RESUMEN
The emergence of the H7N9 influenza virus in humans in Eastern China has raised concerns that a new influenza pandemic could occur. Here, we used a ferret model to evaluate the infectivity and transmissibility of A/Shanghai/2/2013 (SH2), a human H7N9 virus isolate. This virus replicated in the upper and lower respiratory tracts of the ferrets and was shed at high titers for 6 to 7 days, with ferrets showing relatively mild clinical signs. SH2 was efficiently transmitted between ferrets via direct contact, but less efficiently by airborne exposure. Pigs were productively infected by SH2 and shed virus for 6 days but were unable to transmit the virus to naïve pigs or ferrets. Under appropriate conditions, human-to-human transmission of the H7N9 virus may be possible.
Asunto(s)
Enfermedades Transmisibles Emergentes/transmisión , Enfermedades Transmisibles Emergentes/virología , Gripe Humana/transmisión , Gripe Humana/virología , Orthomyxoviridae/patogenicidad , Animales , Modelos Animales de Enfermedad , Hurones , Humanos , Gripe Humana/patología , Orthomyxoviridae/clasificación , Orthomyxoviridae/genética , Sistema Respiratorio/patología , Sistema Respiratorio/virología , Sus scrofaRESUMEN
Cortical spreading depression (CSD) has been shown to have neuroprotective effects when administered in advance of cerebral ischemia. The mechanism by which CSD induces its neuroprotective effect however remains to be elucidated. Since MAP kinases have been shown to impart neuroprotection in ischemic preconditioning paradigms, we attempted to determine the role CSD may have in the activation of MAPK. We show that CSD is capable of increasing the phosphorylation of ERK in a MEK-dependent manner. This phosphorylation is, however, transient, as phosphorylated ERK levels return to control levels 45 min after 2 h of CSD elicitation. Immunohistochemical analysis reveals that the phosphorylated form of ERK is located ubiquitously in cells of the CSD-treated cortex while CSD-elicited MEK phosphorylation resides solely in the nuclei. These data suggest that CSD may act via the MAP kinase pathways to mediate preconditioning.
Asunto(s)
Isquemia Encefálica/enzimología , Supervivencia Celular/fisiología , Corteza Cerebral/enzimología , Depresión de Propagación Cortical/fisiología , Precondicionamiento Isquémico , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neuronas/enzimología , Regulación hacia Arriba/fisiología , Animales , Isquemia Encefálica/fisiopatología , Compartimento Celular , Núcleo Celular/metabolismo , Corteza Cerebral/fisiopatología , Citoplasma/metabolismo , Lateralidad Funcional/fisiología , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Potent, selective, and structurally new inhibitors of the Fe(II) enzyme Escherichia coli peptide deformylase (PDF) were obtained by rational optimization of the weakly binding screening hit (5-chloro-2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-acetic acid hydrazide (1). Three-dimensional structural information, gathered from Ni-PDF complexed with 1, suggested the preparation of two series of related hydroxamic acid analogues, 2-(2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-N-hydroxy-acetamides (A) and 2-(2,2-dioxo-1,4-dihydro-2H-2lambda(6)-benzo[1,2,6]thiadiazin-3-yl)-N-hydroxy-acetamides (B), among which potent PDF inhibitors (37, 42, and 48) were identified. Moreover, two selected compounds, one from each series, 36 and 41, showed good selectivity for PDF over several endoproteases including matrix metalloproteases. However, these compounds showed only weak antibacterial activity.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Escherichia coli/enzimología , Ácidos Hidroxámicos/síntesis química , Inhibidores de Proteasas/síntesis química , Quinazolinas/síntesis química , Tiadiazinas/síntesis química , Aminopeptidasas/química , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Tiadiazinas/química , Tiadiazinas/farmacologíaAsunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Enzimas/química , Enzimas/metabolismo , Investigación/tendencias , Animales , Inhibidores Enzimáticos/farmacocinética , Humanos , Modelos Moleculares , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacocinéticaRESUMEN
Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Inhibidores Enzimáticos/síntesis química , Ácidos Hidroxámicos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Chlamydophila pneumoniae/efectos de los fármacos , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/metabolismo , Haemophilus influenzae/efectos de los fármacos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacología , Modelos Moleculares , Moraxella catarrhalis/efectos de los fármacos , Mycoplasma pneumoniae/efectos de los fármacos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Infecciones del Sistema Respiratorio/microbiología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A computational algorithm was used to design automatically novel thrombin inhibitors that are available from a single-step chemical reaction. The compounds do not contain amide bonds, are achiral and have a molecular weight below 400. Of the 10 compounds that were synthesized, five bind to thrombin with a Ki in the nanomolar range. Subsequent X-ray structure determination of the thrombin-inhibitor complex for the best compound (Ki = 95 nM) confirms the predicted binding mode. The novel algorithm is applicable to a broad range of chemical reactions.
