RESUMEN
Nicotinamide phosphoribosyltransferase is a key metabolic enzyme that is a potential target for oncology. Utilizing publicly available crystal structures of NAMPT and in silico docking of our internal compound library, a NAMPT inhibitor, 1, obtained from a phenotypic screening effort was replaced with a more synthetically tractable scaffold. This compound then provided an excellent foundation for further optimization using crystallography driven structure based drug design. From this approach, two key motifs were identified, the (S,S) cyclopropyl carboxamide and the (S)-1-N-phenylethylamide that endowed compounds with excellent cell based potency. As exemplified by compound 27e such compounds could be useful tools to explore NAMPT biology in vivo.
Asunto(s)
Amidas/farmacología , Ciclopropanos/farmacología , Citocinas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Adenosina/análogos & derivados , Amidas/síntesis química , Amidas/química , Cristalografía por Rayos X , Ciclopropanos/síntesis química , Ciclopropanos/química , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Nicotinamida Fosforribosiltransferasa/metabolismo , Fenotipo , Relación Estructura-ActividadRESUMEN
Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.
Asunto(s)
Fosfatidilcolinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/farmacología , Glucemia/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Hígado Graso/tratamiento farmacológico , Hígado Graso/enzimología , Células HeLa , Homeostasis/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Resistencia a la Insulina/fisiología , Ligandos , Lipogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilcolinas/farmacología , Unión Proteica , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Hipoglucemiantes/farmacología , PPAR gamma/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Líquidos Corporales/efectos de los fármacos , Células COS , Chlorocebus aethiops , Grasas de la Dieta/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/química , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Obesidad/inducido químicamente , Obesidad/metabolismo , Osteogénesis/efectos de los fármacos , PPAR gamma/agonistas , PPAR gamma/química , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Rosiglitazona , Tiazolidinedionas/efectos adversos , Tiazolidinedionas/farmacología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Aumento de Peso/efectos de los fármacosRESUMEN
In addition to delivering a haploid genome to the egg, sperm have additional critical functions, including egg activation, origination of the zygote centrosome and delivery of paternal factors. Despite this, existing knowledge of the molecular basis of sperm form and function is limited. We used whole-sperm mass spectrometry to identify 381 proteins of the Drosophila melanogaster sperm proteome (DmSP). This approach identified mitochondrial, metabolic and cytoskeletal proteins, in addition to several new functional categories. We also observed nonrandom genomic clustering of sperm genes and underrepresentation on the X chromosome. Identification of widespread functional constraint on the proteome indicates that sexual selection has had a limited role in the overall evolution of D. melanogaster sperm. The relevance of the DmSP to the study of mammalian sperm function and fertilization mechanisms is demonstrated by the identification of substantial homology between the DmSP and proteins of the mouse axoneme accessory structure.
Asunto(s)
Drosophila melanogaster/genética , Proteoma , Espermatozoides/metabolismo , Animales , Mapeo Cromosómico , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Evolución Molecular , Femenino , Genoma de los Insectos , Masculino , Proteoma/genética , Proteoma/aislamiento & purificación , Espermatogénesis/genéticaRESUMEN
Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Proteínas/química , Animales , Hormonas/química , Humanos , Hidrógeno/química , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Quinasas/química , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores Citoplasmáticos y Nucleares/químicaRESUMEN
Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both RORalpha and RORgamma. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) inhibited transactivation activity of RORalpha and RORgamma but not RORbeta. T0901317 was found to directly bind to RORalpha and RORgamma with high affinity (K(i) = 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed RORalpha/gamma-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by RORalpha at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine RORalpha/gamma function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.
Asunto(s)
Hidrocarburos Fluorados/farmacología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Receptores de Ácido Retinoico/agonistas , Sulfonamidas/farmacología , Línea Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Unión Proteica/fisiología , Receptores de Ácido Retinoico/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often benefits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening of billions of compounds, a hunt for new drug modalities, as well as emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technologies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implementing various assay types to select high quality leads for drug development.
