Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels.

ABSTRACT: K(+) channels, a superfamily of ?80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K(+) channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K(+) channels and identified new activators and inhibitors for biological research on channel function and for medicinal development. We further engineered a hERG (human ether-à-go-go-related gene) channel, which, when used in LFA, provides a highly sensitive (zero false negatives on 50 hERG-sensitive drugs) and highly specific (zero false positives on 50 hERG-insensitive drugs), low-cost hERG safety assay.

SUBMITTER: Su Z

PROVIDER: S-EPMC4878532 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels.

Su Zhenwei Z Brown Emily C EC Wang Weiwei W MacKinnon Roderick R

Proceedings of the National Academy of Sciences of the United States of America 20160418 20

K(+) channels, a superfamily of ∼80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K(+) channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K(+) channels and identified new activators and inhibitors ...[more]

PMID: 27091997

Similar Datasets

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds.

Project description:Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

| S-EPMC7143316 | biostudies-literature

High throughput screening technologies for ion channels.

Project description:Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications.

| S-EPMC4722985 | biostudies-other

Novel method for high-throughput colony PCR screening in nanoliter-reactors.

Project description:We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20,000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100,000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.

| S-EPMC2677890 | biostudies-other

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors.

Project description:Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.

| S-EPMC6627609 | biostudies-literature

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.

Project description:DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

| S-EPMC4417145 | biostudies-literature

A High-Throughput Screening Identifies MICU1 Targeting Compounds.

Project description:Mitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU) complex, a highly selective channel of the inner mitochondrial membrane (IMM). Here, we screen a library of 44,000 non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca2+ influx. Docking simulations reveal that these molecules directly bind a specific cleft in MICU1, a key element of the MCU complex that controls channel gating. Accordingly, in MICU1-silenced or deleted cells, the inhibitory effect of the two compounds is lost. Moreover, MCU-i4 and MCU-i11 fail to inhibit mitochondrial Ca2+ uptake in cells expressing a MICU1 mutated in the critical amino acids that forge the predicted binding cleft. Finally, these compounds are tested ex vivo, revealing a primary role for mitochondrial Ca2+ uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs.

| S-EPMC7034061 | biostudies-literature

High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

Project description:The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions.

| S-EPMC4551901 | biostudies-literature

Convenient, sensitive and high-throughput method for screening botanic origin.

Project description:In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

| S-EPMC4066250 | biostudies-literature

High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.

Project description:The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

| S-EPMC4344505 | biostudies-literature

Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

Project description:A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

| S-EPMC3134069 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data