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1.
Nat Rev Drug Discov ; 17(7): 471-492, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29880918

RESUMEN

Cryo-electron microscopy (cryo-EM) of non-crystalline single particles is a biophysical technique that can be used to determine the structure of biological macromolecules and assemblies. Historically, its potential for application in drug discovery has been heavily limited by two issues: the minimum size of the structures it can be used to study and the resolution of the images. However, recent technological advances - including the development of direct electron detectors and more effective computational image analysis techniques - are revolutionizing the utility of cryo-EM, leading to a burst of high-resolution structures of large macromolecular assemblies. These advances have raised hopes that single-particle cryo-EM might soon become an important tool for drug discovery, particularly if they could enable structural determination for 'intractable' targets that are still not accessible to X-ray crystallographic analysis. This article describes the recent advances in the field and critically assesses their relevance for drug discovery as well as discussing at what stages of the drug discovery pipeline cryo-EM can be useful today and what to expect in the near future.


Asunto(s)
Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Animales , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Sustancias Macromoleculares/química
2.
Nat Rev Drug Discov ; 15(10): 679-98, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27516170

RESUMEN

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.


Asunto(s)
Fenómenos Biofísicos/efectos de los fármacos , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Animales , Fenómenos Biofísicos/fisiología , Calorimetría/métodos , Calorimetría/tendencias , Descubrimiento de Drogas/tendencias , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/tendencias , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/tendencias
3.
ChemMedChem ; 10(9): 1511-21, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26259992

RESUMEN

Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.


Asunto(s)
Bioquímica/métodos , Biofisica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Descubrimiento de Drogas/métodos , Espectroscopía de Resonancia Magnética , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Espectrometría de Masa por Ionización de Electrospray/métodos
4.
PLoS Pathog ; 9(9): e1003645, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24086136

RESUMEN

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/ß HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.


Asunto(s)
Epigénesis Genética , Proteínas del Helminto/química , Histona Desacetilasas/química , Pliegue de Proteína , Schistosoma mansoni/enzimología , Animales , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Estructura Secundaria de Proteína , Schistosoma mansoni/genética
5.
J Chem Inf Model ; 53(4): 836-51, 2013 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-23537132

RESUMEN

This paper describes the use and validation of S4MPLE in Fragment-Based Drug Design (FBDD)--a strategy to build drug-like ligands starting from small compounds called fragments. S4MPLE is a conformational sampling tool based on a hybrid genetic algorithm that is able to simulate one (conformer enumeration) or more molecules (docking). The goal of the current paper is to show that due to the judicious design of genetic operators, S4MPLE may be used without any specific adaptation as an in silico FBDD tool. Such fragment-to-lead evolution involves either growing of one or linking of several fragment-like binder(s). The native ability to specifically "dock" a substructure that is covalently anchored to its target (here, some prepositioned fragment formally part of the binding site) enables it to act like dedicated de novo builders and differentiates it from most classical docking tools, which may only cope with non-covalent interactions. Besides, S4MPLE may address growing/linking scenarios involving protein site flexibility, and it might also suggest "growth" moves by bridging the ligand to the site via water-mediated interactions if H2O molecules are simply appended to the input files. Therefore, the only development overhead required to build a virtual fragment→ligand growing/linking strategy based on S4MPLE were two chemoinformatics programs meant to provide a minimalistic management of the linker library. The first creates a duplicate-free library by fragmenting a compound database, whereas the second builds new compounds, attaching chemically compatible linkers to the starting fragments. S4MPLE is subsequently used to probe the optimal placement of the linkers within the binding site, with initial restraints on atoms from initial fragments, followed by an optimization of all kept poses after restraint removal. Ranking is mainly based on two criteria: force-field potential energy and RMSD shifts of the original fragment moieties. This strategy was applied to several examples from the FBDD literature with good results over several monitored criteria: ability to generate the optimized ligand (or close analogs), good ranking of analogs among decoy compounds, and accurate predictions of expected binding modes of reference ligands. Simulations included "classical" covalent growing/linking, more challenging ones involving binding site conformational changes, and growth with optional recognition of putatively favorable water-mediated interactions.


