RESUMO
The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Biotecnologia/métodos , Cristalização/métodos , Imunoglobulina G/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Técnicas de Cultura de Células , Simulação por Computador , Cricetinae , Cricetulus , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Modelos Moleculares , Temperatura , TrometaminaRESUMO
Iclaprim is a novel dihydrofolate reductase (DHFR) inhibitor belonging to the 2,4-diaminopyrimidine class of antibiotics, of which trimethoprim (TMP) is the most well known representative. Iclaprim exhibits potent bactericidal activity against major Gram-positive pathogens, notably methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) phenotypes, including TMP-resistant strains. The inhibition properties of racemic iclaprim and of the two enantiomers, termed AR-101 and AR-102, towards S. aureus wild-type DHFR and TMP-resistant F98Y mutant DHFR were determined and compared. Similar to TMP, AR-101, AR-102 and iclaprim are all competitive inhibitors with respect to the substrate dihydrofolate. Iclaprim, AR-101 and AR-102 demonstrated little or no difference in activity towards these enzymes and were significantly more potent than TMP. The crystal structures of S. aureus DHFR and F98Y mutant DHFR were determined as ternary complexes with NADPH and either AR-101, AR-102 or iclaprim. The binding modes of the inhibitors were analysed and compared. The X-ray crystallographic data explain the binding modes of all molecules well and can be used to rationalize the equipotent affinity of AR-101, AR-102 and iclaprim, which is also reflected in their antibacterial properties.
Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Staphylococcus aureus Resistente à Meticilina/enzimologia , Proteínas Mutantes/metabolismo , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/química , Antibacterianos/metabolismo , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Isomerismo , Resistência a Meticilina , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , NADP/química , NADP/metabolismo , Estrutura Terciária de Proteína , Pirimidinas/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/metabolismoRESUMO
OBJECTIVES: Iclaprim is a novel 2,4-diaminopyrimidine that exhibits potent, rapid bactericidal activity against major Gram-positive pathogens, including methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus, and is currently in clinical development for the treatment of complicated skin and skin structure infections. An understanding of the known mechanism of resistance to trimethoprim led to the design of this new inhibitor, with improved affinity towards dihydrofolate reductase (DHFR) from S. aureus and clinically useful activity against S. aureus including isolates resistant to trimethoprim. The objective of this study was to characterize the mode of action of iclaprim and its inhibitory properties against DHFR. METHODS: The mode of action of iclaprim was assessed by enzymatic analysis, direct binding studies, macromolecular synthesis profiles, synergy and antagonism studies to define its role as an inhibitor of DHFR. The binding properties of iclaprim to DHFR were compared with those of trimethoprim by X-ray crystallography. RESULTS: The enzymatic properties, direct binding and X-ray crystallographic studies delineated the mode of interaction with DHFR and the reason for the increased affinity of iclaprim towards the enzyme. The effect of iclaprim on bacterial physiology suggests that iclaprim behaves as a classical antibacterial DHFR inhibitor, as previously documented for trimethoprim. CONCLUSIONS: Iclaprim binds and inhibits bacterial DHFR in a similar manner to trimethoprim. However, the increased hydrophobic interactions between iclaprim and DHFR account for increased affinity and, unlike trimethoprim, enable iclaprim to inhibit even the resistant enzyme with nanomolar affinity, thus overcoming the mechanism of trimethoprim resistance. The increased antibacterial activity and lower propensity for resistance make iclaprim a clinically promising and useful inhibitor.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Antibacterianos/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Pirimidinas/metabolismo , Trimetoprima/metabolismo , Trimetoprima/farmacologiaRESUMO
OBJECTIVE: The serine protease MT-SP1/matriptase plays an important role in cell migration and matrix degradation. Hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and protease-activated receptor 2 (PAR-2) have been identified as in vitro substrates of MT-SP1/matriptase. Because PAR-2 is expressed in endothelial cells and contributes to inflammatory processes, we sought to investigate the effects of MT-SP1/matriptase on endothelial cytokine expression and analyzed MT-SP1/matriptase expression in vascular cells and atherosclerotic lesions. METHODS AND RESULTS: In endothelial cells, recombinant MT-SP1/matriptase dose-dependently induced interleukin (IL)-8 and IL-6 mRNA and protein expression dependent on its proteolytic activity. MT-SP1/matriptase time-dependently induced phosphorylation of p38 MAPK and p42/44 MAPK. Inhibitor experiments revealed that p38 MAPK and PKCalpha were necessary for IL-8 induction. PAR-2 downregulation abolished and PAR-2 overexpression augmented MT-SP1/matriptase-induced IL-8 expression as evidence for PAR-2 signaling. In human atherectomies, MT-SP1/matriptase was expressed in blood cells adherent to the endothelium. Concordantly, basal MT-SP1/matriptase expression was detected in isolated monocytes. Coincubation of monocytes and endothelial cells resulted in an increased IL-8 release, which was reduced after downregulation of endothelial PAR-2 and monocytic MT-SP1/matriptase. CONCLUSION: MT-SP1/matriptase induces release of proinflammatory cytokines in endothelial cells through activation of PAR-2. MT-SP1/matriptase is expressed in monocytes, thus, interaction of monocytic MT-SP1/matriptase with endothelial PAR-2 may contribute to atherosclerosis.
