RESUMO
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Variância , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Deutério/análise , Medição da Troca de Deutério/instrumentação , Hidrogênio/análise , Ligantes , Espectrometria de Massas/instrumentação , Peptídeos/análise , Proteínas/química , Receptores de Calcitriol/química , Reprodutibilidade dos TestesRESUMO
Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure-activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.
Assuntos
Matriz Óssea/efeitos dos fármacos , Colágeno/metabolismo , Fêmur/efeitos dos fármacos , Cloridrato de Raloxifeno/farmacologia , Animais , Matriz Óssea/metabolismo , Colágeno/química , Cães , Durapatita/química , Fêmur/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Masculino , Piperidinas/química , Polilisina/química , Polilisina/metabolismo , Ligação Proteica , Cloridrato de Raloxifeno/metabolismo , Receptores de Estrogênio/metabolismo , Eletricidade Estática , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor-ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile.
Assuntos
Bioquímica/métodos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Linhagem Celular Tumoral , Análise por Conglomerados , Cristalografia por Raios X , Interpretação Estatística de Dados , Humanos , Ligantes , Espectrometria de Massas/métodos , Modelos Biológicos , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Ligação Proteica , Distribuição TecidualRESUMO
Here we present the use of hydrogen-deuterium exchange (HDX) mass spectrometry in analyzing the estrogen receptor beta ligand binding domain (ERbeta LBD) in the absence and presence of a variety of chemical compounds with different binding modes and pharmacological properties. Previously, we reported the use of HDX as a method for predicting the tissue selectivity of ERalpha ligands. HDX profiles of ERalpha LBD in complex with ligand could differentiate compounds of the same chemotype. In contrast, similar analysis of ERbeta LBD showed correlation to the compound chemical structures but little correlation with compound tissue selectivity. The different HDX patterns observed for ERbeta LBD when compared to those for ERalpha LBD bound to the same chemical compounds serve as an indication that ERbeta LBD undergoes a different structural response to the same ligand when compared to ERalpha LBD. The conformational dynamics revealed by HDX for ERbeta LBD together with those for ERalpha LBD shed light on ER ligand interactions and offer new structural insights. The compound-specific perturbations in HDX kinetics observed for each of the two isoforms should aid the development of subtype-selective ER ligands.
Assuntos
Medição da Troca de Deutério , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Linhagem Celular , Cristalografia por Raios X , Medição da Troca de Deutério/métodos , Estradiol/metabolismo , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/química , Genisteína/metabolismo , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMO
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.
Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Descoberta de Drogas/economia , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Desenho de Equipamento , Análise de Injeção de Fluxo/economia , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério/economia , Espectrometria de Massa com Troca Hidrogênio-Deutério/instrumentação , Ligantes , Proteínas/químicaRESUMO
Molecular characterization of the binding epitope of IL-23R and its cognate cytokine IL-23 is paramount to understand the role in autoimmune diseases and to support the discovery of new inhibitors of this protein-protein interaction. Our results revealed that HDX-MS was able to identify the binding epitope of IL-23R:IL-23, which opened the way to evaluate a peptide macrocycle described in the literature as disrupter of this autoimmune target. Thus, the characterization of the interactions of this chemotype by HDX-MS in combination with computational approaches was achieved. To our knowledge, this is the first reported structural evidence regarding the site where a small compound binds to IL-23R.
RESUMO
To date, IL-17A antibodies remain the only therapeutic approach to correct the abnormal activation of the IL-17A/IL-17R signaling complex. Why is it that despite the remarkable success of IL-17 antibodies, there is no small molecule antagonist of IL-17A in the clinic? Here we offer a unique approach to address this question. In order to understand the interaction of IL-17A with its receptor, we combined peptide discovery using phage display with HDX, crystallography, and functional assays to map and characterize hot regions that contribute to most of the energetics of the IL-17A/IL-17R interaction. These functional maps are proposed to serve as a guide to aid in the development of small molecules that bind to IL-17A and block its interaction with IL-17RA.
Assuntos
Colífagos/metabolismo , Interleucina-17/metabolismo , Peptídeos/metabolismo , Receptores de Interleucina-17/metabolismo , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Células HT29 , Humanos , Interleucina-17/química , Modelos Moleculares , Receptores de Interleucina-17/química , Ressonância de Plasmônio de SuperfícieRESUMO
Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness, and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.
Assuntos
Medição da Troca de Deutério , Espectrometria de Massas , Proteínas/química , Teorema de Bayes , Ligantes , Simulação de Dinâmica Molecular , Método de Monte CarloRESUMO
The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.
