RESUMEN
Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was â¼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was â¼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (â¼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.
Asunto(s)
Conducta Animal/efectos de los fármacos , Técnicas de Sustitución del Gen , Isoquinolinas/farmacología , Locomoción/efectos de los fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Taquifilaxis , Adamantano/análogos & derivados , Adamantano/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Benzopiranos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Isoquinolinas/efectos adversos , Masculino , Ratones , Transporte de Proteínas/efectos de los fármacos , Receptores de Dopamina D1/agonistasRESUMEN
Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu2 receptor. The hydroxyacetophenone scaffold is a second series of mGlu2 PAMs we have identified. This series of molecules are potent mGlu2 potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.
Asunto(s)
Descubrimiento de Drogas , Trastornos Migrañosos/tratamiento farmacológico , Receptores de Leucotrienos/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Sulfonamidas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Perros , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/químicaRESUMEN
There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.
Asunto(s)
Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Purinas/síntesis química , Purinas/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Animales , Transporte Biológico Activo , Células CHO , Canales de Calcio , Cricetulus , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , Células de Riñón Canino Madin Darby , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Dimensión del Dolor/efectos de los fármacos , Ratas , Relación Estructura-Actividad , Canal Catiónico TRPA1RESUMEN
Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting ß-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Tanquirasas/antagonistas & inhibidores , Administración Oral , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Tanquirasas/metabolismoRESUMEN
The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Algoritmos , Anticuerpos/química , Tampones (Química) , Interpretación Estadística de Datos , Proteína Potenciadora del Homólogo Zeste 2 , Inmunoensayo de Polarización Fluorescente , Humanos , Indicadores y Reactivos , Metilación , Péptidos/química , Proteínas Recombinantes/química , Estándares de Referencia , Reproducibilidad de los Resultados , EstreptavidinaRESUMEN
Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Células Precursoras de Linfocitos B/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT5/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that, when genetically altered by mutation, amplification, chromosomal translocation or inversion, has been shown to play an oncogenic role in certain cancers. Small molecule inhibitors targeting the kinase activity of ALK have proven to be effective therapies in certain ALK-driven malignancies and one such inhibitor, crizotinib, is now approved for the treatment of EML4-ALK-driven, non-small cell lung cancer. In neuroblastoma, activating point mutations in the ALK kinase domain can drive disease progression, with the two most common mutations being F1174L and R1275Q. We report here crystal structures of the ALK kinase domain containing the F1174L and R1275Q mutations. Also included are crystal structures of ALK in complex with novel small molecule ALK inhibitors, including a classic type II inhibitor, that stabilize previously unobserved conformations of the ALK activation loop. Collectively, these structures illustrate a different series of activation loop conformations than has been observed in previous ALK crystal structures and provide insight into the activating nature of the R1275Q mutation. The novel active site topologies presented here may also aid the structure-based drug design of a new generation of ALK inhibitors.
Asunto(s)
Benzoxazoles/química , Mutación Missense , Neuroblastoma/genética , Pirazoles/química , Piridinas/química , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Quinasa de Linfoma Anaplásico , Animales , Unión Competitiva , Dominio Catalítico , Línea Celular , Crizotinib , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Neuroblastoma/enzimología , Unión Proteica , Proteolisis , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Spodoptera , Homología Estructural de ProteínaRESUMEN
A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Administración Oral , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Disponibilidad Biológica , Línea Celular Tumoral , Estabilidad de Medicamentos , Humanos , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/farmacocinética , Imidazoles/farmacología , Concentración 50 Inhibidora , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Ratas , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Especificidad por SustratoRESUMEN
The Ubiquitin Proteasome Pathway (UPP) has become a target rich pathway for therapeutic intervention as its role in pathogenic disease is better understood. In particular many E3 ligases within this pathway have been implicated in cancer, inflammation and metabolic diseases. It has been of great interest to develop biochemical assays to identify inhibitors of catalytic E3 ubiquitination activity as a means of abrogating the disease mechanism. Here we describe a homogeneous biochemical assay that utilizes native ubiquitin and Tandem-repeated Ubiquitin-Binding Entities (TUBEs) as a detection technology for ubiquitination activity. We developed a TUBEs based proximity AlphaLisa® assay for Mdm2, which is an E3 ligase that negatively regulates p53 tumor suppressor protein. We further demonstrate that this assay strategy or design can also be applied to the development of assays to detect autoubiquitination of other E3 ligases that are also of interest for therapeutic intervention. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Asunto(s)
Poliubiquitina/química , Poliubiquitina/metabolismo , Proteínas Recombinantes/análisis , Secuencias Repetidas en Tándem , Proteínas Ubiquitinadas/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Recombinantes/metabolismo , Proteínas Ubiquitinadas/metabolismo , UbiquitinaciónRESUMEN
Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC(50) = 0.174 µM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.
