Functional Assay of Voltage-Gated Sodium Channels Using Membrane Potential-Sensitive Dyes

Felix, John P.; Williams, Brande S.; Priest, Birgit T.; Brochu, Richard M.; Dick, Ivy E.; Warren, Vivien A.; Yan, Lizhen; Slaughter, Robert S.; Kaczorowski, Gregory J.; Smith, McHardy M.; García, María L.

doi:10.1089/1540658041410696

Cited by 79 publications

(66 citation statements)

References 22 publications

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“…In many of these cases, where voltage cannot be used to activate the channels, 12,13 small-molecule activators such as veratridine or deltamethrin are used to increase the open probability of the channel to evoke a significant response that can be detected. [14][15][16] Two membrane potential dyes are widely used for HTS: the FLIPR Membrane Potential dye (Molecular Devices, Sunnyvale, CA) that uses a fluorescence indicator in combination with a quencher and voltage sensor probes (VSPs) that rely on fluorescence-resonance energy transfer (FRET) between a voltage-sensing oxonol acceptor and a fluorescent membrane-bound coumarin dye. 17 In the current study, we compared the performance of these membrane potential dyes, along with a sodium-sensing dye, in assay development and found that these dye systems could only detect certain subsets of Na V 1.7 inhibitors using standard data analysis methods.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

et al. 2016

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The Na V 1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding Na V 1.7, resulting in either lossof-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of Na V 1.7. A full-deck high-throughput screen was developed for both Na V 1.7 and cardiac Na V 1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in Na V 1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with Na V 1.7 pharmacological selectivity over Na V 1.5 as confirmed in electrophysiology.

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Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

et al. 2016

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“…8,10,11 The latter is often based on the membrane potential changes following ion fluxes. 12,13 Using surrogate ionic fluxes, several large HTS campaigns have been recently described in published reports 14,15 and in a public database (PubChem AIDs 1456, 1511, 1672, 1918, 2156, and 2239). Examples of HTS campaigns using direct measurement of ionic current have been less commonly reported.…”

mentioning

confidence: 99%

Profiling Diverse Compounds by Flux- and Electrophysiology-Based Primary Screens for Inhibition of Human Ether-à-go-go Related Gene Potassium Channels

Zou

Babcock

et al. 2010

ASSAY and Drug Development Technologies

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Compound effects on cloned human Ether-à-go-go related gene (hERG) potassium channels have been used to assess the potential cardiac safety liabilities of drug development candidate compounds. In addition to radioactive ligand displacement tests, two other common approaches are surrogate ion-based flux assays and electrophysiological recordings. The former has much higher throughput, whereas the latter measures directly the effects on ionic currents. Careful characterization in earlier reports has been performed to compare the relative effectiveness of these approaches for known hERG blockers, which often yielded good overall correlation. However, cases were reported showing significant and reproducible differences in potency and/or sensitivity by the two methods. This raises a question concerning the rationale and criteria on which an assay should be selected for evaluating unknown compounds. To provide a general basis for considering assays to profile large compound libraries for hERG activity, we have conducted parallel flux and electrophysiological analyses of 2,000 diverse compounds, representative of the 300,000 compound collection of NIH Molecular Library Small Molecular Repository (MLSMR). Our results indicate that at the conventional testing concentration 1.0 mM, the overlap between the two assays ranges from 32% to 50% depending on the hit selection criteria. There was a noticeable rate of false negatives by the thallium-based assay relative to electrophysiological recording, which may be greatly reduced under modified comparative conditions. As these statistical results identify a preferred method for cardiac safety profiling of unknown compounds, they suggest an efficient method combining flux and electrophysiological assays to rapidly profile hERG liabilities of large collection of naive compounds.

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“…[137][138][139][140] The interaction of Compound 4 with Na v 1.7, Na v 1.8, and Na v 1.5 channels was further characterized in whole cell electrophysiology. Minimal block was observed when compound 4 was applied to hNa v 1.7 channels at Ϫ100 mV.…”

Section: Novel Na V 17 Blockers From Merckmentioning

confidence: 99%

“…Cell based, functional, fluorescence, or ion flux-based assays are typically the assays of choice for high throughput sodium channel screening. 137,[142][143][144][145] Although well suited to the screening of large compound collections, these assays have a number of significant limitations that may ultimately impact the probability of identifying subtype selective sodium channel blockers. For example, although accurate for the most part, assays reliant on membrane potential dyes are prone to artifacts.…”

Section: Future Directionsmentioning

confidence: 99%

“…Numerous studies have demonstrated the high sensitivity of commonly used cell-based functional assays to local anesthetics and related compounds. 137,[142][143][144] However, few studies have rigorously explored the sensitivity of commonly used assays for mechanistically novel sodium channel modulators, such as the gating modifier spider toxins protoxin I and protoxin II, for example (see below). Although activity of protoxin I and protoxin II has been demonstrated in a voltage ion-probe reader (VIPR) FRET-based assay, the potency of these peptides appeared highly dependent on the duration of the assay.…”

Section: Future Directionsmentioning

confidence: 99%

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Sodium Channel Blockers for the Treatment of Neuropathic Pain

2009

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Summary:Drugs that block voltage-gated sodium channels are efficacious in the management of neuropathic pain. Accordingly, this class of ion channels has been a major focus of analgesic research both in academia and in the pharmaceutical/biotechnology industry. In this article, we review the history of the use of sodium channel blockers, describe the current status of sodium channel drug discovery, highlight the challenges and hurdles to attain sodium channel subtype selectivity, and review the potential usefulness of selective sodium channel blockers in neuropathic pain.

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Functional Assay of Voltage-Gated Sodium Channels Using Membrane Potential-Sensitive Dyes

Cited by 79 publications

References 22 publications

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

Profiling Diverse Compounds by Flux- and Electrophysiology-Based Primary Screens for Inhibition of Human Ether-à-go-go Related Gene Potassium Channels

Sodium Channel Blockers for the Treatment of Neuropathic Pain

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