RESUMEN
INTRODUCTION: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. METHODS: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. RESULTS: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). DISCUSSION: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Bioensayo/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Calibración , Evaluación Preclínica de Medicamentos/métodos , Electrocardiografía/métodos , HumanosRESUMEN
INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.
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Canal de Potasio ERG1/antagonistas & inhibidores , Medición de Riesgo/métodos , Torsades de Pointes/inducido químicamente , Teorema de Bayes , Simulación por Computador , Humanos , Modelos Biológicos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Seguridad , Torsades de Pointes/fisiopatologíaRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(ß,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac â¼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.
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Adenosina Trifosfato/análogos & derivados , Adenilil Imidodifosfato/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/agonistas , Mutación Missense , Adenosina Trifosfato/farmacología , Sitios de Unión , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HEK293 , Humanos , Hidrólisis , Activación del Canal Iónico , Potenciales de la Membrana , Técnicas de Placa-Clamp , Dominios y Motivos de Interacción de ProteínasRESUMEN
Skeletonized zirconium phosphonate surfaces are used to support planar lipid bilayers and are shown to be viable substrates for studying transmembrane proteins. The skeletonized surfaces provide space between the bilayer and the solid support to enable protein insertion and avoid denaturation. The skeletonized zirconium octadecylphosphonate surfaces were prepared using Langmuir-Blodgett techniques by mixing octadecanol with octadecylphosphonic acid. After zirconation of the transferred monolayer, rinsing the coating with organic solvent removes the octadecanol, leaving holes in the film ranging from â¼50 to â¼500 nm in diameter, depending on the octadecanol content. Upon subsequent deposition of a lipid bilayer, either by vesicle fusion or by Langmuir-Blodgett/Langmuir-Schaefer techniques, the lipid assemblies span the holes providing reservoirs beneath the bilayer. The viability of the supported bilayers as model membranes for transmembrane proteins was demonstrated by examining two approaches for incorporating the proteins. The BK channel protein inserts directly into a preformed bilayer on the skeletonized surface, in contrast to a bilayer on a nonskeletonized film, for which the protein associates only weakly. As a second approach, the integrin α(5)ß(1) was reconstituted in lipid vesicles, and its inclusion in supported bilayers on the skeletonized surface was achieved by vesicle fusion. The integrin retains its ability to recognize the extracellular matrix protein fibronectin when supported on the skeletonized film, again in contrast to the response if the bilayer is supported on a nonskeletonized film.
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Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Circonio/química , Tamaño de la Partícula , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be "rescued" by culturing cells at 26-28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Citosol/química , Mutación , Adenosina Trifosfato/química , Transporte Biológico , Línea Celular , Citosol/metabolismo , Calor , Humanos , Modelos Genéticos , Nucleótidos/genética , Técnicas de Placa-Clamp , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , TemperaturaRESUMEN
Lipid bilayers are of interest in applications where a cell membrane mimicking environment is desired. The performance of the lipid bilayer is largely dependent on the physical and chemical properties of the component lipids. Lipid bilayers consisting of phytanoyl lipids have proven to be appropriate choices since they exhibit high mechanical and chemical stability. In addition, such bilayers have high electrical resistances. Two different phytanoyl lipids, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and various combinations of the two have been investigated with respect to their behavior in aqueous solutions, their interactions with solid surfaces, and their electrical stability. Dynamic light scattering, nuclear magnetic resonance diffusion, and cryogenic transmission electron microscopy measurements showed that pure DPhPC as well as mixtures of DPhPC and DPhPE consisting of greater than 50% (mol%) DPhPC formed unilamellar vesicles. If the total lipid concentration was greater than 0.15g/l, then the vesicles formed solid-supported bilayers on plasma-treated gold and silica surfaces by the process of spontaneous vesicle adsorption and rupture, as determined by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. The solid-supported bilayers exhibited a high degree of viscoelasticity, probably an effect of relatively high amounts of imbibed water or incomplete vesicle fusion. Lipid compositions consisting of greater than 50% DPhPE formed small flower-like vesicular structures along with discrete liquid crystalline structures, as evidenced by cryogenic transmission electron microscopy. Furthermore, electrophysiology measurements were performed on bilayers using the tip-dip methodology and the bilayers' capacity to retain its electrical resistance towards an applied potential across the bilayer was evaluated as a function of lipid composition. It was shown that the lipid ratio significantly affected the bilayer's electrical stability, with pure DPhPE having the highest stability followed by 3DPhPC:7DPhPE and 7DPhPC:3DPhPE in decreasing order. The bilayer consisting of 5DPhPC:5DPhPE had the lowest stability towards the applied electrical potential.
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Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Microscopía por Crioelectrón/métodos , Electroquímica/métodos , Electrofisiología/métodos , Oro/química , Luz , Lípidos/química , Espectroscopía de Resonancia Magnética/métodos , Microscopía de Fuerza Atómica/métodos , Microscopía Electrónica de Transmisión/métodos , Tecnicas de Microbalanza del Cristal de Cuarzo , Dispersión de Radiación , Propiedades de SuperficieRESUMEN
The mechanosensitive (MS) ion channel is gated by changes in bilayer deformation. It is functional without the presence of any other proteins and gating of the channel has been successfully achieved using conventional patch clamping techniques where a voltage has been applied together with a pressure over the membrane. Here, we have for the first time analyzed the large conducting (MscL) channel in a supported membrane using only an external electrical field. This was made possible using a newly developed technique utilizing a tethered lipid bilayer membrane (tBLM), which is part of an engineered microelectronic array chip. Single ion channel activity characteristic for MscL was obtained, albeit with lower conductivity. The ion channel was gated using solely a transmembrane potential of 300 mV. Computations demonstrate that this amount of membrane potential induces a membrane tension of 12 dyn/cm, equivalent to that calculated to gate the channel in patch clamp from pressure-induced stretching of the bilayer. These results strengthen the supposition that the MscL ion channel gates in response to stress in the lipid membrane rather than pressure across it. Furthermore, these findings illustrate the possibility of using the MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.