Asunto(s)
Acetolactato Sintasa/química , Algoritmos , Factor Xa/química , Proteínas HSP90 de Choque Térmico/química , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Sitios de Unión , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Ligandos , Conformación Molecular , Simulación del Acoplamiento Molecular , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Interfaz Usuario-Computador
6.
Life Sci ; 89(7-8): 259-68, 2011 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-21736880

RESUMEN

AIMS: We have developed biochemical and cell based assays to characterize small therapeutic molecules that inhibit the DNA damage checkpoint enzyme, Chk1 (Checkpoint kinase 1). MAIN METHODS: To prepare a screen of large chemical libraries, we purified the full-length and the catalytic domain versions of human Chk1. We characterized their properties and then selected full-length Chk1 as the variant most suitable for screening. We then identified and characterized structurally different Chk1 inhibitors in cell based-assays by measuring cytotoxicity and checkpoint bypass activity. KEY FINDINGS: We treated human cells with topoisomerase I inhibitors and demonstrated that at the time of Chk1 inhibitor addition, the cells have damaged DNA and activated Chk1. One Chk1 inhibitor, the indolocarbazole S27888, was active in the checkpoint bypass assay. SIGNIFICANCE: Knowing that the protein kinase inhibitory properties are different for each inhibitor, it seems that only a limited range of inhibitory activity is tolerated by cells. Chk1 has an essential role in determining how cancer cells respond to genotoxic treatments, therefore, inhibitors of this protein kinase are of great medical interest.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carbazoles/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas Quinasas/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas Quinasas/genética , Spodoptera/citología
7.
Comb Chem High Throughput Screen ; 14(6): 500-20, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21521152

RESUMEN

Fragment-based screening is an emerging technology which is used as an alternative to high-throughput screening (HTS), and often in parallel. Fragment screening focuses on very small compounds. Because of their small size and simplicity, fragments exhibit a low to medium binding affinity (mM to µM) and must therefore be screened at high concentration in order to detect binding events. Since some issues are associated with high-concentration screening in biochemical assays, biophysical methods are generally employed in fragment screening campaigns. Moreover, these techniques are very sensitive and some of them can give precise information about the binding mode of fragments, which facilitates the mandatory hit-to-lead optimization. One of the main advantages of fragment-based screening is that fragment hits generally exhibit a strong binding with respect to their size, and their subsequent optimization should lead to compounds with better pharmacokinetic properties compared to molecules evolved from HTS hits. In other words, fragments are interesting starting points for drug discovery projects. Besides, the chemical space of low-complexity compounds is very limited in comparison to that of drug-like molecules, and thus easier to explore with a screening library of limited size. Furthermore, the "combinatorial explosion" effect ensures that the resulting combinations of interlinked binding fragments may cover a significant part of "drug-like" chemical space. In parallel to experimental screening, virtual screening techniques, dedicated to fragments or wider compounds, are gaining momentum in order to further reduce the number of compounds to test. This article is a review of the latest news in both experimental and in silico virtual screening in the fragment-based discovery field. Given the specificity of this journal, special attention will be given to fragment library design.


Asunto(s)
Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/química , Animales , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas Pequeñas/farmacología
8.
Curr Opin Pharmacol ; 9(5): 622-8, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19581126

RESUMEN

Biophysical methods are currently involved in drug design in two ways: the qualitative detection of small molecule binding to a target (hit identification), and the quantitative determination of physical parameters associated to binding (hit-to-lead progression). In the first case, efforts have been made toward miniaturization, automation, and speed-up of the screening process allowing a higher throughput. In the second one, sophisticated applications have been developed to derive detailed relevant information. Preferably, several methods are used in combination to avoid bias and/or limitations associated with a single one, often together with computational methods. New developments should allow important systems overlooked so far to be studied: membrane proteins, intrinsically unstructured proteins, as well as in-cell studies.


Asunto(s)
Biofisica , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Ligandos , Proteínas/química , Animales , Automatización de Laboratorios , Sitios de Unión , Biología Computacional , Humanos , Miniaturización , Estructura Molecular , Conformación Proteica , Proteínas/metabolismo , Proteómica , Relación Estructura-Actividad
9.
Curr Biol ; 18(11): 825-30, 2008 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-18514519

RESUMEN

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.


Asunto(s)
Evolución Biológica , Cordados no Vertebrados/crecimiento & desarrollo , Metamorfosis Biológica/fisiología , Receptores de Hormona Tiroidea/fisiología , Hormonas Tiroideas/fisiología , Animales
10.
Pharmacol Rev ; 58(4): 798-836, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17132856

RESUMEN

Half of the members of the nuclear receptors superfamily are so-called "orphan" receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.


