RESUMEN
Steroid hormone receptors play a crucial role in the development and characterization of the majority of breast cancers. These receptors canonically function through homodimerization, but physical interactions between different hormone receptors play a key role in cell functions as well. The estrogen receptor (ERα) and progesterone receptor (PR), for example, are involved in a complex set of interactions known as ERα/PR crosstalk. Here, we developed a valuable panel of nuclear receptor expression plasmids specifically for use in NanoBRET assays to assess nuclear receptor homo- and heterodimerization. We demonstrate the utility of this assay system by assessing ERα/PR physical interaction in the context of the endocrine therapy resistance-associated ERα Y537S mutation. We identify a role of the ERα Y537S mutation beyond that of constitutive activity of the receptor; it also increases ERα/PR crosstalk. In total, the NanoBRET assay provides a novel avenue for investigating hormone receptor crosstalk. Future research may use this system to assess the effects of other clinically significant hormone receptor mutations on hormone receptor crosstalk.
RESUMEN
Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1. Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles.
Asunto(s)
Neoplasias de la Mama , Receptor alfa de Estrógeno/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Ligandos , Mutación , Pirrolidinas , Moduladores Selectivos de los Receptores de Estrógeno/química , TetrahidronaftalenosRESUMEN
Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).
Asunto(s)
Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Compuestos Orgánicos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Humanos , Compuestos Orgánicos/química , Relación Estructura-ActividadRESUMEN
Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA's selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.
Asunto(s)
Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Receptor alfa de Estrógeno/química , Indoles/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/genética , Femenino , Fulvestrant/farmacología , Humanos , Indoles/química , Ligandos , Células MCF-7 , Proteínas Mutantes/metabolismo , Mutación/genética , Piperazinas/farmacología , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Estructura Secundaria de Proteína , Piridinas/farmacología , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/química , Relación Estructura-Actividad , Tamoxifeno/farmacologíaRESUMEN
Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and KDM5B have been identified as potential anti-cancer drug targets. Using structural information from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed. Structure-activity relationship (SAR) exploration resulted in the identification of compound 33, an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in the breast cancer cell line ZR-75-1.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Femenino , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Modelos Moleculares , Estructura Molecular , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteína 2 de Unión a Retinoblastoma/metabolismo , Relación Estructura-ActividadRESUMEN
Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive breast cancer behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone.Experimental Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary breast cancers. The resulting subset of GR-targeted genes was analyzed in two independent ER-negative breast cancer cohorts to derive and then validate the GR activity signature (GRsig).Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig that comprised n = 74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predict probability of relapse in a separate Validation cohort (HR = 1.9; P = 0.012).Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive breast cancers most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome for these high-risk patients. Clin Cancer Res; 24(14); 3433-46. ©2018 AACR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Transcriptoma , Animales , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Pronóstico , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Receptores de Estrógenos/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The bromodomain and extra-terminal (BET) family of epigenetic proteins has attracted considerable attention in drug discovery given its involvement in regulating gene transcription. Screening a focused small molecule library based on the bromodomain pharmacophore resulted in the identification of 2-methylisoquinoline-1-one as a novel BET bromodomain-binding motif. Structure guided SAR exploration resulted in >10,000-fold potency improvement for the BRD4-BD1 bromodomain. Lead compounds exhibited excellent potencies in both biochemical and cellular assays in MYC-dependent cell lines. Compound 36 demonstrated good physicochemical properties and promising exposure levels in exploratory PK studies.
Asunto(s)
Diseño de Fármacos , Isoquinolinas/química , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Simulación de Dinámica Molecular , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound 6 (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.
RESUMEN
Guided by co-crystal structural information obtained from a different series we were exploring, a scaffold morphing and SBDD approach led to the discovery of the 1,4-disubstituted indazole series as a novel class of GKAs that potently activate GK in enzyme and cell assays. anti-diabetic OGTT efficacy was demonstrated with 29 in a rodent models of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Descubrimiento de Drogas , Activadores de Enzimas/farmacología , Glucoquinasa/metabolismo , Indazoles/farmacología , Administración Oral , Regulación Alostérica/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/administración & dosificación , Activadores de Enzimas/química , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Indazoles/administración & dosificación , Indazoles/química , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Estructura Molecular , Bloqueadores de los Canales de Potasio/farmacología , Relación Estructura-ActividadRESUMEN
Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Imidazoles/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/síntesis química , Animales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Células HT29 , Xenoinjertos , Humanos , Imidazoles/química , Imidazoles/farmacología , Ratones , Estructura Molecular , Pteridinas/química , Pteridinas/farmacología , Relación Estructura-Actividad , Quinasa Tipo Polo 1RESUMEN
Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.
Asunto(s)
Azepinas/química , Azepinas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/farmacocinética , Ácido 4-Aminobenzoico/farmacología , Administración Oral , Adulto , Azepinas/farmacocinética , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Inhibidores de Proteínas Quinasas/farmacocinética , Adulto Joven , Quinasa Tipo Polo 1RESUMEN
The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer. Despite its success, the duration of response is often limited. For previous antiandrogens, one mechanism of resistance is mutation of the androgen receptor (AR). To prospectively identify AR mutations that might confer resistance to enzalutamide, we performed a reporter-based mutagenesis screen and identified a novel mutation, F876L, which converted enzalutamide into an AR agonist. Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment. Molecular dynamics simulations performed on antiandrogen-AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12. This model then provided the rationale for a focused chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide. DOI:http://dx.doi.org/10.7554/eLife.00499.001.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Diseño de Fármacos , Mutación , Antagonistas de Andrógenos/química , Línea Celular , Resistencia a Antineoplásicos/genética , Humanos , Masculino , Simulación de Dinámica Molecular , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyrazolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase inhibitory activities both in cell-free (IC50=0.64µM) and in cellular assays (IC50=7.1µM). These results clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.