Asunto(s)
Descubrimiento de Drogas/métodos , Compuestos Orgánicos/análisis , Ensayos Analíticos de Alto Rendimiento , Humanos , Bibliotecas de Moléculas Pequeñas/análisisRESUMEN
Human carbonic anhydrase II (HCA II) is a monomeric zinc-containing metalloenzyme that catalyzes the hydration of CO(2) to form bicarbonate and a proton. The properties of the zinc have been extensively elucidated in catalysis but less well studied as a contributor to structure and stability. Apo-HCA II (without zinc) was prepared and compared to holo-HCA II: in crystallographic structural features, in backbone amide H/D exchange, and in thermal stability. The removal of zinc from the active site has no effect on either the topological fold of the enzyme or the ordered water network in the active site. However, the removal of the zinc alters the collective electrostatics of the apo-HCA II that result in the following differences from that of the holoenzyme: (1) the main thermal unfolding transition of the apo-HCA II is lowered by 8 degrees C, (2) the relative increase in thermal mobility of atoms of the apo-HCA II was not observed in the vicinity of the active site but manifested on the surface of the enzyme, and (3) the side chain of His 64, the proton shuttle residue that sits on the rim of the active site, is oriented outward and is associated with additional ordered "external" waters, as opposed to a near equal inward and outward orientation in the holo-HCA II.
Asunto(s)
Apoproteínas/química , Anhidrasa Carbónica II/química , Solventes/química , Zinc , Apoproteínas/metabolismo , Anhidrasa Carbónica II/metabolismo , Catálisis , Cristalografía por Rayos X , Estabilidad de Enzimas , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Electricidad Estática , Zinc/química , Zinc/metabolismoRESUMEN
Binding to helix 12 of the ligand-binding domain of PPARgamma is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARgamma with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARgamma transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.
Asunto(s)
PPAR gamma/agonistas , PPAR gamma/química , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Medición de Intercambio de Deuterio , Ligandos , Modelos Moleculares , PPAR gamma/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The tumor antigens simian virus 40 small t antigen (ST) and polyomavirus small and medium T antigens mediate cell transformation in part by binding to the structural A subunit of protein phosphatase 2A (PP2A). The replacement of B subunits by tumor antigens inhibits PP2A activity and prolongs phosphorylation-dependent signaling. Here we show that ST mediates PP2A A/C heterodimer transfer onto the ligand-activated androgen receptor (AR). Transfer by ST is strictly dependent on the agonist-activated conformation of AR, occurs within minutes of the addition of androgen to cells, and can occur in either the cytoplasm or the nucleus. The binding of ST changes the conformation of the A subunit, and ST rapidly dissociates from the complex upon PP2A A/C heterodimer binding to AR. PP2A is transferred onto the carboxyl-terminal half of AR, and the phosphatase activity is directed to five phosphoserines in the amino-terminal activation function region 1, with a corresponding reduction in transactivation. Thus, ST functions as a transfer factor to specify PP2A targeting in the cell and modulates the transcriptional activity of AR.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Subunidades de Proteína/metabolismo , Receptores Androgénicos/metabolismo , Virus 40 de los Simios/metabolismo , Andrógenos/metabolismo , Animales , Células COS , Núcleo Celular , Chlorocebus aethiops , Citoplasma , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata , Conformación Proteica , Proteína Fosfatasa 2 , Transducción de Señal/fisiología , Activación Transcripcional/fisiología , Células Tumorales CultivadasRESUMEN
The peroxisome proliferator-activated receptor is a member of the nuclear receptor superfamily of transcriptional regulators. Regulation of the nuclear receptors occurs through changes to the structure and dynamics of the ligand-binding domain. Therefore, the need has arisen for a rapid method capable of detecting changes in the dynamics of nuclear receptors following ligand binding. We recently described how solution-phase amide hydrogen/deuterium exchange (HDX) provides a biophysical technique for probing changes in protein dynamics induced by ligand interaction. Building from this platform, we have optimized the robustness of the differential HDX experiment by minimizing systematic errors, and have increased the efficiency of the chromatographic separation through the use of high-pressure liquid chromatography. Using knowledge gained previously from comprehensive HDX experiments of PPARgamma, a modest throughput method to probe changes in the dynamics of key regions of the receptor was developed. A collection of ten synthetic and endogenous PPARgamma ligands were characterized with this new method requiring approximately 24 h of analysis. This is a dramatic improvement over the 10 d of analysis that would have been required with our previous approach for comprehensive differential HDX analysis. In addition to demonstrating the utility of this approach, the study presented here is the first to measure changes to the dynamics of PPARgamma upon the binding of putative endogenous ligands.
