Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Gen Physiol ; 155(8)2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37318452

RESUMO

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter and modulated by the side chains that surround it. While crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels is less clear. Here, we combined electrophysiological recordings with molecular dynamics simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. We found that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both of these variables on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the "vertical" orientation, in which E55 forms a hydrogen bond with D64 (as in KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, in the "horizontal" orientation, K+ conductance is closer to that of MthK WT; although selectivity filter stability is lowered, resulting in more frequent inactivation. Surprisingly, inactivation in MthK WT and V55E is associated with a widening of the selectivity filter, unlike what is observed for KcsA and reminisces recent structures of inactivated channels, suggesting a conserved inactivation pathway across the potassium channel family.


Assuntos
Ativação do Canal Iônico , Potássio , Conformação Proteica , Potássio/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio/metabolismo , Simulação de Dinâmica Molecular , Íons/metabolismo , Proteínas de Bactérias/metabolismo
2.
J Immunol ; 206(5): 1067-1076, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33483346

RESUMO

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.


Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Quimiocina CCL20/imunologia , Animais , Doenças Autoimunes/imunologia , Membrana Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Imunoglobulina G/imunologia , Inflamação/imunologia , Macaca fascicularis , Células Th17/imunologia
3.
J Gen Physiol ; 152(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32342093

RESUMO

Polyamines such as spermidine and spermine are found in nearly all cells, at concentrations ranging up to 0.5 mM. These cations are endogenous regulators of cellular K+ efflux, binding tightly in the pores of inwardly rectifying K+ (Kir) channels in a voltage-dependent manner. Although the voltage dependence of Kir channel polyamine blockade is thought to arise at least partially from the energetically coupled movements of polyamine and K+ ions through the pore, the nature of physical interactions between these molecules is unclear. Here we analyze the polyamine-blocking mechanism in the model K+ channel MthK, using a combination of electrophysiology and computation. Spermidine (SPD3+) and spermine (SPM4+) each blocked current through MthK channels in a voltage-dependent manner, and blockade by these polyamines was described by a three-state kinetic scheme over a wide range of polyamine concentrations. In the context of the scheme, both SPD3+ and SPM4+ access a blocking site with similar effective gating valences (0.84 ± 0.03 e0 for SPD3+ and 0.99 ± 0.04 e0 for SPM4+), whereas SPM4+ binds in the blocked state with an ∼20-fold higher affinity than SPD3+ (Kd = 28.1 ± 3.1 µM for SPD3+ and 1.28 ± 0.20 µM for SPM4+), consistent with a free energy difference of 1.8 kcal/mol. Molecular simulations of the MthK pore in complex with either SPD3+ or SPM4+ are consistent with the leading amine interacting with the hydroxyl groups of T59, at the selectivity filter threshold, with access to this site governed by outward movement of K+ ions. These coupled movements can account for a large fraction of the voltage dependence of blockade. In contrast, differences in binding energetics between SPD3+ and SPM4+ may arise from distinct electrostatic interactions between the polyamines and carboxylate oxygens on the side chains of E92 and E96, located in the pore-lining helix.


Assuntos
Poliaminas , Canais de Potássio Corretores do Fluxo de Internalização , Fenômenos Eletrofisiológicos , Cinética , Potássio , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Espermidina , Espermina
4.
PLoS One ; 15(4): e0231655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32325480

RESUMO

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Quimiocina CCL20/imunologia , Proteínas do Sistema Complemento/imunologia , Doenças do Complexo Imune/tratamento farmacológico , Doenças do Complexo Imune/imunologia , Imunoconjugados/uso terapêutico , Animais , Anticorpos Monoclonais/toxicidade , Doença Crônica , Cristalização , Determinação de Ponto Final , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Macaca fascicularis
5.
J Pharm Sci ; 108(4): 1415-1422, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30465782

RESUMO

Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography-mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.


Assuntos
Anticorpos Monoclonais/farmacocinética , Mapeamento de Peptídeos/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/química , Biotransformação , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Controle de Qualidade , Ratos
6.
Biotechnol Bioeng ; 114(6): 1208-1214, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28112396

RESUMO

Recombinant monoclonal antibody (mAb) products are widely produced in the pharmaceutical industry using Chinese hamster ovary (CHO) cells. Host cell proteins (HCPs) are one of many process-related impurities generated during the production of mAb products. The multi-analyte HCP enzyme linked immunosorbent assay (ELISA) is the industry standard accepted assay to measure clearance of HCPs from recombinant protein therapeutics. While similar platform processes are used for expression and purification, varying amounts of HCPs are found in final drug substances for different mAb products. Through the use of novel ELISA formats, an HCP-mAb product interaction was identified using a variety of ELISAs to investigate the underlying protein-protein interaction. This result provides evidence to explain why some mAb products have HCPs that are not cleared during purification. This ELISA technique can be used as a high-throughput screening tool to identify conditions to improve clearance of difficult HCPs during downstream processing purification. In addition, an interaction was observed to occur between anti-CHO HCP antibodies and mAb products. However, this interaction only occurs under denaturing conditions, and does not interfere with the HCP quantitation obtained by ELISA, and it was demonstrated that the cross-reactive anti-CHO HCP antibodies can be removed by affinity purification. Biotechnol. Bioeng. 2017;114: 1208-1214. © 2017 Wiley Periodicals, Inc.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Células CHO , Cricetulus , Ensaios de Triagem em Larga Escala/métodos , Ligação Proteica
7.
Proc Natl Acad Sci U S A ; 111(17): E1713-22, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24733889

RESUMO

K(+) efflux through K(+) channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K(+) channel. Either Mg(2+) or Ca(2+) can reduce K(+) efflux through MthK channels. However, Ca(2+), but not Mg(2+), can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca(2+) ions can partially dehydrate, enabling selective accessibility of Ca(2+) to a site at the entry to the selectivity filter. Ca(2+) binding at the site interacts with K(+) ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.


Assuntos
Ativação do Canal Iônico , Canais de Potássio/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Íons/metabolismo , Modelos Biológicos , Simulação de Dinâmica Molecular , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Força Próton-Motriz , Termodinâmica
8.
J Gen Physiol ; 136(5): 569-79, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20937694

RESUMO

Voltage-dependent K(+) channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K(+) channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca(2+) or Ba(2+), suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K(+)] (47 mV per 10-fold increase in [K(+)]), suggesting that K(+) binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K(+) ≈ Rb(+) > Cs(+) > Na(+) > Li(+) ≈ NMG(+). Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K(+)] using kinetic schemes in which the open-conductive state is stabilized by K(+) binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K(+) dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K(+)-sensitive inactivation gating, a property that may be common to other K(+) channels.


Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Células Procarióticas/fisiologia , Animais , Fenômenos Eletrofisiológicos , Escherichia coli , Bicamadas Lipídicas , Lítio/farmacologia , Técnicas de Patch-Clamp , Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Sódio/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...