RESUMEN
The influenza virus (IV) is a highly contagious virus causing seasonal global outbreaks affecting annually up to 20% of the world's population and leading to 250,000-500,000 deaths worldwide. Current vaccines have variable effectiveness, and, in particular during a pandemic outbreak, they are probably not available in the amounts needed to protect the world population. Therefore we need effective small molecule drugs to combat an IV infection and that can be produced, in case of pandemic, rapidly and in large quantities. Unfortunately, natural occurring IV becomes more and more resistant to current anti-IV drugs. And thus, there is an urgent need for development of alternative agents with new mechanisms of action. This review provides an overview of the pharmacology and effectiveness of new anti-IV agents, focusing on inhibition mechanisms directed against virus-host interactions.
Asunto(s)
Antivirales/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/fisiología , Animales , Antivirales/uso terapéutico , Interacciones Farmacológicas , Humanos , Gripe Humana/inmunología , Gripe Humana/virología , Orthomyxoviridae/genética , Orthomyxoviridae/inmunologíaRESUMEN
Binding of tissue-type plasminogen activator (tPA) to amyloid and denatured proteins is reported in a number of studies. The binding site has been mapped previously to the finger domain of tPA. In this study, tPA and truncated tPA constructs, lacking the finger domain, were tested for their ability to bind to Aß and AIAPP amyloid-like fibrils. Surface plasmon resonance experiments and pull-down assays clearly show that indeed tPA binds, but that the finger domain is not essential. Another possible binding mechanism via the lysine binding site on the kringle 2 domain was also not crucial for the binding. Immuno-electron microscopy studies show that tPA binds to fibril sides. This study shows that, besides the finger domain, other domains in tPA are involved in amyloid binding.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Fragmentos de Péptidos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Sitios de Unión , Técnicas Biosensibles , Humanos , Lisina/metabolismo , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.
Asunto(s)
Papaína/química , Péptidos/química , Resonancia por Plasmón de Superficie/instrumentación , Azidas/química , Reacción de Cicloadición , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Óxidos de Nitrógeno/química , Papaína/metabolismo , Péptidos/metabolismo , Propiedades de SuperficieRESUMEN
Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FcεRI) mediated mast cell degranulation. Once FcεRI is stimulated, intracellular ITAM motifs of the receptor are diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2 domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C(12)-chain. In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is convenient for combining different cargos with different carriers and has the advantage that the cargo and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the mediator release up to 140%, indicating that ITAM mimics may have the ability to activate non-receptor bound Syk.
Asunto(s)
Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Mastocitos/química , Mastocitos/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Supervivencia Celular , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Ratas , Quinasa SykRESUMEN
Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether MTL can be avoided by experimental design. In this framework also rules are presented to convert SPR signals (RU or millidegree) to mass/surface unit. The chapter concludes with an overview of commercially available SPR equipment.
Asunto(s)
Resonancia por Plasmón de Superficie/métodos , Cinética , Metales/química , Física , Resonancia por Plasmón de Superficie/instrumentación , Propiedades de Superficie , TemperaturaRESUMEN
Surface plasmon resonance (SPR) is one of the leading tools in biomedical research. The challenge in its use is the controlled positioning of one of the components of an interaction on a carefully designed surface. Many attempts in interaction analysis fail due to the non-functional or unsuccessful immobilization of a reactant onto the complex matrix of that surface. The most common technique for linking ligands covalently to a hydrophilic solid surface is amine coupling via reactive esters. In this chapter detailed methods and problem discussions will be given to assist in fast decision analysis to optimize immobilization and regeneration. Topics in focus are different coupling techniques for small and large molecules, streptavidin-biotin sandwich immobilization, and optimizing regeneration conditions.
Asunto(s)
Aminas/química , Carbodiimidas/química , Succinimidas/química , Resonancia por Plasmón de Superficie/métodos , Aldehídos/química , Aminación , Biotina/química , Proteínas Inmovilizadas/análisis , Proteínas Inmovilizadas/química , Oxidación-Reducción , Estreptavidina/química , Propiedades de SuperficieRESUMEN
The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Cinética , Ligandos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores de IgE/química , Espectrometría de Masa por Ionización de Electrospray , Resonancia por Plasmón de Superficie , Quinasa Syk , Termodinámica , Dominios Homologos srcRESUMEN
Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding.
Asunto(s)
Precursores Enzimáticos/química , Proteínas Tirosina Quinasas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bazo/enzimología , Dominios Homologos src , Humanos , Péptidos y Proteínas de Señalización Intracelular , Modelos Químicos , Fosforilación , Conformación Proteica , Quinasa SykRESUMEN
Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src- and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Diseño de Fármacos , Proteína Oncogénica pp60(v-src)/química , Fosfopéptidos/química , Resonancia por Plasmón de Superficie , Dominios Homologos src , Proteína Adaptadora GRB2 , Cinética , Modelos Moleculares , Oligopéptidos/química , Unión Proteica , TermodinámicaRESUMEN
Growth factor receptor-bound protein 2 (Grb2) is an extensively studied adaptor protein involved in cell signaling. Grb2 is a highly flexible protein composed of a single SH2 domain flanked by two SH3 domains. Here we report on the structural dynamic effects upon interaction of a phosphopeptide ligand derived from the recognition sequence of the Shc adaptor protein with (i) the isolated SH2 domain of Grb2 (Grb2 SH2) and (ii) the full-length Grb2 protein. From kinetic studies using surface plasmon resonance, it was deduced that a conformation change occurred in the SH2 protein as well as the full-length Grb2 after binding. Measurements of hydrogen/deuterium exchange (HDX) in the isolated SH2 domain and full-length Grb2 protein as monitored by electrospray mass spectrometry, showed that binding reduces the overall flexibility of the proteins, possibly via slightly different mechanisms for the single SH2 domain and the full-length Grb2 protein.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Fosfotirosina/metabolismo , Proteínas/química , Proteínas/metabolismo , Dominios Homologos src , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Medición de Intercambio de Deuterio , Proteína Adaptadora GRB2 , Cinética , Ligandos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Adaptadoras de la Señalización Shc , Espectrometría de Masa por Ionización de Electrospray , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Resonancia por Plasmón de SuperficieRESUMEN
A library of pentapeptides containing the sequence -Y-X-Y- based on rational design was screened with six different lectins. Sequences were identified that modulate galectin binding to its natural carbohydrate ligand. SPR showed inhibition values 2-3 times stronger than galactose and NMR studies suggested real carbohydrate mimicry.
Asunto(s)
Galactosa/metabolismo , Lectinas/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Galactosa/química , Lectinas/química , Ligandos , Datos de Secuencia Molecular , Oligopéptidos/química , Unión Proteica/fisiologíaRESUMEN
Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Diseño de Fármacos , Fosfopéptidos/síntesis química , Fosfopéptidos/farmacología , Proteínas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Dominios Homologos src , Ciclización , Proteína Adaptadora GRB2 , Modelos Moleculares , Estructura Molecular , Fosfopéptidos/química , Fosforilación/efectos de los fármacos , Unión Proteica , Conformación Proteica , Proteínas/metabolismo , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity divalent constructs that can successfully interfere with crucial protein-protein interactions.
Asunto(s)
Alquinos/síntesis química , Benzoatos/síntesis química , Dominios Homologos src/efectos de los fármacos , Unión Competitiva , Diseño de Fármacos , Indicadores y Reactivos , Ligandos , Resonancia por Plasmón de SuperficieRESUMEN
The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56(lck) was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pK(a) shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the -2 to the -1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pK(a) value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the -2 state the binding energy is 1.8 kcal/mol more favourable than for the -1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.