RESUMO
SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (â¼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , DNA de Cadeia Simples , Guanosina Trifosfato/metabolismo , Cinética , Proteínas Monoméricas de Ligação ao GTP/genética , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/químicaRESUMO
A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Compostos Bicíclicos com Pontes/química , Hepatócitos/metabolismo , Cetonas/química , Fígado/metabolismo , Polímeros/química , Compostos Bicíclicos com Pontes/metabolismo , Cristalografia por Raios X , Portadores de Fármacos/química , Humanos , Cetonas/metabolismo , Fígado/citologia , Modelos Moleculares , Estrutura Molecular , Polímeros/metabolismoRESUMO
Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.
Assuntos
Regulação da Expressão Gênica , Glucoquinase/metabolismo , Glucose/química , Sítio Alostérico , Catálise , Ativação Enzimática , Homeostase , Humanos , Hipoglicemia/patologia , Cinética , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação Proteica , Espalhamento de Radiação , Raios XRESUMO
Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Gravidez , Feminino , Humanos , Animais , Anticorpos Antivirais , Anticorpos Neutralizantes , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/genética , Glicoproteínas , Sigmodontinae , Proteínas Virais de Fusão/genéticaRESUMO
Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.
Assuntos
Fígado , Membranas Mitocondriais , Humanos , Fígado/metabolismo , Membranas Mitocondriais/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/química , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Sequência de AminoácidosRESUMO
Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.
Assuntos
Hipercolesterolemia/genética , Mutação de Sentido Incorreto/fisiologia , Serina Endopeptidases/metabolismo , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Hipercolesterolemia/etiologia , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ligação Proteica/genética , Conformação Proteica , Receptores de LDL/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/química , Ésteres do Colesterol/química , Modelos Moleculares , Fosfatidilcolinas/química , Triglicerídeos/química , Animais , Sítios de Ligação , Células CHO , Proteínas de Transferência de Ésteres de Colesterol/genética , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mutação Puntual , Ligação Proteica , Conformação ProteicaRESUMO
Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region.
Assuntos
Quimiocina CCL20/metabolismo , Receptores CCR6/química , Receptores CCR6/metabolismo , Quimiocina CCL20/química , Quimiocina CCL20/genética , Microscopia Crioeletrônica , Humanos , Ligantes , Ligação Proteica , Receptores CCR6/genética , Receptores Acoplados a Proteínas G , Transdução de SinaisRESUMO
We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.
Assuntos
Anticorpos/farmacologia , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Anticorpos/química , Membrana Celular/metabolismo , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Polímeros , Propilaminas , Ligação Proteica , Conformação ProteicaRESUMO
A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC(50) = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.
Assuntos
Inibidores da Dipeptidil Peptidase IV/farmacologia , Hipoglicemiantes/farmacologia , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Administração Oral , Animais , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/síntese química , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Cães , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirrolidinas/síntese química , Pirrolidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.
Assuntos
Aminopiridinas/síntese química , Aminopiridinas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aminopiridinas/efeitos adversos , Animais , Disponibilidade Biológica , Ácidos Carboxílicos/química , Inibidores do Citocromo P-450 CYP3A/síntese química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Descoberta de Drogas , Meia-Vida , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/efeitos adversos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/sangueRESUMO
Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.
Assuntos
Receptor alfa de Estrogênio/química , Pirrolidinas/química , Tetra-Hidronaftalenos/química , Cristalografia por Raios X , Receptor alfa de Estrogênio/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirrolidinas/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Tetra-Hidronaftalenos/metabolismoRESUMO
Inhibitors of the glucagon-like peptide-1 (GLP-1) degrading enzyme dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes in animal models and in human subjects. A novel series of cis-2,5-dicyanopyrrolidine alpha-amino amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. 1-({[1-(Hydroxymethyl)cyclopentyl]amino}acetyl)pyrrolidine-2,5-cis-dicarbonitrile (1c) is an achiral, slow-binding (time-dependent) inhibitor of DPP-IV that is selective for DPP-IV over other DPP isozymes and proline specific serine proteases, and which has oral bioavailability in preclinical species and in vivo efficacy in animal models. The mode of binding of the cis-2,5-dicyanopyrrolidine moiety was determined by X-ray crystallography. The hydrochloride salt of 1c was further profiled for development as a potential new treatment for type 2 diabetes.
Assuntos
Inibidores de Adenosina Desaminase , Adenosina Desaminase/química , Dipeptidil Peptidase 4/química , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/química , Hipoglicemiantes/síntese química , Nitrilas/síntese química , Pirrolidinas/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Injeções Intravenosas , Masculino , Camundongos , Modelos Moleculares , Nitrilas/química , Nitrilas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Recent studies in adipose tissue, pancreas, muscle, and macrophages suggest that MAP4K4, a serine/threonine protein kinase may be a viable target for antidiabetic drugs. As part of the evaluation of MAP4K4 as a novel antidiabetic target, a tool compound, 16 (PF-6260933) and a lead 17 possessing excellent kinome selectivity and suitable properties were delivered to establish proof of concept in vivo. The medicinal chemistry effort that led to the discovery of these lead compounds is described herein together with in vivo pharmacokinetic properties and activity in a model of insulin resistance.
RESUMO
Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.
Assuntos
Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Colesterol/metabolismo , Fator de Crescimento Epidérmico/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese , Peptídeos/síntese química , Peptídeos/química , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic ß-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativadores de Enzimas/síntese química , Glucoquinase/metabolismo , Hepatócitos/metabolismo , Hipoglicemiantes/síntese química , Imidazóis/síntese química , Ácidos Nicotínicos/síntese química , Sítio Alostérico , Animais , Glicemia/metabolismo , Cães , Ativadores de Enzimas/farmacocinética , Ativadores de Enzimas/farmacologia , Haplorrinos , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Imidazóis/farmacocinética , Imidazóis/farmacologia , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Masculino , Modelos Moleculares , Ácidos Nicotínicos/farmacocinética , Ácidos Nicotínicos/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
A series of pyrrolidine based inhibitors of dipeptidyl peptidase IV were developed from a high throughput screening hit for the treatment of type 2 diabetes. Potency, selectivity, and pharmacokinetic properties were optimized resulting in the identification of a pre-clinical candidate for further profiling.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV , Flúor/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Animais , Cristalografia por Raios X , Dipeptidil Peptidase 4/química , Cães , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Pirrolidinas/síntese química , Pirrolidinas/farmacocinética , Ratos , EstereoisomerismoRESUMO
Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.
Assuntos
Catepsinas/metabolismo , Sequência de Bases , Catepsinas/antagonistas & inibidores , Catepsinas/química , Catepsinas/isolamento & purificação , Cristalografia por Raios X , Primers do DNA , Humanos , Modelos Moleculares , Inibidores de Proteases/farmacologia , Conformação Proteica , Especificidade por SubstratoRESUMO
In this communication, we wish to describe the discovery of a novel series of 6-azauracil-based thyromimetics that possess up to 100-fold selectivities for binding and functional activation of the beta(1)-isoform of the thyroid receptor family. Structure-activity relationship studies on the 3,5- and 3'-positions provided compounds with enhanced TR beta affinity and selectivity. Key binding interactions between the 6-azauracil moiety and the receptor have been determined through of X-ray crystallographic analysis.