Asunto(s)
Antitrombinas/química , Biblioteca de Péptidos , Algoritmos , Antitrombinas/síntesis química , Conformación Proteica , Difracción de Rayos XRESUMEN
We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.
Asunto(s)
Factor VII/genética , Factor VII/metabolismo , Mutación , Tromboplastina/metabolismo , Sustitución de Aminoácidos , Animales , Arginina/genética , Coagulación Sanguínea , Deficiencia del Factor VII/genética , Glicina/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAsunto(s)
Deficiencia del Factor VII/fisiopatología , Factor VII/fisiología , Variación Genética , Secuencia de Aminoácidos , Factor VII/química , Factor VII/genética , Deficiencia del Factor VII/genética , Humanos , Datos de Secuencia Molecular , Fenotipo , Procesamiento Proteico-Postraduccional , Relación Estructura-Actividad , Tromboplastina/fisiologíaRESUMEN
The tissue factor:factor VIIa (TF:F.VIIa) complex is the physiological initiator of blood coagulation and plays a role both in normal hemostasis and in various thrombotic disorders. The TF:F.VIIa crystal structure presented here shows the two molecules in association as a complex and provides a ready structural explantation for most of the complementary observations on the complex obtained by other techniques. This is only one F.VII structure of a series along the activation pathway going from zymogen F.VII, through F.VIIa alone, towards that in the ternary complex with a macromolecular substrate such as F.IX or F.X. To fully understand the activation of F.VIIa by TF will require further structural work, but in the meantime this structure may be used in the search for more effective and more specific anticoagulants.
Asunto(s)
Factor VIIa/química , Tromboplastina/química , Coagulación Sanguínea/fisiología , Catálisis , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
BACKGROUND: The serine protease thrombin is central in the processes of hemostasis and thrombosis. To be useful, thrombin inhibitors should combine potency towards thrombin with selectivity towards other related enzymes such as trypsin. We previously reported the structure-based design of thrombin inhibitors with rigid, bicyclic core structures. These compounds were highly active towards thrombin, but showed only modest selectivity. RESULTS: Here, we describe the rational design of selective thrombin inhibitors starting from the X-ray crystal structure of the complex between the previously generated lead molecule and thrombin. The lead molecule bound with a Ki value of 90nM and a selectivity of 7.8 for thrombin over trypsin. Our design led to inhibitors with improved activity and greatly enhanced selectivity. The binding mode for two of the new inhibitors was determined by X-ray crystallography of their complexes with thrombin. The results confirmed the structures predicted by molecular modeling and, together with the binding assays, provided profound insight into molecular recognition phenomena at the thrombin active site. CONCLUSIONS: A novel class of nonpeptidic, selective thrombin inhibitors has resulted from structure-based design and subsequent improvement of the initial lead molecule. These compounds, which are preorganized for binding to thrombin through a rigid, bicyclic or tricyclic central core, could aid in the development of new antithrombotic drugs. Correlative binding and X-ray structural studies within a series of related, highly preorganized inhibitors, which all prefer similar modes of association to thrombin, generate detailed information on the strength of individual intermolecular bonding interactions and their contribution to the overall free energy of complexation.
Asunto(s)
Antitrombinas/síntesis química , Trombina/antagonistas & inhibidores , Trombina/metabolismo , Antitrombinas/química , Antitrombinas/metabolismo , Antitrombinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Unión Proteica , EstereoisomerismoRESUMEN
The tissue factor:factor VIIa (TF-F.VIIa) complex is considered the physiological initiator of blood coagulation. Besides its role in normal hemostasis, this enzyme complex has been found to play an important role in various thrombotic disorders and thus has become an attractive target for the development of new anticoagulants. Recently, significant progress has been made in regard to structural and molecular aspects of TF-VIIa-initiated coagulation. A rather complete picture on how tissue factor binds to factor VIIa has emerged and is discussed in detail in this review. Also, the combined data of the TF-F.VIIa crystal structure, of naturally occurring F.VII variants, and of mutagenesis studies provide a framework to discuss molecular aspects of the tissue factor-mediated enhancement of F.VIIa catalytic efficiency and the recognition of macromolecular substrates. F.VIIa as a member of the serine protease family has an active site homologous to other coagulation factors. The release of the coordinates of the crystal structures of F.X and F.IX, together with the earlier determined thrombin structure, now allows a detailed comparison of these active centers with respect to the development of specific and potent active site inhibitors. This structural and molecular information about the TF-F.VIIa complex and other coagulation enzymes adds to our understanding of blood coagulation and should further the development of new classes of anticoagulants. (Trends Cardiovasc Med 1997;7:316-324). © 1997, Elsevier Science Inc.