Assuntos
DNA/química , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , DNA/genética , DNA/metabolismo , Medição da Troca de Deutério , Dimerização , Humanos , Hidrogênio , Ligantes , Espectrometria de Massas , Osteocalcina/genética , Osteocalcina/metabolismo , Ligação Proteica , Receptores de Calcitriol/genética , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMO
A selective estrogen receptor modulator (SERM) for the potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol, LSN2120310, potently binds ERalpha and ERbeta and is an antagonist in MCF-7 breast adenocarcinoma and Ishikawa uterine cancer cell lines. The compound is a potent estrogen antagonist in the rat uterus. In ovariectomized rats, the compound lowers cholesterol, maintains bone mineral density, and is efficacious in a morphine dependent rat model of hot flush efficacy.
Assuntos
Benzopiranos/síntese química , Antagonistas de Estrogênios/síntese química , Fogachos/tratamento farmacológico , Naftalenos/síntese química , Moduladores Seletivos de Receptor Estrogênico/síntese química , Adenocarcinoma , Animais , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacologia , Benzopiranos/química , Benzopiranos/farmacologia , Densidade Óssea/efeitos dos fármacos , Neoplasias da Mama , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colesterol/sangue , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Morfina/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Ovariectomia , Ratos , Ratos Sprague-Dawley , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Estereoisomerismo , Neoplasias Uterinas , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimentoRESUMO
Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.
Assuntos
Receptor beta de Estrogênio/agonistas , Flavonoides/síntese química , Hiperplasia Prostática/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/síntese química , Animais , Sítios de Ligação , Cristalografia por Raios X , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/química , Estrogênios , Flavonoides/química , Flavonoides/farmacologia , Humanos , Ligantes , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Próstata/efeitos dos fármacos , Próstata/patologia , Hiperplasia Prostática/patologia , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Relação Estrutura-AtividadeRESUMO
Computational assessment of the IL-17A structure identified two distinct binding pockets, the ß-hairpin pocket and the α-helix pocket. The ß-hairpin pocket was hypothesized to be the site of binding for peptide macrocycles. Support for this hypothesis was obtained using HDX-MS which revealed protection to exchange only within the ß-hairpin pocket. This data represents the first direct structural evidence of a small molecule binding site on IL-17A that functions to disrupt the interaction with its receptor.
Assuntos
Medição da Troca de Deutério , Interleucina-17/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Espectrometria de Massas , Peptídeos Cíclicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Interleucina-17/metabolismo , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Conformação Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Relação Estrutura-AtividadeRESUMO
The use of selective estrogen receptor modulators for the treatment of estrogen-dependent diseases in premenopausal women has been hindered by undesirable ovarian stimulation and associated risks of ovarian cysts. We have identified a selective estrogen receptor modulator compound (LY2066948) that is a strong estrogen antagonist in the uterus yet has minimal effects on the ovaries of rats. LY2066948 binds with high affinity to both estrogen receptors and has potent estrogen antagonist activity in human uterine and breast cancer cells. Oral administration of LY2066948 to immature rats blocked uterine weight gain induced by ethynyl estradiol with an ED50 of 0.07 mg/kg. Studies in mature rats demonstrated that LY2066948 decreases uterine weight by 51% after 35 d treatment, confirming potent uterine antagonist activity over several estrous cycles. This strong uterine response contrasted with the minimal effects on the ovaries: serum estradiol levels remained within the normal range, whereas histologic evaluation showed granulosa cell hyperplasia in few of the rats. Bone studies demonstrated that LY2066948 prevented ovariectomy-induced bone loss and treatment of ovary-intact rats caused no bone loss, confirming estrogen receptor agonist skeletal effects. Collectively, these data show that LY2066948 exhibits a tissue-specific profile consistent with strong antagonist activity in the uterus, agonist activity in bone, and minimal effects in the ovaries.
Assuntos
Osso e Ossos/fisiologia , Naftalenos/farmacologia , Indução da Ovulação , Piperidinas/farmacologia , Receptores de Estrogênio/fisiologia , Útero/fisiologia , Animais , Osso e Ossos/efeitos dos fármacos , Linhagem Celular Tumoral , Etinilestradiol/farmacologia , Feminino , Humanos , Cinética , Ovariectomia , Ratos , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/efeitos dos fármacos , Maturidade Sexual , Útero/efeitos dos fármacosRESUMO
The design of a novel selective estrogen receptor modulator (SERM) for the potential treatment of uterine leiomyoma is described. 16 (LY2066948-HCl) binds with high affinity to estrogen receptors alpha and beta (ERalpha and ERbeta, respectively) and is a potent uterine antagonist with minimal effects on the ovaries as determined by serum biomarkers and histologic evaluation.
