RESUMEN
Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed "AggScore" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.
Asunto(s)
Modelos Biológicos , Agregado de Proteínas , Proteínas/química , Algoritmos , Péptidos beta-Amiloides/química , Anticuerpos/química , Hormona del Crecimiento/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica , Solubilidad , Electricidad EstáticaRESUMEN
Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).
Asunto(s)
Alanina/metabolismo , Anticuerpos Monoclonales/metabolismo , Mapeo Epitopo/métodos , Epítopos/metabolismo , Interleucina-23/metabolismo , Reacciones Antígeno-Anticuerpo , Clonación Molecular , Medición de Intercambio de Deuterio , Fragmentos Fab de Inmunoglobulinas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Mutagénesis , Oxidación-Reducción , Unión ProteicaRESUMEN
Chemical modifications can potentially change monoclonal antibody's (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen-deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody-antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.
Asunto(s)
Anticuerpos Monoclonales/química , Medición de Intercambio de Deuterio , Espectrometría de Masas , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Ácido Aspártico/química , Células CHO , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/metabolismo , Cricetinae , Cricetulus , Deuterio/química , Ensayo de Inmunoadsorción Enzimática , Hidrógeno/química , Isomerismo , Cinética , Metionina/química , Oxidación-Reducción , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.
Asunto(s)
Biotecnología , Disulfuros/química , Péptidos/química , Péptidos/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Modelos Moleculares , Péptidos/genética , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
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
A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.
Asunto(s)
Cromatografía Liquida/métodos , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Células CHO , Cricetulus , Fragmentos Fc de Inmunoglobulinas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein-protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal ß-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.
Asunto(s)
Amidas/química , Deuterio/química , Hidrógeno/química , Unión Proteica , Secuencia de Aminoácidos , Enlace de Hidrógeno , Modelos Moleculares , Proteínas/química , SolubilidadRESUMEN
Glucokinase (GK) is a key regulator of glucose homeostasis, and its small-molecule activators represent a promising opportunity for the treatment of type 2 diabetes. Several GK activators have been advanced into clinical trials and have demonstrated promising efficacy; however, hypoglycemia represents a key risk for this mechanism. In an effort to mitigate this hypoglycemia risk while maintaining the efficacy of the GK mechanism, we have investigated a series of amino heteroaryl phosphonate benzamides as ''partial" GK activators. The structure-activity relationship studies starting from a "full GK activator" 11, which culminated in the discovery of the "partial GK activator" 31 (BMS-820132), are discussed. The synthesis and in vitro and in vivo preclinical pharmacology profiles of 31 and its pharmacokinetics (PK) are described. Based on its promising in vivo efficacy and preclinical ADME and safety profiles, 31 was advanced into human clinical trials.
Asunto(s)
Azetidinas , Diabetes Mellitus Tipo 2 , Hipoglucemia , Organofosfonatos , Azetidinas/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucoquinasa , Humanos , Hipoglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Organofosfonatos/farmacología , Organofosfonatos/uso terapéuticoRESUMEN
We describe the proceedings and conclusions from the "Workshop on Applications of Protein Models in Biomedical Research" (the Workshop) that was held at the University of California, San Francisco on 11 and 12 July, 2008. At the Workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) the requirements and challenges for different applications, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.
Asunto(s)
Investigación Biomédica/métodos , Modelos Moleculares , Proteínas/química , Animales , Investigación Biomédica/tendencias , Química Farmacéutica/métodos , Bases de Datos de Proteínas , Descubrimiento de Drogas/métodos , Enzimas/química , Directrices para la Planificación en Salud , Humanos , Conformación Proteica , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Programas InformáticosRESUMEN
The success of structure-based drug design relies on accurate protein modeling where one of the key issues is the modeling and refinement of loops. This study takes a critical look at modeled loops, determining the effect of re-sampling side-chains after the loop conformation has been generated. The results are evaluated in terms of backbone and side-chain conformations with respect to the native loop. While models can contain loops with high quality backbone conformations, the side-chain orientations could be poor, and therefore unsuitable for ligand docking and structure-based design. In this study, we report on the ability to model loop side-chains accurately using a variety of commercially available algorithms that include rotamer libraries, systematic torsion scans and knowledge-based methods.