RESUMEN
Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extracellular domain of tissue factor. In the complex, factor VIIa adopts an extended conformation. This structure provides a basis for understanding many molecular aspects of the initiation of coagulation.
Asunto(s)
Factor VIIa/química , Tromboplastina/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Factor IX/metabolismo , Factor VIIa/metabolismo , Factor X/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Relación Estructura-Actividad , Especificidad por Sustrato , Subtilisinas , Tromboplastina/metabolismoRESUMEN
Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 x 0.12 x 0.6 mm and showed diffraction to a resolution of 3 A.
Asunto(s)
Factor VIIa/química , Fragmentos de Péptidos/química , Tromboplastina/química , Cristalización , Factor VIIa/antagonistas & inhibidores , Factor VIIa/metabolismo , Espectrometría de Masas , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serpinas/química , Subtilisinas/farmacología , Tromboplastina/efectos de los fármacos , Tromboplastina/genética , Tromboplastina/metabolismoRESUMEN
Thrombin, a serine protease, plays a central role in the initiation and propagation of thrombotic events. An extensive search for new thrombin inhibitors was performed, using an unconventional approach. Screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of (aminoiminomethyl)piperidine (amidinopiperidine) as a weak, but intrinsically selective, thrombin inhibitor. Elaboration of this molecule provided compounds which inhibit thrombin with Ki's in the range of 20-50 nM and with selectivities of 1000-4000 against trypsin. These inhibitor compounds show a new and unexpected binding mode to thrombin. Modification of the central building block and then of one of the hydrophobic substituents led to the discovery of a new family of thrombin inhibitors which has reverted to the former binding mode to thrombin. This last class of compounds shows inhibitory activities in the picomolar range, low toxicity, and a short plasma half life which favors its use for an intravenous application. From this series of thrombin inhibitors, 19f(Ro 46-6240) was selected for clinical development as an antithrombotic agent for intravenous administration.
Asunto(s)
Antitrombinas/síntesis química , Piperidinas/síntesis química , Aminoácidos/química , Aminoácidos/farmacología , Animales , Antitrombinas/farmacología , Ácido Aspártico/síntesis química , Ácido Aspártico/farmacología , Diseño de Fármacos , Semivida , Humanos , Piperidinas/farmacología , Ratas , Relación Estructura-Actividad , Inhibidores de Tripsina/farmacologíaRESUMEN
To probe the ligand receptor interface, a number of point mutations were introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis. The mutated proteins were expressed in Escherichia coli and analyzed for selective binding to recombinant 55- and 75-kDa TNF receptors in competition with radiolabeled wild-type TNF alpha. Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, whereas mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor. Mutation of the conserved Tyr87 resulted in a dramatic loss of binding activity to both receptors. The selectivity for one or the other receptor type was found to be enhanced by combining two or three point mutations, the effects of the single mutations with respect to receptor selectivity being at least additive. A combination of the mutations Arg32-->Trp and Ser86-->Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor. In contrast, combining the Asp143-->Asn and Ala145-->Arg mutations (D143N-A145R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-fold. In functional assays, selective activation of the 55-kDa TNF receptor by the R32W-S86T mutant elicited a full cytotoxic response in human KYM-1 cells and secretion of interleukin 6 and granulocyte-macrophate colony-stimulating factor in human umbilical vein endothelial cells. In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A145R mutant as well as with agonistic antibodies failed to induce these responses.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Sitios de Unión , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor (TNF) receptor with human TNF beta has been determined at 2.85 A resolution. The complex has three receptor molecules bound symmetrically to one TNF beta trimer. The receptor fragment, a very elongated end to end assembly of four similar folding domains, binds in the groove between two adjacent TNF beta subunits. The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation. The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor (NGF)/TNF receptor family as a whole.
Asunto(s)
Linfotoxina-alfa/química , Linfotoxina-alfa/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Receptores de Superficie Celular/genética , Receptores de Factor de Crecimiento Nervioso/química , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.
Asunto(s)
ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Simulación por Computador , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Difracción de Rayos XRESUMEN
A complex of tumour necrosis factor-beta with the soluble extracellular domain of the human 55 kDa TNF receptor has been crystallized. Crystals of the complex were grown using polyethylene glycol 4000 as the precipitating agent in the presence of beta-octyl glucoside. The receptor-ligand complex crystallizes in a cubic space group and diffracts to 2.85 A.