Novel Neuroprotective K+ Channel Inhibitor Identified by High-Throughput Screening in Yeast

Zaks-Makhina, Elena; Kim, Yonjung; Aizenman, Elias; Levitan, Edwin S.

doi:10.1124/mol.65.1.214

Cited by 66 publications

(76 citation statements)

References 29 publications

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“…10 Likewise, a novel high-affinity Kv2.1 blocker prevents oxidant-induced neuronal apoptosis. 11 Nonetheless, the molecular mechanism underlying the apoptotic increase in channel activity remains unknown. In the present study, we tested the hypothesis that de novo membrane insertion of Kv2.1-encoded channels, rather than a modification of previously resident surface channels, is responsible for the pronounced current surge observed during oxidant-induced apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Apoptotic surface delivery of K+ channels

et al. 2005

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Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K þ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K þ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K þ current.

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Section: Introductionmentioning

confidence: 99%

Apoptotic surface delivery of K+ channels

et al. 2005

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“…17 Modulators of these channels have been reported, 17,18 some of which were identified via HTS compound screens using flux-based assays. [19][20][21] The primary approach has involved the use of surrogate ions such as rubidium (Rb þ ) or thallium (Tl þ ), which may be detected by atomic absorption spectrometry 22,23 or fluorescent dyes. 8,10,11 With the advent of automated patch-clamps, 6,[24][25][26][27] it is now possible to consider whether the more direct measurements of channel electrophysiological activity obtained from the automated patch-clamp justifies its higher implementation cost in high-throughput screens.…”

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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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“…In addition, Marjanovic et al employed a secondary enzymatic screen to further validate the specificity and selectivity of their hits and identify a small subset of AAC2 specific inhibitors that conform to Lipinski's rule of five criteria for drug-like characteristics (Lipinski et al 2001). This simple approach of screening for phenotypic changes in a complemented S. cerevisiae strain has also been used to identify a novel human K + channel inhibitor (Zaks-Makhina et al 2004). An HTS assay was designed where the K + -dependent growth phenotype of K + channel mutants was rescued by mammalian Kir2.1 channel expression.…”

Section: Y E a S T A S A S U B S T I T U T I O N P L A T F O R Mmentioning

confidence: 99%

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

Norcliffe¹,

Álvarez-Ruíz

Martín-Plaza

et al. 2013

Parasitology

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Novel Neuroprotective K+ Channel Inhibitor Identified by High-Throughput Screening in Yeast

Cited by 66 publications

References 29 publications

Apoptotic surface delivery of K+ channels

Apoptotic surface delivery of K+ channels

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

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

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