Asunto(s)
Deuterio/química , Hidrógeno/química , Proteínas/metabolismo , Cromatografía Líquida de Alta Presión , Ligandos , Espectrometría de Masas , Unión ProteicaRESUMEN
Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.
Asunto(s)
Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Cromatografía Liquida , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus del Dengue/genética , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/normas , Evaluación Preclínica de Medicamentos/normas , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana/normas , Reproducibilidad de los Resultados , Bibliotecas de Moléculas PequeñasRESUMEN
AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and ß subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the ß subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and ß subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Regulación Alostérica , Compuestos de Bifenilo , Dominio Catalítico , Medición de Intercambio de Deuterio , Activación Enzimática , Activadores de Enzimas/química , Humanos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Pironas/química , Tiofenos/químicaRESUMEN
The nuclear receptor (NR) superfamily is composed of 48 members in humans and includes receptors for steroid hormones, thyroid hormone, various lipids and oxysterols. This superfamily has been a rich source of drug targets for myriad diseases including inflammation, cancer, and metabolic disorders. Approximately half of the superfamily have well characterized natural ligands whereas the remaining receptors are considered orphan receptors and remain a focus of a number of investigators assessing their ability to be regulated by ligands. Here, we review recent discoveries that yield important insight into the druggability of three orphan nuclear receptors: the retinoic acid receptor-like orphan receptors (RORs), peroxisome proliferator-activated receptor γ (PPARγ), and liver receptor homolog-1 (LRH-1).
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Metabólicas/tratamiento farmacológico , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Enfermedades Autoinmunes/metabolismo , Humanos , Enfermedades Metabólicas/metabolismo , Receptores Nucleares Huérfanos/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacología , Fosfatidilcolinas/uso terapéutico , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/metabolismo , Esteroles/química , Esteroles/farmacología , Esteroles/uso terapéutico , Tiazolidinedionas/química , Tiazolidinedionas/farmacología , Tiazolidinedionas/uso terapéuticoRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) is involved in expression of genes that control glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione (TZD) class of antidiabetic drugs. However, despite their clinical use these drugs are associated with numerous adverse effects, which are related to their full activation of PPARγ transcriptional responses. PPARγ partial agonists are the focus of development efforts towards second-generation PPARγ modulators with favorable pharmacology, potent insulin sensitization without the severe full agonists' adverse effects. In order to identify novel PPARγ partial agonist lead compounds, we developed a virtual screening protocol based on three-dimensional ligand-shape similarity and docking. Prioritization gave 235 compounds for experimental screening from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR)-a chemical library containing 340 000 compounds. Seven novel potent partial agonists were confirmed in cell-based transactivation and competitive binding assays. Our results illustrate a well-designed virtual screening campaign successfully identifying novel lead compounds as potential entry points for the development of antidiabetic drugs.
Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes , PPAR gamma/agonistas , PPAR gamma/metabolismo , Bibliotecas de Moléculas Pequeñas , Animales , Sitios de Unión/efectos de los fármacos , Células COS , Chlorocebus aethiops , Diabetes Mellitus/fisiopatología , Ensayos Analíticos de Alto Rendimiento , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Ligandos , Modelos Moleculares , PPAR gamma/genética , Conformación Proteica/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacología , Interfaz Usuario-ComputadorRESUMEN
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.