Assuntos
Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Leiomioma/tratamento farmacológico , Naftalenos/síntese química , Ovário/efeitos dos fármacos , Piperidinas/síntese química , Moduladores Seletivos de Receptor Estrogênico/síntese química , Neoplasias Uterinas/tratamento farmacológico , Útero/efeitos dos fármacos , Animais , Sítios de Ligação , Disponibilidade Biológica , Linhagem Celular , Proliferação de Células , Estradiol/sangue , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/agonistas , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Modelos Moleculares , Naftalenos/química , Naftalenos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/metabolismo , Piperidinas/química , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Relação Estrutura-Atividade , Útero/anatomia & histologia , Útero/citologia , Útero/metabolismoRESUMO
The estrogen receptor is a regulator of a wide range of physiological functions, including the female reproductive system, in addition to bone, cardiovascular and CNS function. ER ligands have been approved for the treatment of menopausal symptoms, breast cancer and osteoporosis, however the search continues for new modulators of ER function with improved properties. Progress in medicinal chemistry programs has resulted in the identification of structurally diverse molecules with unique biological properties. Recent advances in the design and synthesis of these non-steroidal and steroidal estrogen receptor ligands is reviewed. The relationship between the structural features of the ligand and receptor function is also discussed.
Assuntos
Moduladores de Receptor Estrogênico/química , Receptores de Estrogênio/metabolismo , Animais , Ligação Competitiva , Moduladores de Receptor Estrogênico/farmacologia , Humanos , Ligantes , Estrutura Molecular , Receptores de Estrogênio/fisiologia , Relação Estrutura-AtividadeRESUMO
Tamoxifen has been shown to be active in vitro against Leishmania and effective in the treatment for leishmaniasis in murine models. Through the screening of a compound library of estrogen receptor modulator analogs, we identified the major characteristics required for antileishmanial activity. To overcome the difficulties presented by tamoxifen's propensity for E/Z isomerization, we used the 2-arylbenzothiophene compound BTP as a more stable alternative. Directed screening of a small compound library based on BTP led to active compounds against Leishmania. Subsequent structure-activity data for the synthetic 2-arylbenzothiophenes evaluated in this study indicate that optimal antileishmanial potency is dependent on the presence of two basic side chains. In addition, the primary structural features required for estrogen receptor binding, the phenols, are not required for inhibiting parasitic growth. Significantly, the most active antileishmanial benzothiophenes lack the pharmacophore for estrogen receptor activity and therefore address potential concerns about the undesirable effects of using selective estrogen receptor modulators in women and children with leishmaniasis. Three compounds selected from the screening have shown consistent activity against all species and stages of Leishmania in vitro although improvements in selectivity are needed. These compounds represent viable starting points for further optimization as antileishmanial agents.
Assuntos
Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Tiofenos/química , Tiofenos/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Células VeroRESUMO
The discovery, pharmacology, and biophysical characterization of an ERα selective benzothiophene (BTPα) is described. BTPα (4) is a high affinity ligand with 140-fold greater selectivity for ERα (K(i)=0.25 nM) over ERbeta (K(i)=35 nM). In rodent models of estrogen action, BTPα blocks the effects of estrogen in the uterus but mimics the effects estrogen on bone. The basis of ERα selectivity for BTPα was evaluated by using protein crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry. HDX data supports that the n-butyl chain of BTPα stabilizes helix 7 in ERα relative to that of ERß which we propose leads to an enhancement of affinity to the alpha receptor sub-type.
RESUMO
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.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptores de Calcitriol/química , Receptores X de Retinoides/química , Alitretinoína , Sítios de Ligação , Di-Hidroxicolecalciferóis/química , Humanos , Ligantes , Modelos Moleculares , Coativador 1 de Receptor Nuclear/química , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Receptores X de Retinoides/agonistas , Receptores X de Retinoides/metabolismo , Tretinoína/químicaRESUMO
We report the synthesis and characterization of novel 3-aryl indoles as potent and efficacious progesterone receptor (PR) antagonists with potential for the treatment of uterine fibroids. These compounds demonstrated excellent selectivity over other steroid nuclear hormone receptors such as the mineralocorticoid receptor (MR). They were prepared from 2-bromo-6-nitro indole in four to six steps using a Suzuki cross-coupling as the key step. Compound 8f was orally active in the complement 3 model of progesterone antagonism in the rat uterus and demonstrated partial antagonism in the McPhail model of progesterone activity.