Asunto(s)
Amidas/síntesis química , Antineoplásicos/síntesis química , Piperidinas/síntesis química , Pirimidinas/síntesis química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Amidas/química , Amidas/farmacología , Quinasa de Linfoma Anaplásico , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estructura Molecular , Piperidinas/química , Piperidinas/farmacología , Conformación Proteica , Pirimidinas/química , Pirimidinas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Secuencia de Aminoácidos , Inhibidores Enzimáticos/metabolismo , Inmunoensayo/métodos , Lisina/metabolismo , Metilación , Datos de Secuencia Molecular , Péptidos/metabolismoRESUMEN
Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.
Asunto(s)
Adenosina Trifosfato/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Transferencia Resonante de Energía de Fluorescencia/métodos , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Quinasa 4 Dependiente de la Ciclina/metabolismo , Humanos , Cinética , Espectrometría de Masas , Modelos MolecularesRESUMEN
Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Tienopiridinas/síntesis química , Animales , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Humanos , Janus Quinasa 1/química , Janus Quinasa 2/química , Leucocitos Mononucleares/efectos de los fármacos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Porcinos , Tienopiridinas/química , Tienopiridinas/farmacologíaRESUMEN
Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.
Asunto(s)
Bioensayo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Línea Celular , Proliferación Celular/efectos de los fármacos , Crizotinib , Inhibidores Enzimáticos/farmacología , Humanos , Piperidinas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Pirazoles , Piridinas/farmacología , Proteínas Tirosina Quinasas ReceptorasRESUMEN
The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1. Smurf1 is a critical regulator of TGF-beta and bone morphogenic protein signaling, and has been demonstrated to play a role in regulating cell polarity through the degradation of RhoA. We set out to identify novel Smurf1 substrates involved in the regulation of the aforementioned pathways. Proof-of-principle experiments with known Smurf1 substrates demonstrated efficient ubiquitination thereby validating this approach. Assaying a human protein microarray for ubiquitination with Smurf1 and the partner E2 ubiquitin ligase Ubch5 or Ubch7 identified 89 potential substrates of the Smurf1 E3 activity, which spanned a number of different biological pathways. Substrates identified utilizing protein microarray technology have been validated in vitro. Here we demonstrate the utility of this approach for identifying substrates of particular E2/E3 complexes.
Asunto(s)
Análisis por Matrices de Proteínas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Galphaq-coupled GPCRs are activated. Although Galphai- and Galphas-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Galphaprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.
Asunto(s)
Calcio/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Fluorometría/métodos , Receptores Acoplados a Proteínas G/metabolismo , AnimalesRESUMEN
A series of alpha-amidosulfones were found to be potent and selective agonists of CB(2). The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.
Asunto(s)
Receptor Cannabinoide CB2/agonistas , Sulfonas/química , Amidas/química , Amidas/farmacología , Animales , Humanos , Microsomas Hepáticos , Farmacocinética , Ratas , Relación Estructura-Actividad , Sulfonas/farmacologíaRESUMEN
The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC50 > 10 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.
Asunto(s)
Aminoquinolinas/síntesis química , Oxadiazoles/síntesis química , Receptor Cannabinoide CB2/agonistas , Administración Oral , Aminoquinolinas/administración & dosificación , Aminoquinolinas/farmacocinética , Animales , Disponibilidad Biológica , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Modelos Moleculares , Oxadiazoles/administración & dosificación , Oxadiazoles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.
Asunto(s)
Microsomas Hepáticos/metabolismo , Oxadiazoles , Receptor Cannabinoide CB2/agonistas , Animales , Técnicas Químicas Combinatorias , Humanos , Conformación Molecular , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.