RESUMEN
A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.
Asunto(s)
Mapeo de Interacción de Proteínas/normas , Secuencia de Aminoácidos , Área Bajo la Curva , Proteínas Bacterianas/química , Endorribonucleasas/química , Enzimas Inmovilizadas/química , Estudios de Evaluación como Asunto , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Estándares de Referencia , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3â Å resolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.
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Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/química , Secuencia de Aminoácidos , Calorimetría , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/aislamiento & purificación , Ligando Inductor de Apoptosis Relacionado con TNF/aislamiento & purificación , TermodinámicaRESUMEN
A biological solids reduction process that uses a side-stream anaerobic reactor (Cannibal process) was investigated to better understand why variations in solids reduction occur in field installations. Field observations indicated that single stage, high sludge age processes would frequently not provide a low solids yield. To study this, laboratory sequencing batch reactors were operated, one as a conventional activated sludge system and the other two with a side-stream anaerobic reactors. The control and one anaerobic side-stream system were operated with a 5-minute feed cycle to mimic a plug flow system. The other anaerobic side-stream system operated with a 4-hour feed cycle to simulate complete mix. The 5 minutes feed Cannibal system generated up to 80%. less biological solids than the control. The 4 hours feed Cannibal system produced less biological solids than the conventional activated sludge system but remained higher than for system with a 5-minute feed time.
Asunto(s)
Reactores Biológicos , Eliminación de Residuos Líquidos/métodos , Anaerobiosis , Bacterias/metabolismo , Filtración , Hierro , Oxígeno/química , Oxígeno/metabolismo , Factores de TiempoRESUMEN
IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.
Asunto(s)
Terapia Biológica , Deuterio/química , Hidrógeno/química , Interleucina-23/química , Biología Computacional , Humanos , Interleucina-23/metabolismo , Espectrometría de Masas/métodos , Unión Proteica , Conformación Proteica , Receptores de Interleucina/químicaRESUMEN
The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.á
Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Receptores ErbB/antagonistas & inhibidores , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.
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Sulfato de Amonio/química , Anticuerpos/química , Animales , Línea Celular , Descubrimiento de Drogas , Humanos , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , SolubilidadRESUMEN
Solids retention time (SRT) is a critical parameter for the performance of anaerobic digesters (AD) in wastewater treatment plants. AD SRT should increase when active biomass is input to the AD by recycling anaerobic sludge via the wastewater-treatment tanks, creating a hybrid aerobic/anaerobic system. When 85% of the flow through the AD was recycled in pilot-scale hybrid systems, the AD SRT increased by as much as 9-fold, compared to a parallel system without anaerobic-sludge recycle. Longer AD SRTs resulted in increased hydrolysis and methanogenesis in the AD: net solids yield decreased by 39-96% for overall and 23-94% in the AD alone, and AD methane yield increased 1.5- to 5.5-fold. Microbial community assays demonstrated higher, more consistent Archaea concentrations in all tanks in the wastewater-treatment system with anaerobic-sludge recycle. Thus, multiple lines of evidence support that AD-sludge recycle increased AD SRT, solids hydrolysis, and methane generation.
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Bacterias Anaerobias/fisiología , Reactores Biológicos/microbiología , Aguas del Alcantarillado/microbiología , Diseño de Equipo , Análisis de Falla de Equipo , Proyectos PilotoRESUMEN
The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.
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Calorimetría/estadística & datos numéricos , Resonancia por Plasmón de Superficie/estadística & datos numéricos , Industria Farmacéutica , Unión Proteica , Mapeo de Interacción de Proteínas/estadística & datos numéricos , Encuestas y Cuestionarios , Termodinámica , VolumetríaRESUMEN
Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.
Asunto(s)
Benchmarking , Mapeo de Interacción de Proteínas/normas , Resonancia por Plasmón de Superficie/normas , Proteínas Bacterianas/química , Unión Competitiva , Humanos , Proteínas Inmovilizadas/química , Interferometría/normas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Estándares de Referencia , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/química , Espectrometría de Masa por Ionización de Electrospray/normasRESUMEN
OBJECTIVES: Drug-excipient binding can affect in-vitro drug release. Literature suggests that drug-excipient ionic binding interaction that is not disrupted by physiological salt concentration in the dissolution medium can impact a drug's oral bioavailability. We investigated whether nondisruption of interaction by physiological salt concentration was an adequate predictor of its biorelevance using the binding of a model amine high dose drug brivanib alaninate (BA) to croscarmellose sodium (CCS) as an example. METHODS: BA was formulated into an immediate release tablet using CCS as disintegrant by a wet granulation process. In-vitro drug release was carried out as a function of pH and buffer concentration of the medium. BA-CCS binding was studied in buffer solution and data fitted to a Langmuir isotherm. A simulation model and an isothermal titration calorimetry method were developed to assess the bioavailability risk and strength of drug-excipient binding interaction, independent of physiological salt concentration consideration. KEY FINDINGS: BA-CCS binding was pH-dependent, reversible, ionic, and not disrupted by increasing the buffer concentration in the dissolution medium. Absorption simulation predictions of no effect of CCS binding on BA's bioavailability were confirmed by a monkey pharmacokinetic study. CONCLUSIONS: A pH-dependent and reversible weak drug-excipient binding interaction is unlikely to affect the oral bioavailability of high dose drugs.