Asunto(s)
Meclizina/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide/metabolismo , Sitio Alostérico , Animales , Sitios de Unión , Receptor de Androstano Constitutivo , Humanos , Ratones , Modelos Moleculares , Coactivador 1 de Receptor Nuclear/química , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/química , Receptores X Retinoide/química , Termodinámica , Activación TranscripcionalRESUMEN
Retinoids are potent forms of vitamin A and are involved in a broad range of physiological processes and the pharmacological effects of retinoids are primarily mediated by the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Several natural and synthetic RAR modulators have proven to be clinically useful for a number of therapeutic indications including cancer, psoriasis, and diabetes. Unfortunately, these agents lead to a number of significant side effects. Most synthetic retinoid ligands are based on the retinoid scaffold and thus have similarities to the natural ligand with all previously disclosed RAR ligands having a carboxylic acid that makes a critical ionic bridge within the ligand binding domain of the receptors. The potential therapeutic value offered from RAR modulation provides the impetus to identify novel ligands based on unique scaffolds that may offer improved toxicity and pharmacokinetic profiles. Here we describe the identification of an atypical RAR inverse agonist that represents the first non-acid, non-retinoid direct modulator of RAR receptor subfamily. SR-0065 functions as a pan-RAR inverse agonist suppressing the basal activity of RARα, RARß, and RARγ, as well as inhibiting agonist-induced RAR activity. SR-0065 treatment enhanced receptor interaction with a peptide representative of the corepressor SMRT, and in cells SR-0065 enhances recruitment of SMRT to the promoter of the RARγ dependent gene, Cyp26A1. The acid form of SR-0065, SR-1758, was inactive in all assays. Thus, SR-0065 represents a new class of non-acid, non-retinoid RAR modulator that may be used as a point to initiate development of improved RAR-targeted drugs.
Asunto(s)
Dioxanos/farmacología , Quinolonas/farmacología , Receptores de Ácido Retinoico/agonistas , Receptores X Retinoide/agonistas , Animales , Línea Celular , Dioxanos/química , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Ratones , Estructura Molecular , Quinolonas/química , Relación Estructura-ActividadRESUMEN
The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Receptores de Calcitriol/química , Receptores X Retinoide/química , Alitretinoína , Sitios de Unión , Dihidroxicolecalciferoles/química , Humanos , Ligandos , Modelos Moleculares , Coactivador 1 de Receptor Nuclear/química , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Receptores X Retinoide/agonistas , Receptores X Retinoide/metabolismo , Tretinoina/químicaRESUMEN
Here we describe an integrated software platform titled HD Desktop designed specifically to enhance the analysis of hydrogen/deuterium exchange (HDX) mass spectrometry data. HD Desktop integrates tools for data extraction with visualization components within a single web-based application. The interface design enables users to navigate from the peptide view to the sample and experiment levels, tracking all manipulations while updating the aggregate graphs in real time. HD Desktop is integrated with a relational database designed to provide performance enhancements, as well as a robust model for data storage and retrieval. Additional features of the software include retention time determination, which is achieved with the use of theoretical isotope fitting; here, we assume that the best theoretical fit will occur at the correct retention time for any given peptide. Peptide data consolidation for the rendering of data in 2D was realized by automating known and novel approaches. Designed to address broad needs of the HDX community, the platform presented here provides an efficient and manageable workflow for HDX data analysis and is freely available as a web tool at the project home page http://hdx.florida.scripps.edu.
Asunto(s)
Medición de Intercambio de Deuterio , Almacenamiento y Recuperación de la Información/métodos , Espectrometría de Masas , Programas Informáticos , Interfaz Usuario-Computador , Sistemas de Administración de Bases de Datos , Bases de Datos como Asunto , Internet , Péptidos/análisis , Proteínas/análisisRESUMEN
Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285-295, 302-316, and 437-457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.