, which led to potent inhibitors of HSP90 (25-30).
Asunto(s)
Amidas/química , Benzodiazepinas/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Indoles/química , Oxazepinas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Amidas/metabolismo , Amidas/farmacología , Animales , Azepinas/química , Benzamidas/química , Benzamidas/metabolismo , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacología , Sitios de Unión , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/metabolismo , Indoles/metabolismo , Indoles/farmacología , Células de Riñón Canino Madin Darby , Simulación del Acoplamiento Molecular , Oxazepinas/metabolismo , Oxazepinas/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismoRESUMEN
Chemokine ligand 2 (CCL2) mediates chemotaxis of monocytes to inflammatory sites via interaction with its G protein-coupled receptor CCR2. Preclinical animal models suggest that the CCL2-CCR2 axis has a critical role in the development and maintenance of inflammatory disease states (e.g., multiple sclerosis, atherosclerosis, insulin resistance, restenosis, and neuropathic pain), which can be treated through inhibition of the CCR2 receptor. However, in clinical trials high-affinity inhibitors of CCR2 have often demonstrated a lack of efficacy. We have previously described a new approach for the design of high-affinity CCR2 antagonists, by taking their residence time (RT) on the receptor into account. Here, we report our findings on both structure-affinity relationship (SAR) and structure-kinetic relationship (SKR) studies for a series of 3-((inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists. SAR studies showed that this class of compounds tolerates a vast diversity of substituents on the indenyl ring with only small changes in affinity. However, the SKR is affected greatly by minor modifications of the structure. The combination of SAR and SKR in the hit-to-lead process resulted in the discovery of a new high-affinity and long-residence-time CCR2 antagonist (compound 15a, Ki = 2.4 nM; RT = 714 min).
Asunto(s)
Quimiocina CCL2/antagonistas & inhibidores , Ciclopentanos/síntesis química , Animales , Línea Celular Tumoral , Quimiocina CCL2/genética , Ciclopentanos/química , Ciclopentanos/farmacología , Humanos , Cinética , Estructura Molecular , Unión Proteica , Estereoisomerismo , Relación Estructura-Actividad , Factores de Tiempo , TransfecciónRESUMEN
Extensive phase II metabolism of an advanced PKCε inhibitor resulted in sub-optimal pharmacokinetics in rat marked by elevated clearance. Synthesis of the O-glucuronide metabolite as a standard was followed by three distinct strategies to specifically temper phase II metabolic degradation of the parent molecule. In this study, it was determined that the introduction of proximal polarity to the primary alcohol generally curbed O-glucuronidation and improved PK and physical chemical properties while maintaining potency against the target. Utilization of a Jacobsen hydrolytic kinetic resolution to obtain optically enriched final compounds is also discussed.
Asunto(s)
Glucurónidos/farmacología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Perros , Relación Dosis-Respuesta a Droga , Glucurónidos/química , Glucurónidos/metabolismo , Estructura Molecular , Proteína Quinasa C-epsilon/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.
Asunto(s)
Ciclopentanos/síntesis química , Indanos/síntesis química , Indenos/síntesis química , Receptores CCR2/antagonistas & inhibidores , Unión Competitiva , Línea Celular Tumoral , Ciclopentanos/química , Ciclopentanos/farmacología , Humanos , Indanos/química , Indanos/farmacología , Indenos/química , Indenos/farmacología , Cinética , Ligandos , Estereoisomerismo , Relación Estructura-Actividad , Tetrahidronaftalenos/síntesis química , Tetrahidronaftalenos/química , Tetrahidronaftalenos/farmacologíaRESUMEN
Efforts to substitute the cyclopropane ring in a series of aryl cyclopropylnitriles led to the discovery of an operationally simple one-pot method for Knoevenagel condensation and subsequent Corey-Chaykovsky cyclopropanation giving diastereomerically pure products as a racemic mixture of enantiomers. Method development and results for variably substituted aryl acetonitriles and aldehydes in the reaction are reported. A concise synthesis of (±)-bicifadine in two steps is provided to demonstrate the utility of the method.
Asunto(s)
Aldehídos/química , Ciclopropanos/química , Nitrilos/química , Estructura Molecular , EstereoisomerismoRESUMEN
In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Modelos Químicos , Modelos Moleculares , Señales de Clasificación de Proteína , Staphylococcus aureus/enzimología , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico/fisiología , Cisteína Endopeptidasas/metabolismo , Mapeo Peptídico/métodos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína/fisiología , Staphylococcus aureus/patogenicidadRESUMEN
Many virulence factors in gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom-Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the beta6/beta7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the beta6/beta7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Calcio/fisiología , Peptidil Transferasas/metabolismo , Staphylococcus aureus/enzimología , Regulación Alostérica , Sustitución de Aminoácidos , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cisteína Endopeptidasas , Cartilla de ADN , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Peptidil Transferasas/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , VirulenciaRESUMEN
L-Threonine 2 was converted in seven steps into the protected aminomercaptoalcohol 8, a threonine mimic. This compound 8 was coupled to various oligopeptides to produce two different tetrapeptide analogues, for example, 11 and 17, which were shown to inhibit the Sortase enzymes (SrtA and SrtB) via covalent attachment of the thiol groups of 11 and 17 to the catalytically active cysteine residue of the Sortase enzymes.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Butanoles/síntesis química , Butanoles/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Treonina/análogos & derivados , Butanoles/química , Cromatografía Líquida de Alta Presión , Cisteína Endopeptidasas , Inhibidores Enzimáticos/química , Estructura Molecular , Treonina/químicaRESUMEN
Phosphothioates may provide metabolic stability when compared to their phosphate counterparts, while retaining the potency and efficacy as agonists at sphingosine-1-phosphate (S1P) G-protein coupled receptors. Unlike their phosphate precursors, phosphothioate compounds with S1P-receptor profiles similar to that of FTY720, an emerging immunomodulator, were shown to evoke prolonged lymphopenia in vivo. Analysis of mouse plasma concentrations for a series of related alcohol/phosphate/phosphothioate compounds showed the conversion of the phosphate to alcohol. These preliminary data highlight the importance of metabolic regulation of S1P receptor ligands.