Asunto(s)
Alanina/análogos & derivados , Carboximetilcelulosa de Sodio/química , Excipientes/química , Triazinas/farmacocinética , Administración Oral , Alanina/administración & dosificación , Alanina/química , Alanina/farmacocinética , Animales , Disponibilidad Biológica , Tampones (Química) , Calorimetría/métodos , Interacciones Farmacológicas , Concentración de Iones de Hidrógeno , Macaca fascicularis , Masculino , Modelos Biológicos , Comprimidos , Triazinas/administración & dosificación , Triazinas/químicaRESUMEN
Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 µM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Fibronectinas/química , Fragmentos de Péptidos/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Receptores ErbB/metabolismo , Femenino , Humanos , Immunoblotting , Cinética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Panitumumab , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The protein therapeutics market is one of the highest growing segments of the pharmaceutical industry with an estimated global market value of $77 billion by 2011 (Global Protein Therapeutics Market report by RNCOS: Delhi, India, 2009). This growth has been fueled by several advantages that protein drugs can offer such as higher specificity, reduced side effects, and faster development time compared to small molecule drugs. Major pharmaceutical companies are strategically shifting gears toward protein therapeutics and gradually increasing the biologics portion of their pipelines. Consequently, in the present pharmaceutical industry, there is a rapid growth in the number and types of protein structural mass spectrometry analyses, particularly during the discovery phase where an abundance of new drug candidates are being investigated. This perspective article discusses the role of protein structural mass spectrometry during the discovery of protein therapeutics with focus on recombinant protein production quality control and structural biology applications. The current challenges in technologies associated with this field and the analytical prospects for the future direction will be also discussed.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas Recombinantes/química , Evaluación Preclínica de Medicamentos , Glicosilación , Espectrometría de Masas/normas , Peso Molecular , Estructura Terciaria de Proteína , Control de Calidad , Proteínas Recombinantes/genética , Proteínas Recombinantes/normasRESUMEN
Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed. Here we demonstrate that mass spectrometry analysis of the time course of limited proteolysis reactions provides new information that is self-sufficient to identify all proteolytic fragments. The method uses a non-specific protease like subtilisin and exploits information contained in the time-resolved dataset such as: increased likelihood of identifying larger fragments generated during initial proteolysis solely by their masses, additivity of the masses of two mutually exclusive sequence regions that generate the full-length molecule (or an already assigned subfragment), and analyses of the proteolytic subfragment patterns that are facilitated by having established the initial cleavage sites. We show that the identities of the observed proteolytic fragments can be determined by LC/MS alone because enough constraints exist in the time-resolved dataset. For a medium-sized protein, it takes about 8 h to complete the study, a significant improvement over the traditional SDS-PAGE and N-terminal sequencing method, which usually takes several days. We illustrate this method with application to the catalytic domain of mitogen-activated protein kinase-activated protein kinase-2, and compare the results with N-terminal sequencing data and the known X-ray crystal structure.
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Cromatografía Líquida de Alta Presión/métodos , Proteína Quinasa 1 Activada por Mitógenos/química , Péptido Hidrolasas/química , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Activación Enzimática , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2' (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Oligopéptidos/farmacología , Piridinas/farmacología , Sustitución de Aminoácidos , Sulfato de Atazanavir , Unión Competitiva/efectos de los fármacos , Rastreo Diferencial de Calorimetría , Catálisis , Farmacorresistencia Viral , Proteasa del VIH/química , Calor , Modelos Moleculares , Modelos Estructurales , TemperaturaRESUMEN
CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.
Asunto(s)
Proteínas Bacterianas/química , Metiltransferasas/química , Streptococcus pneumoniae/enzimología , Homología Estructural de Proteína , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
Asunto(s)
Proteínas Bacterianas/fisiología , Genes Esenciales/fisiología , Azúcares de Nucleósido Difosfato/metabolismo , Streptococcus pneumoniae/genética , Transaminasas/fisiología , Secuencia de Aminoácidos , Benzopiranos/metabolismo , Dimerización , Furanos/metabolismo , Ligandos , Datos de Secuencia Molecular , Fosfato de Piridoxal/metabolismo , Piridoxamina/metabolismo , Streptococcus pneumoniae/enzimologíaRESUMEN
Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.
Asunto(s)
Receptores de Factor Estimulante de Colonias de Granulocito/química , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Animales , Bencimidazoles/farmacología , Células de la Médula Ósea/metabolismo , Calorimetría , Línea Celular , Dicroismo Circular , Citocinas/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Ácido Edético/farmacología , Guanidinas/farmacología , Iones , Ligandos , Ratones , Modelos Químicos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Ultracentrifugación , ZincRESUMEN
The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.
Asunto(s)
Entropía , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/química , VIH-1/inmunología , Receptores del VIH/metabolismo , Afinidad de Anticuerpos , Sitios de Unión , Antígenos CD4/química , Antígenos CD4/metabolismo , Calorimetría , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Modelos Moleculares , Pruebas de Neutralización , Conformación Proteica , Receptores del VIH/químicaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.