Assuntos
Células Endoteliais/metabolismo , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Receptor PAR-2/metabolismo , Serina Endopeptidases/farmacologia , Aterosclerose/enzimologia , Aterosclerose/etiologia , Catálise , Células Cultivadas , Citocinas/biossíntese , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Humanos , Interleucina-6/genética , Interleucina-8/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C-alfa/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/sangue , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 A resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 A crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.
Assuntos
Aldose-Cetose Isomerases/química , Antimaláricos/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Complexos Multienzimáticos/química , NADP/química , Oxirredutases/química , Conformação Proteica , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Antimaláricos/metabolismo , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fosfomicina/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação ProteicaRESUMO
Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.
Assuntos
Aminopeptidases/química , Inibidores Enzimáticos/química , Staphylococcus aureus/enzimologia , Triazóis/química , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Apoenzimas , Sítios de Ligação , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares , Metionil Aminopeptidases , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
High-resolution crystal structures of Staphylococcus aureus methionine aminopeptidase I in complex with various keto heterocycles and aminoketones were determined, and the intermolecular ligand interactions with the enzyme are reported. The compounds are effective inhibitors of the S. aureus enzyme because of the formation of an uncleavable tetrahedral intermediate upon binding. The electron densities unequivocally show the enzyme-catalyzed transition-state analogue mimicking that for amide bond hydrolysis of substrates.
Assuntos
Aminas/química , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/química , Cetonas/química , Piridinas/química , Staphylococcus aureus/enzimologia , Tiazóis/química , Sítios de Ligação , Cristalografia por Raios X , Ciclopropanos/química , Metionil Aminopeptidases , Modelos Moleculares , Estrutura MolecularRESUMO
Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation.
Assuntos
Anticorpos/isolamento & purificação , Etanol/química , Animais , Células CHO , Cromatografia em Gel , Cromatografia por Troca Iônica , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Proteínas Recombinantes/isolamento & purificação , Proteína Estafilocócica A/químicaRESUMO
Endothelin-converting enzyme I (ECE-1) is a mammalian type II integral membrane zinc-containing endopeptidase. ECE-1 catalyzes the final step in the biosynthesis of endothelins in a rate-limiting fashion, through post-translational conversion of the biologically inactive big endothelins. Endothelin-1 overproduction has been implicated in a heterogeneous list of diseases including systemic and pulmonary hypertension, stroke and asthma, cardiac and renal failure. Therefore, ECE-1 is a prime therapeutic target for the regulation of endothelin-1 production in vivo and there is considerable interest in selective inhibitors of this enzyme. Here, we present the crystal structure of the extracellular domain (residues 90-770) of human ECE-1 (C428S) with the generic metalloprotease inhibitor phosphoramidon determined at 2.38 A resolution. The structure is closely related to that of human NEP, providing essential information for a detailed understanding of ligand-binding, specificity determinants as well as selectivity criteria. Selective inhibitors of ECE-1s should have beneficial effects for the treatment of diseases in which an overproduction of ETs plays a pathogenic role.
Assuntos
Ácido Aspártico Endopeptidases/química , Glicopeptídeos/química , Metaloendopeptidases/química , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Enzimas Conversoras de Endotelina , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neprilisina/química , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato/efeitos dos fármacos , Propriedades de Superfície/efeitos dos fármacos , Zinco/metabolismoRESUMO
Neutral endopeptidase (NEP) is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor, as well as the incretin hormone glucagon-like peptide 1 (GLP-1), which is a potent stimulator of insulin secretion. The activity of GLP-1 is also rapidly abolished by the serine protease dipeptidyl peptidase IV (DPP-IV), which led to an elevated interest in inhibitors of this enzyme for the treatment of type II diabetes. A dual NEP/DPP-IV inhibitor concept is proposed, offering an alternative strategy for the treatment of type 2 diabetes. Here, the synthesis and crystal structures of the soluble extracellular domain of human NEP (residues 52-749) complexed with the NEP, competitive and potent dual NEP/DPP-IV inhibitor MCB3937 are described.
Assuntos
Inibidores de Adenosina Desaminase , Inibidores da Dipeptidil Peptidase IV , Glicoproteínas/antagonistas & inibidores , Neprilisina/antagonistas & inibidores , Neprilisina/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Adenosina Desaminase/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glicoproteínas/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Neprilisina/química , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Zinco/químicaRESUMO
Malonyl-CoA-acyl carrier protein transacylase (FabD; EC 2.3.1.39) is a key enzyme in the fatty-acid biosynthesis pathway of bacteria, catalyzing the transfer of a malonyl moiety from malonyl-CoA to holo acyl carrier protein (ACP), generating malonyl-ACP and free CoASH. Malonyl-ACP, which is the product of this reaction, is the key building block for de novo fatty-acid biosynthesis. Various binary complex structures of the Escherichia coli enzyme are presented, including that of the natural substrate malonyl-CoA, indicating the functional role of the highly conserved amino acids Gln11, Ser92, Arg117 and His201 and the stabilizing function of the preformed oxyanion hole during the enzymatic reaction. Based on the presented structural data, a possible new catalytic enzyme mechanism is discussed. The data obtained could be used in aiding the process of rational inhibitor design.
Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Sítios de Ligação , Catálise , Cristalografia por Raios X , Ácido Graxo Sintase Tipo II , Malonil Coenzima A/química , Modelos Químicos , Especificidade por SubstratoRESUMO
CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome c maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.