Asunto(s)
Receptores de Lisoesfingolípidos/agonistas , Tionucleótidos/síntesis química , Tionucleótidos/farmacología , Espectroscopía de Resonancia Magnética , Tionucleótidos/químicaRESUMEN
The novel immunosuppressant FTY720 has been demonstrated to elicit immunomodulating effects via interaction with the G-protein coupled receptor S1P(1). FTY720 induced agonism at the S1P(3) receptor, however, has been shown to result in mild bradycardia, a minor side-effect of initial FTY720 therapy. This report describes the synthesis of several potent 4(5)-phenylimidazole-based S1P(1) receptor agonists that are accompanied by poor agonist activity at S1P(3). For instance, compound 20 displayed an EC(50)=4.7+/-1.3 nM at the S1P(1) receptor and EC(50)=780+/-1.3 nM at the S1P(3) receptor using a [gamma-(35)S]GTP-binding assay as compared to phospho-FTY720 (S1P(1): EC(50)=1.3+/-1.3nM, S1P(3): EC(50)=2.0+/-2.4 nM).
Asunto(s)
Bencimidazoles/síntesis química , Inmunosupresores/síntesis química , Lisofosfolípidos/síntesis química , Glicoles de Propileno/síntesis química , Receptores de Lisoesfingolípidos/agonistas , Esfingosina/análogos & derivados , Animales , Bencimidazoles/farmacología , Sitios de Unión , Bradicardia/tratamiento farmacológico , Células CHO , Cricetinae , Clorhidrato de Fingolimod , Inmunosupresores/farmacología , Dosificación Letal Mediana , Lisofosfolípidos/farmacología , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/síntesis química , Esfingosina/farmacología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte-EC interactions in vivo. METHODS AND RESULTS: We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-alpha in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-alpha-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-alpha plus SEW2871-treated mice had few monocytes bound (P<0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte-EC interactions in response to TNF-alpha. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun. CONCLUSIONS: Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-alpha-mediated monocyte-EC interactions in vivo.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Endotelio Vascular/citología , Lisofosfolípidos/farmacología , Monocitos/citología , Esfingosina/análogos & derivados , Factor de Necrosis Tumoral alfa/metabolismo , Vasculitis/tratamiento farmacológico , Animales , Aorta/citología , Aorta/inmunología , Adhesión Celular/inmunología , Células Cultivadas , Quimiocinas/metabolismo , Selectina E/metabolismo , Endotelio Vascular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Oxadiazoles/farmacología , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Esfingosina/farmacología , Tiofenos/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vasculitis/metabolismo , Vasculitis/prevención & controlRESUMEN
Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structure-activity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P(1) and S1P(3) receptors. The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.
Asunto(s)
Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Fosfoserina/análogos & derivados , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacología , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Clorhidrato de Fingolimod , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Ligandos , Lípidos/química , Linfocitos/metabolismo , Modelos Químicos , Fosfoserina/química , Fosfoserina/farmacología , Glicoles de Propileno/farmacología , Unión Proteica , Transducción de Señal , Relación Estructura-Actividad , TransfecciónRESUMEN
Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. A member of this receptor family, S1P(4), is highly and almost exclusively expressed in the lymphoid system and has been implicated in regulation of cell shape and motility. This report describes the synthesis of several potent benzimidazole based S1P(4) receptor selective agonists. For instance, compound 9b displayed an EC(50)=36 nM at the S1P(4) receptor using a [gamma-(35)S]GTP binding assay as compared to an EC(50)=37 nM for the endogenous ligand. We also report the effects of altering stereochemistry at the C2 position, methylation at the C1 and C2 position, and activity differences between the alcohol and phosphate head groups of the analogues.
Asunto(s)
Bencimidazoles/síntesis química , Lisofosfolípidos/síntesis química , Receptores de Lisoesfingolípidos/agonistas , Esfingosina/análogos & derivados , Esfingosina/síntesis química , Humanos , Relación Estructura-ActividadRESUMEN
Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. This report describes the synthesis of several potent S1P receptor agonists. For instance, compound 9c displayed an EC(50)=8.6 nM at the S1P(1) receptor using a [gamma-35S]GTP binding assay as compared to an EC(50)=4.5 nM for the endogenous ligand. We also report the effects associated with introduction of a phenyl ring between the 'linker' and 'lipophilic tail' regions of the analogues, for example total loss of activity at S1P(2) and increased agonism at S1P(5).