RESUMO
Tyrosine kinase 2 (TYK2) is a nonreceptor tyrosine kinase that belongs to the JAK family also comprising JAK1, JAK2, and JAK3. TYK2 is an attractive target for various autoimmune diseases as it regulates signal transduction downstream of IL-23 and IL-12 receptors. Selective TYK2 inhibition offers a differentiated clinical profile compared to currently approved JAK inhibitors. However, selectivity for TYK2 versus other JAK family members has been difficult to achieve with small molecules that inhibit the catalytically active kinase domain. Successful targeting of the TYK2 pseudokinase domain as a strategy to achieve isoform selectivity was recently exemplified with deucravacitinib. Described herein is the optimization of selective TYK2 inhibitors targeting the pseudokinase domain, resulting in the discovery of the clinical candidate ABBV-712 (21).
Assuntos
Doenças Autoimunes , TYK2 Quinase , Humanos , Janus QuinasesRESUMO
Mechanism of G protein-coupled receptor (GPCR) activation and their modulation by functionally distinct ligands remains elusive. Using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry, we examined the ligand-induced changes in conformational states and stability within the beta-2-adrenergic receptor (ß(2)AR). Differential HDX reveals ligand-specific alterations in the energy landscape of the receptor's conformational ensemble. The inverse agonists timolol and carazolol were found to be most stabilizing even compared with the antagonist alprenolol, notably in intracellular regions where G proteins are proposed to bind, while the agonist isoproterenol induced the largest degree of conformational mobility. The partial agonist clenbuterol displayed conformational effects found in both the inverse agonists and the agonist. This study highlights the regional plasticity of the receptor and characterizes unique conformations spanning the entire receptor sequence stabilized by functionally selective ligands, all of which differ from the profile for the apo receptor.
Assuntos
Medição da Troca de Deutério/métodos , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Alprenolol/metabolismo , Sítios de Ligação , Clembuterol/metabolismo , Humanos , Ligação de Hidrogênio , Ligantes , Espectrometria de Massas , Membranas/metabolismo , Peptídeos/metabolismo , Propanolaminas/metabolismo , Ligação Proteica , Estabilidade Proteica , Receptores Adrenérgicos beta 2/metabolismo , Timolol/metabolismoRESUMO
Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.
Assuntos
Antagonistas de Dopamina/química , Receptores de Dopamina D3/química , Salicilamidas/química , Animais , Arginina/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Antagonistas dos Receptores de Dopamina D2 , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de Dopamina D3/antagonistas & inibidores , Proteínas Recombinantes/química , SpodopteraRESUMO
Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.
Assuntos
Receptores CXCR4/química , Animais , Linhagem Celular , Quimiocina CXCL12 , Cristalografia por Raios X , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Proteínas de Membrana , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes/química , Spodoptera , Tioureia/análogos & derivados , Tioureia/químicaRESUMO
To examine the molecular details of ligand activation of G-protein coupled receptors (GPCRs), emphasis has been placed on structure determination of these receptors with stabilizing ligands. Here we present the methodology for receptor dynamics characterization of the GPCR human beta(2) adrenergic receptor bound to the inverse agonist carazolol using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS). The HDX MS profile of receptor bound to carazolol is consistent with thermal parameter observations in the crystal structure and provides additional information in highly dynamic regions of the receptor and chemical modifications demonstrating the highly complementary nature of the techniques. After optimization of HDX experimental conditions for this membrane protein, better than 89% sequence coverage was obtained for the receptor. The methodology presented paves the way for future analysis of beta(2)AR bound to pharmacologically distinct ligands as well as analysis of other GPCR family members.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2 , Sequência de Aminoácidos , Deutério/química , Humanos , Hidrogênio/química , Dados de Sequência Molecular , Propanolaminas/química , Estrutura Terciária de ProteínaRESUMO
The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.
Assuntos
Receptor A2A de Adenosina/química , Antagonistas do Receptor A2 de Adenosina , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Triazinas/química , Triazóis/química , Triptofano/química , PerusRESUMO
The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.
Assuntos
Colesterol/química , Receptores Adrenérgicos beta 2/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Temperatura , Timolol/químicaRESUMO
Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).
Assuntos
Fluorescência , Proteínas de Membrana/química , Receptores de Apelina , Soluções Tampão , Detergentes/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Concentração Osmolar , Desnaturação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Tensoativos/química , Termodinâmica , Temperatura de TransiçãoRESUMO
Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.
Assuntos
Baculoviridae/metabolismo , Conexinas/biossíntese , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/biossíntese , Receptores Adrenérgicos beta 2/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Conexina 26 , Conexinas/genética , Perfilação da Expressão Gênica/instrumentação , Humanos , Receptores Adrenérgicos beta 2/genética , SpodopteraRESUMO
The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-vav/isolamento & purificação , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cisteína/química , DNA Complementar , Glutationa Transferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/análise , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese , Nanotecnologia , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Zinco/químicaRESUMO
A simple and direct analysis of the spatial distribution of childhood leukemia was performed using geographic data from a large case/control study. The data consist of cases of childhood leukemia and their corresponding birth cohort controls located in seven San Francisco Bay Area counties. Both parametric and randomization analyses show no evidence of a non-random spatial pattern of childhood leukemia among six of these counties. The data from San Francisco County, however, produce a moderately small significance probability (0.08) arising from a distance analysis and a significant p-value (0.01) arising from a frequency analysis of concordant case pairs. Although these p-values accurately reflect the probability of the observed spatial pattern occurring by chance alone, these results are based on only four cases of leukemia.
Assuntos
Leucemia/epidemiologia , Adolescente , California/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise por Conglomerados , Humanos , Lactente , Recém-NascidoRESUMO
The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.