Asunto(s)
Algoritmos , Proteínas/química , Homología Estructural de Proteína , Simulación por Computador , Diseño de Fármacos , Modelos MolecularesRESUMEN
We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Triazoles/farmacología , Animales , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Humanos , Proteínas RGS/genética , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
A novel series of [2.2.1]-oxabicyclo imide-based compounds were identified as potent antagonists of the androgen receptor. Molecular modeling and iterative drug design were applied to optimize this series. The lead compound [3aS-(3aalpha,4beta,5beta,7beta,7aalpha)]-4-(octahydro-5-hydroxy-4,7-dimethyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl)-2-iodobenzonitrile was shown to have potent in vivo efficacy after oral dosing in the CWR22 human prostate tumor xenograph model.
Asunto(s)
Antagonistas de Receptores Androgénicos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Isoindoles/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Administración Oral , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Humanos , Isoindoles/síntesis química , Isoindoles/farmacocinética , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Nitrilos/farmacología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Unión Proteica , Receptores Androgénicos/metabolismo , Relación Estructura-Actividad , Compuestos de Tosilo/farmacología , Células Tumorales CultivadasRESUMEN
Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored. On the distal aryl group, a number of substitutions are well tolerated. A crystal structure was obtained for a complex between 11beta-HSD1 and the most potent inhibitor in this series.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Amidas/síntesis química , Amidas/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Amidas/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Conformación Molecular , Estructura Molecular , Piridinas/química , Relación Estructura-ActividadRESUMEN
TL1A, a tumor necrosis factor-like cytokine, is a ligand for the death domain receptor DR3. TL1A, upon binding to DR3, can stimulate lymphocytes and trigger secretion of proinflammatory cytokines. Therefore, blockade of TL1A/DR3 interaction may be a potential therapeutic strategy for autoimmune and inflammatory diseases. Recently, the anti-TL1A monoclonal antibody 1 (mAb1) with a strong potency in blocking the TL1A/DR3 interaction was identified. Here, we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to obtain molecular-level details of mAb1's binding epitope on TL1A. HDX coupled with electron-transfer dissociation MS provided residue-level epitope information. The HDX dataset, in combination with solvent accessible surface area (SASA) analysis and computational modeling, revealed a discontinuous epitope within the predicted interaction interface of TL1A and DR3. The epitope regions span a distance within the approximate size of the variable domains of mAb1's heavy and light chains, indicating it uses a unique mechanism of action to block the TL1A/DR3 interaction.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Medición de Intercambio de Deuterio/métodos , Mapeo Epitopo/métodos , Epítopos/inmunología , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Células CHO , Cricetulus , Humanos , Cinética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Miembro 25 de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunologíaRESUMEN
This paper provides an unbiased comparison of four commercially available programs for loop sampling, Prime, Modeler, ICM, and Sybyl, each of which uses a different modeling protocol. The study assesses the quality of results and examines the relative strengths and weaknesses of each method. The set of loops to be modeled varied in length from 4-12 amino acids. The approaches used for loop modeling can be classified into two methodologies: ab initio loop generation (Modeler and Prime) and database searches (Sybyl and ICM). Comparison of the modeled loops to the native structures was used to determine the accuracy of each method. All of the protocols returned similar results for short loop lengths (four to six residues), but as loop length increased, the quality of the results varied among the programs. Prime generated loops with RMSDs <2.5 A for loops up to 10 residues, while the other three methods met the 2.5 A criteria at seven-residue loops. Additionally, the ability of the software to utilize disulfide bonds and X-ray crystal packing influenced the quality of the results. In the final analysis, the top-ranking loop from each program was rarely the loop with the lowest RMSD with respect to the native template, revealing a weakness in all programs to correctly rank the modeled loops.
Asunto(s)
Modelos Moleculares , Oligopéptidos/química , Estructura Secundaria de Proteína , Programas Informáticos , Algoritmos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Bases de Datos de Proteínas , Disulfuros/química , Hidrógeno/química , Concentración de Iones de Hidrógeno , Conformación ProteicaRESUMEN
A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS-564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, including several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS-564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window.