Asunto(s)
Receptores Citoplasmáticos y Nucleares/fisiología , Transducción de Señal , Animales , Sitios de Unión , Humanos , Ligandos , Mutación , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/clasificación , Receptores Citoplasmáticos y Nucleares/genética , Terminología como Asunto
11.
J Biol Chem ; 279(32): 33639-46, 2004 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-15161930

RESUMEN

The estrogen-related receptor (ERR) gamma behaves as a constitutive activator of transcription. Although no natural ligand is known, ERRgamma is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERRgamma ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERRgamma results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ERalpha LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERRgamma does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERRgamma and ERalpha reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERRgamma relative to ERalpha. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERRgamma and reveal structural differences with ERs that explain why ERRgamma does not bind estradiol or raloxifene and will help to design new selective antagonists.


Asunto(s)
Dietilestilbestrol/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/química , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Sitios de Unión , Cristalización , Dietilestilbestrol/metabolismo , Dimerización , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno , Estrógenos no Esteroides/farmacología , Ratones , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Clorhidrato de Raloxifeno/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes , Tamoxifeno/metabolismo
12.
Biochem Biophys Res Commun ; 315(4): 919-27, 2004 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-14985100

RESUMEN

The retinoid-related orphan receptor gamma (RORgamma) has been shown to function as a positive regulator of transcription in many cell lines. Transcriptional activation by nuclear receptors involves recruitment of co-activators that interact with receptors through their LXXLL motifs (NR box). In this study, we analyze the interaction of RORgamma with the co-activator SRC1 and use a series of LXXLL-containing peptides to probe for changes in the conformation of the co-activator interaction surface of the RORgamma LBD. We demonstrate that the H3-4/H12 co-activator interaction surface of RORgamma displays a selectivity for LXXLL peptides that is distinct from those of other nuclear receptors. LXXLL peptides that interacted with RORgamma efficiently antagonized RORgamma-mediated transcriptional activation. Mutations E502Q and Y500F in H12, and K334A, Q347A, and I348D in H3 and H4 of RORgamma, severely impact the recruitment of LXXLL peptides. The effects of these mutations are consistent with predictions made on the basis of the structure of the RORgamma(LBD) derived through homology modeling. These peptide antagonists provide a useful tool to analyze the conformation changes in the RORgamma(LBD) and to study RORgamma receptor signaling.


Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Péptidos/farmacología , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Hormona Tiroidea/antagonistas & inhibidores , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Genes Reporteros/genética , Histona Acetiltransferasas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Coactivador 1 de Receptor Nuclear , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Péptidos/química , Péptidos/metabolismo , Plásmidos/genética , Mutación Puntual , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Técnicas del Sistema de Dos Híbridos
13.
Biochem Biophys Res Commun ; 312(1): 121-30, 2003 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-14630029

RESUMEN

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.


Asunto(s)
Bromocriptina/química , Bromocriptina/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Análisis Espectral/métodos , Citocromo P-450 CYP3A , Activación Enzimática , Estabilidad de Enzimas , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Microsomas , Transición de Fase , Presión , Unión Proteica , Conformación Proteica , Soluciones , Especificidad por Sustrato
14.
Nat Struct Biol ; 10(10): 820-5, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12958591

RESUMEN

Retinoids regulate gene expression through binding to the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In contrast, no ligands for the retinoic acid receptor-related orphan receptors beta and gamma (ROR beta and gamma) have been identified, yet structural data and structure-function analyses indicate that ROR beta is a ligand-regulated nuclear receptor. Using nondenaturing mass spectrometry and scintillation proximity assays we found that all-trans retinoic acid (ATRA) and several retinoids bind to the ROR beta ligand-binding domain (LBD). The crystal structures of the complex with ATRA and with the synthetic analog ALRT 1550 reveal the binding modes of these ligands. ATRA and related retinoids inhibit ROR beta but not ROR alpha transcriptional activity suggesting that high-affinity, subtype-specific ligands could be designed for the identification of ROR beta target genes. Our results identify ROR beta as a retinoid-regulated nuclear receptor, providing a novel pathway for retinoid action.


Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo , Tretinoina/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares
15.
Hum Mol Genet ; 12(9): 1063-72, 2003 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-12700175

RESUMEN

Mutations in the DAX-1 (NR0B1) gene cause the X-linked form of adrenal hypoplasia congenita (AHC), which is constantly found associated with hypogonadotropic hypogonadism (HHG). DAX-1 encodes an atypical orphan member of the nuclear hormone receptor superfamily. DAX-1 acts at multiple levels to repress the expression of genes involved in steroid hormone metabolism through a potent transcriptional repression domain present in its C-terminus, which is similar to the nuclear receptors' ligand binding domain. All DAX-1 mutations causing AHC/HHG alter the protein C-terminal domain, impairing its nuclear localization and, consequently, its transcriptional repression activity. Here we show that DAX-1 AHC mutants have a misfolded conformation, which correlates with their cytoplasmic retention. Extensive structure-function analysis reveals that the chemical nature of amino acid residues at positions interested by AHC mutations and critical determinants in helix 12 affect DAX-1 nuclear localization and transcriptional silencing. Surprisingly, mutations in a conserved putative corepressor binding surface have a negative effect upon DAX-1 transcriptional repression only when they also affect protein expression levels. These data suggest that a folding defect underlies the impaired function of DAX-1 missense mutants found in AHC/HHG patients and that interactions with transcriptional cofactors different from known corepressors mediate DAX-1 silencing properties.


Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Proteínas de Unión al ADN/genética , Receptores de Ácido Retinoico/genética , Proteínas Represoras/genética , Relación Estructura-Actividad , Hiperplasia Suprarrenal Congénita/metabolismo , Sustitución de Aminoácidos , Receptor Nuclear Huérfano DAX-1 , Proteínas de Unión al ADN/metabolismo , Humanos , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras/metabolismo
16.
Protein Sci ; 12(4): 725-33, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12649431

RESUMEN

Nondenaturing electrospray mass spectrometry (ESI-MS) has been used to reveal the presence of potential ligands in the ligand-binding domain (LBD) of orphan nuclear receptors. This new approach, based on supramolecular mass spectrometry, allowed the detection and identification of fortuitous ligands for the retinoic acid-related orphan receptor beta (RORbeta) and the ultraspiracle protein (USP). These fortuitous ligands were specifically captured from the host cell with the proper stoichiometry. After organic extraction, these molecules have been characterized by classic analytical methods and identified as stearic acid for RORbeta and a phosphatidylethanolamine (PE) for USP, as confirmed by crystallography. These molecules act as "fillers" and may not be the physiological ligands, but they prove to be essential to stabilize the active conformation of the LBD, enabling its crystallization. The resulting crystal structures provide a detailed picture of the ligand-binding pocket, allowing the design of highly specific synthetic ligands that can be used to characterize the function of orphan nuclear receptors. An additional advantage of this new method is that it is not based on a functional test and that it can detect low-affinity ligands.


Asunto(s)
Ligandos , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Ratas , Espectrometría de Masa por Ionización de Electrospray
17.
Mol Cell ; 9(2): 303-13, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11864604

RESUMEN

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor 3 (ERR3) complexed with a steroid receptor coactivator-1 (SRC-1) peptide reveals a transcriptionally active conformation in absence of any ligand. The structure explains why estradiol does not bind ERRs with significant affinity. Docking of the previously reported ERR antagonists, diethylstilbestrol and 4-hydroxytamoxifen, requires structural rearrangements enlarging the ligand binding pocket that can only be accommodated with an antagonist LBD conformation. Mutant receptors in which the ligand binding cavity is filled up by bulkier side chains still interact with SRC-1 in vitro and are transcriptionally active in vivo, but are no longer efficiently inactivated by diethylstilbestrol or 4-hydroxytamoxifen. These results provide structural and functional evidence for ligand-independent transcriptional activation by ERR3.


Asunto(s)
Receptores Citoplasmáticos y Nucleares , Receptores de Estrógenos/fisiología , Tamoxifeno/análogos & derivados , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , Cristalografía por Rayos X , Dietilestilbestrol/metabolismo , Dietilestilbestrol/farmacología , Estradiol/metabolismo , Estradiol/farmacología , Histona Acetiltransferasas , Humanos , Ligandos , Mesocricetus , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Coactivador 1 de Receptor Nuclear , Conformación Proteica , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Tamoxifeno/metabolismo , Tamoxifeno/farmacología , Factores de Transcripción/metabolismo
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