RESUMO
BACKGROUND: Biosimilars provide safety, purity, and potency similar to those of a reference biologic product. METHODS: An array of protein analytical techniques was used to compare the physicochemical properties of proposed biosimilar filgrastim (EP2006), US-approved originator filgrastim, and EU-approved originator filgrastim. Biological characterization involved surface plasmon resonance spectroscopy analyses and in vitro proliferation assays. A randomized, double-blind, two-way crossover, phase I study in healthy volunteers assessed the pharmacodynamics, pharmacokinetics, and safety profiles of EP2006 and US-approved originator filgrastim (administered as a single subcutaneous 10 µg/kg injection). RESULTS: EP2006 and originator filgrastim (US and EU approved) were highly similar with respect to primary, secondary, and tertiary protein structures; mass, size, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro bioactivity. In the phase I study, no statistically significant differences between EP2006 and US-approved originator filgrastim were noted in pharmacodynamic or pharmacokinetic parameters, and all confidence intervals were within the equivalence boundaries. The two products had similar safety profiles. CONCLUSION: These studies provide robust evidence of the structural and functional similarity between the proposed biosimilar filgrastim (EP2006) and the US-approved originator filgrastim.
Assuntos
Medicamentos Biossimilares/farmacologia , Medicamentos Biossimilares/farmacocinética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacocinética , Adolescente , Adulto , Sequência de Aminoácidos , Medicamentos Biossimilares/administração & dosagem , Medicamentos Biossimilares/química , Contagem de Células , Estudos Cross-Over , Método Duplo-Cego , Filgrastim , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/química , Humanos , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Conformação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Adulto JovemRESUMO
Random spin labeling (RSL) is a method for rapid mapping of biomolecular interaction surfaces using an interaction partner with SL and an interaction partner enriched in (13)C or (15)N nuclei for paramagnetic relaxation enhanced NMR-based detection. The SL reaction is conducted in a manner resulting in a heterogeneous reaction product consisting of different populations of the protein carrying a varying number of spin labels at different positions. Preparation of the paramagnetic probe is complete within a few hours and hence much faster than site selective SL. RSL is applicable to tightly interacting systems but shows its particular strength when applied to systems involving weak or transient contacts.
Assuntos
Espectroscopia de Ressonância Magnética , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Ressonância Magnética Nuclear BiomolecularRESUMO
Elongating Escherichia coli RNAP is modulated by NusA protein. The C-terminal domain (CTD) of the RNAP α subunit (αCTD) interacts with the acidic CTD 2 (AR2) of NusA, releasing the autoinhibitory blockade of the NusA S1-KH1-KH2 motif and allowing NusA to bind nascent nut spacer RNA. We determined the solution conformation of the AR2:αCTD complex. The αCTD residues that interface with AR2 are identical to those that recognize UP promoter elements A nusA-ΔAR2 mutation does not affect UP-dependent rrnH transcription initiation in vivo. Instead, the mutation inhibits Rho-dependent transcription termination at phage λtR1, which lies adjacent to the λnutR sequence. The Rho-dependent λtimm terminator, which is not preceded by a λnut sequence, is fully functional. We propose that constitutive binding of NusA-ΔAR2 to λnutR occludes Rho. In addition, the mutation confers a dominant defect in exiting stationary phase.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Fatores de Alongamento de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Fator Rho/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Sítios de Ligação/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Plasmídeos , Ligação Proteica/genética , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fator Rho/química , Fator Rho/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Transfecção , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Paramagnetic relaxation enhancement (PRE) has become a useful and widely applied tool in biomolecular NMR spectroscopy. In particular investigations of large complexes or transient contacts benefit from PRE effects. Frequently such studies involve modification of the biomacromolecules under study. We here present a method for editing NMR spectra by utilizing a soluble gadolinium complex that broadens nuclear spins being at or close to the macromolecule-solvent interface. NOE signals in NOESY spectra are selectively attenuated if surface exposed nuclear spins are involved. HSQC-type spectra with paramagnetic agent contain only signals of the interior of the protein, while the corresponding difference spectra harbor signals allocated to surface spins. Thus, the number of signals can be reduced helping to minimize spectral overlap in large proteins. The method reveals additional information about the localization of spins being helpful for structure determination of large complexes.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Gadolínio DTPA/química , Conformação Proteica , Ubiquitina/químicaRESUMO
The RNA sequences boxA, boxB and boxC constitute the nut regions of phage lambda. They nucleate the formation of a termination-resistant RNA polymerase complex on the lambda chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the lambda N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why lambda requires an additional protein, lambda N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regiões 5' não Traduzidas , Proteínas de Bactérias/química , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , Proteínas de Escherichia coli/química , Genes de RNAr , Dados de Sequência Molecular , Óperon , Fatores de Alongamento de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Fatores de Elongação da TranscriçãoRESUMO
N protein of the Escherichia coli phage lambda (lambdaN) is involved in antitermination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambdaN is an intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambdaN(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambdaN-binding interface, lambdaN(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambdaN(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambdaN(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambdaN peptides showed that only NusA(353-416) is involved in lambdaN binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambdaN(1-53). We also clarified the role of the lambdaN-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambdaN(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambdaN action.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Elongação da TranscriçãoRESUMO
The carboxy-terminal domain of the transcription factor Escherichia coli NusA, NusACTD, interacts with the protein N of bacteriophage lambda, lambdaN, and the carboxyl terminus of the E. coli RNA polymerase alpha subunit, alphaCTD. We solved the solution structure of the unbound NusACTD with high-resolution nuclear magnetic resonance (NMR). Additionally, we investigated the binding sites of lambdaN and alphaCTD on NusACTD using NMR titrations. The solution structure of NusACTD shows two structurally similar subdomains, NusA(353-416) and NusA(431-490), matching approximately two homologous acidic sequence repeats. Further characterization of NusACTD with 15N NMR relaxation data suggests that the interdomain region is only weakly structured and that the subdomains are not interacting. Both subdomains adopt an (HhH)2 fold. These folds are normally involved in DNA-protein and protein-protein interactions. NMR titration experiments show clear differences of the interactions of these two domains with alphaCTD and lambdaN, in spite of their structural similarity.