Asunto(s)
Antineoplásicos/química , Óxidos S-Cíclicos/química , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Compuestos Heterocíclicos con 3 Anillos/química , Inhibidores de Proteínas Quinasas/química , Tiazinas/química , Sitio Alostérico , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Óxidos S-Cíclicos/síntesis química , Óxidos S-Cíclicos/metabolismo , Evaluación Preclínica de Medicamentos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Tiazinas/síntesis química , Tiazinas/metabolismoRESUMEN
Guided by co-crystal structures of compounds 15, 22 and 30, an SBDD approach led to the discovery of the 6-methyl pyridone series as a novel class of GKAs that potently activate GK in enzyme and cell assays. Anti-diabetic OGTT efficacy was demonstrated with 54 in a mouse model of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Diseño de Fármacos , Activadores de Enzimas/farmacología , Glucoquinasa/metabolismo , Hipoglucemiantes/farmacología , Piridonas/farmacología , Animales , Cristalografía por Rayos X , Diabetes Mellitus Experimental/tratamiento farmacológico , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/administración & dosificación , Activadores de Enzimas/química , Prueba de Tolerancia a la Glucosa , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Estructura Molecular , Piridonas/administración & dosificación , Piridonas/química , Relación Estructura-ActividadRESUMEN
Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.
Asunto(s)
Proteínas Bacterianas/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Desoxirribonucleasa IV (Fago T4-Inducido)/química , Thermotoga maritima/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , ADN/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Escherichia coli , Humanos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Unión Proteica , Estructura Secundaria de Proteína , Homología Estructural de ProteínaRESUMEN
Focal adhesion kinase (FAK) regulates cell survival and proliferation pathways. Here we report the discovery of a highly selective series of 1,5-dihydropyrazolo[4,3-c][2,1]benzothiazines that demonstrate a novel mode of allosteric inhibition of FAK. These compounds showed slow dissociation from unphosphorylated FAK and were noncompetitive with ATP after long preincubation. Co-crystal structural analysis revealed that the compounds target a novel allosteric site within the C-lobe of the kinase domain, which induces disruption of ATP pocket formation leading to the inhibition of kinase activity. The potency of allosteric inhibition was reduced by phosphorylation of FAK. Coupled SAR analysis revealed that N-substitution of the fused pyrazole is critical to achieve allosteric binding and high selectivity among kinases.
Asunto(s)
Descubrimiento de Drogas , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Tiazinas/farmacología , Regulación Alostérica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazoles/síntesis química , Pirazoles/química , Relación Estructura-Actividad , Tiazinas/síntesis química , Tiazinas/químicaRESUMEN
Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRalpha. These compounds are GW3965, F(3)methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRalpha ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRbeta homodimer and LXRalpha:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.
Asunto(s)
Receptores Nucleares Huérfanos/química , Transducción de Señal , Benzoatos/química , Benzoatos/metabolismo , Bencilaminas/química , Bencilaminas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Isoxazoles/química , Isoxazoles/metabolismo , Ligandos , Receptores X del Hígado , Modelos Moleculares , Receptores Nucleares Huérfanos/metabolismo , Fenilacetatos/química , Fenilacetatos/metabolismo , Alineación de Secuencia , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three-metal ion mechanism for damage binding and incision. The 1.10-A resolution DNA-free and the 2.45-A resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-A resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV alpha-beta triose phosphate isomerase (TIM) barrel and APE1 four-layer alpha-beta folds of the apurinic-apyrimidinic endonuclease families.
Asunto(s)
Desoxirribonucleasa IV (Fago T4-Inducido)/metabolismo , Escherichia coli/enzimología , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido)/química , Desoxirribonucleasa IV (Fago T4-Inducido)/genética , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11beta-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11beta-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11beta-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11beta-HSD1 activities and will ultimately facilitate the development of specific inhibitors.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasas/química , Glucocorticoides/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular , Cortisona/química , Cristalografía por Rayos X , Cisteína/química , Diabetes Mellitus Tipo 2/metabolismo , Dimerización , Disulfuros/química , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Humanos , Hidrocortisona/química , Cinética , Espectrometría de Masas , Modelos Moleculares , Mutagénesis , Obesidad/metabolismo , Oxígeno/metabolismo , Mutación Puntual , Prolina/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
STI-571 (Gleevec) is a highly successful cancer drug due to its activity as an inhibitor of the Abelson cytoplasmic tyrosine kinase (Abl), which is constitutively active in a majority of patients with chronic myelogenous leukemia. STI-571 also inhibits two type III receptor tyrosine kinases, c-Kit and platelet-derived growth factor receptor, and functions by targeting inactive conformations of these kinases. This review focuses on recent developments in X-ray co-crystal structure analyses of STI-571 bound to Abl and the c-Kit receptor tyrosine kinase domain, and also three other relevant kinase inhibitor co-crystal structures. The similar structural features of these inactive kinases suggest they will be useful for the successful drug discovery and development of specific and targeted gene-based cancer drugs.