Asunto(s)
Imidazoles/síntesis química , Imidazoles/farmacología , Músculo Esquelético/efectos de los fármacos , Próstata/efectos de los fármacos , Pirroles/síntesis química , Pirroles/farmacología , Receptores Androgénicos/metabolismo , Testosterona/análogos & derivados , Envejecimiento/metabolismo , Animales , Aromatasa/metabolismo , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , Dihidrotestosterona/química , Dihidrotestosterona/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Imidazoles/metabolismo , Ligandos , Hormona Luteinizante/sangre , Masculino , Músculo Esquelético/fisiología , Orquiectomía , Próstata/fisiología , Estructura Terciaria de Proteína , Pirroles/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/química , Receptores Androgénicos/genética , Testosterona/química , Testosterona/metabolismo , Transcripción Genética/fisiologíaRESUMEN
A novel series of imidazolin-2-ones were designed and synthesized as highly potent, orally active and muscle selective androgen receptor modulators (SARMs), with most of the compounds exhibiting low nM in vitro potency in androgen receptor (AR) binding and functional assays. Once daily oral treatment with the lead compound 11a (AR Ki = 0.9 nM, EC50 = 1.8 nM) for 14 days induced muscle growth with an ED50 of 0.09 mg/kg, providing approximately 50-fold selectivity over prostate growth in an orchidectomized rat model. Pharmacokinetic studies in rats demonstrated that the lead compound 11a had oral bioavailability of 65% and a plasma half-life of 5.5 h. On the basis of their preclinical profiles, the SARMs in this series are expected to provide beneficial anabolic effects on muscle with minimal androgenic effects on prostate tissue.
Asunto(s)
Anabolizantes/síntesis química , Imidazoles/síntesis química , Músculo Esquelético/efectos de los fármacos , Pirroles/síntesis química , Receptores Androgénicos/metabolismo , Administración Oral , Anabolizantes/farmacocinética , Anabolizantes/farmacología , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Semivida , Imidazoles/farmacocinética , Imidazoles/farmacología , Masculino , Modelos Moleculares , Músculo Esquelético/anatomía & histología , Orquiectomía , Próstata/anatomía & histología , Próstata/efectos de los fármacos , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Variants of monoclonal antibody containing an extra light chain have been reported in protein products. Due to potential impact on potency and immunogenicity, it is important to understand the formation mechanism of such variants so that appropriate control strategies can be implemented to assure product quality. In a model monoclonal antibody, we observed a size variant with an extra light chain noncovalently associated with the monomer (later named as "1.2mer"). The interaction between monomer and the extra light chain was characterized by native spray and hydrogen-deuterium exchange mass spectrometry techniques. The goal is to understand the nature of the noncovalent interaction, to map out the interaction interface and regions of potential conformational distortions. In addition, computational modeling was used to aid in binding site identification. The combined results identify the interaction interface to be located in the heavy chain region 38-57 and in the extra light chain region 30-50. To the best of our knowledge, this study is the first to characterize noncovalent interaction of a size variant comprising an antibody monomer and an extra light chain. Structural knowledge generated in this research work is invaluable for process development and construct design of antibody-based biopharmaceuticals.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Espectrometría de Masas/métodos , Animales , Sitios de Unión de Anticuerpos , Células CHO , Cromatografía en Gel , Cricetulus , Deuterio/análisis , Medición de Intercambio de Deuterio/métodos , Humanos , Hidrógeno/análisis , Modelos Moleculares , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Homología Estructural de ProteínaRESUMEN
Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.Graphical Abstract.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Espectrometría de Masas/métodos , Agregado de Proteínas , Medición de Intercambio de Deuterio/métodos , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
A new crystal structure of the pituitary glycoprotein human follicle-stimulating hormone (hFSH) in complex with a fragment of the human FSH receptor (hFSHR(1-250)) ectodomain has been determined. The hFSH receptor is a Class A rhodopsin-like G-protein-coupled receptor (GPCR). The structure serves as a template for the modeling of the related luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) receptors and explains why these hormones do not cross-react with the FSH receptor. Unexpectedly, the FSH receptor ectodomain in complex with FSH is a dimer but, unlike other cysteine-knot cytokines, FSH does not participate in a receptor-receptor tether. A host of biochemical data can now be placed in the context of the new structure, and will provide new insights that could lead to the development of small-molecule contraceptives and more-active or bifunctional versions of hormones for FSH receptor and other related receptors.