Use of Escin as a Perforating Agent on the IonWorks Quattro Automated Electrophysiology Platform

Morton, Michael J.; Main, Martin J.

doi:10.1177/1087057112456599

Cited by 11 publications

(8 citation statements)

References 23 publications

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“…Most notably, IonWorks Quattro (IWQ, the predecessor to IWB) has been used in Ca v 1.2 recording with results comparable to those described here. For instance, Morton and Main 17 reported a verapamil IC 50 value of 3.3 μM (at a holding potential of −60 mV), similar to the 4.3 μM IC 50 value reported here ( Table 4 , prepulse −50 mV, holding potential, −90 mV). Interestingly, Cao et al 18 also reported a verapamil IC 50 value of 4.0 μM, but at a holding potential of −90 mV, whereas we observed a marked decrease in potency (IC 50 =24.2 μM, Table 4 , prepulse and holding potential, −90 mV) under these conditions.…”

Section: Discussionsupporting

confidence: 85%

Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays

Kuryshev

Brown²,

Duzic³

et al. 2014

ASSAY and Drug Development Technologies

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Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks™ Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug–channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.

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Section: Discussionsupporting

confidence: 85%

Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays

Kuryshev

Brown²,

Duzic³

et al. 2014

ASSAY and Drug Development Technologies

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“…It was hypothesized that the natural products eugenol (EUG), β-escin, and curcumin (CUR) may act synergistically in combination. This was based on their related, reported mechanisms of action: in causing lipid peroxidation and disruption of cell membrane integrity (EUG), pore formation within the cell membrane (β-escin), and interactions with ABC drug transporters and ERG3 gene downregulation, leading to decreased membrane ergosterol and permeability (CUR) ( Morton and Main, 2013 ; Moghadamtousi et al, 2014 ; Marchese et al, 2017 ). Checkerboard assays measuring growth of the model yeast Saccharomyces cerevisiae were used to assess synergy.…”

Section: Resultsmentioning

confidence: 99%

Discovery of Natural Products With Antifungal Potential Through Combinatorial Synergy

Augostine

Avery

2022

Front. Microbiol.

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The growing prevalence of antifungal drug resistance coupled with the slow development of new, acceptable drugs and fungicides has raised interest in natural products (NPs) for their therapeutic potential and level of acceptability. However, a number of well-studied NPs are considered promiscuous molecules. In this study, the advantages of drug–drug synergy were exploited for the discovery of pairwise NP combinations with potentiated antifungal activity and, potentially, increased target specificity. A rational approach informed by previously known mechanisms of action of selected NPs did not yield novel antifungal synergies. In contrast, a high-throughput screening approach with yeast revealed 34 potential synergies from 800 combinations of a diverse NP library with four selected NPs of interest (eugenol, EUG; β-escin, ESC; curcumin, CUR; berberine hydrochloride, BER). Dedicated assays validated the most promising synergies, namely, EUG + BER, CUR + sclareol, and BER + pterostilbene (PTE) [fractional inhibitory concentrations (FIC) indices ≤ 0.5 in all cases], reduced to as low as 35 (BER) and 7.9 mg L–1 (PTE). These three combinations synergistically inhibited a range of fungi, including human or crop pathogens Candida albicans, Aspergillus fumigatus, Zymoseptoria tritici, and Botrytis cinerea, with synergy also against azole-resistant isolates and biofilms. Further investigation indicated roles for mitochondrial membrane depolarization and reactive oxygen species (ROS) formation in the synergistic mechanism of EUG + BER action. This study establishes proof-of-principle for utilizing high-throughput screening of pairwise NP interactions as a tool to find novel antifungal synergies. Such NP synergies, with the potential also for improved specificity, may help in the management of fungal pathogens.

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“…β-Escin is the major active component in extracts of horse chestnut seeds and has been reported to show certain antifungal properties. , The compound interacts with sterol molecules creating pores in the cell membrane . Paromomycin is known to cause mRNA mistranslation.…”

Section: Resultsmentioning

confidence: 99%

Repurposing Nonantifungal Approved Drugs for Synergistic Targeting of Fungal Pathogens

et al. 2020

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With the spread of drug resistance, new antimicrobials are urgently needed. Here, we set out to tackle this problem by high-throughput exploration for novel antifungal synergies among combinations of approved, nonantifungal drugs; a novel strategy exploiting the potential of alternative targets, low chemicals usage and low development risk. We screened the fungal pathogen Candida albicans by combining a small panel of nonantifungal drugs (all in current use for other clinical applications) with 1280 compounds from an approved drug library. Screens at sublethal concentrations of the antibiotic paromomycin (PM), the antimalarial primaquine (PQ), or the anti-inflammatory drug ibuprofen (IF) revealed a total of 17 potential strong, synergistic interactions with the library compounds. Susceptibility testing with the most promising combinations corroborated marked synergies [fractional inhibitory concentration (FIC) indices ≤0.5] between PM + β-escin, PQ + celecoxib, and IF + pentamidine, reducing the MICs of PM, PQ, and IF in C. albicans by >64-, 16-, and 8-fold, respectively. Paromomycin + β-escin and PQ + celecoxib were effective also against C. albicans biofilms, azole-resistant clinical isolates, and other fungal pathogens. Actions were specific, as no synergistic effect was observed in mammalian cells. Mode of action was investigated for one of the combinations, revealing that PM + β-escin synergistically increase the error-rate of mRNA translation and suggesting a different molecular target to current antifungals. The study unveils the potential of the described combinatorial strategy in enabling acceleration of drug-repurposing discovery for combatting fungal pathogens.

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Use of Escin as a Perforating Agent on the IonWorks Quattro Automated Electrophysiology Platform

Cited by 11 publications

References 23 publications

Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays

Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays

Discovery of Natural Products With Antifungal Potential Through Combinatorial Synergy

Repurposing Nonantifungal Approved Drugs for Synergistic Targeting of Fungal Pathogens

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