title: A high-throughput screening campaign for detection of ca(2+)-activated k(+) channel activators and inhibitors using a fluorometric imaging plate reader-based tl(+)-influx assay.
abstract: The intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) has been proposed to play many physiological roles, and modulators of KCa3.1 activity are potentially interesting as new drugs. In order to identify new chemical scaffolds, high-throughput screening (HTS) assays are needed. In the current study, we present an HTS assay that has been optimized for the detection of inhibitors as well as activators of KCa3.1 in a combined assay. We used HEK293 cells heterologously expressing KCa3.1 in a fluorescence-based Tl(+) influx assay, where the permeability of potassium channels to Tl(+) is taken advantage of. We found the combined activator-and-inhibitor assay to be robust and insensitive to dimethyl sulfoxide (up to 1%), and conducted an HTS campaign of 217,119 small molecules. In total, 224 confirmed activators and 312 confirmed inhibitors were found, which corresponded to a hit rate of 0.10% and 0.14%, respectively. The confirmed hits were further characterized in a fluorometric imaging plate reader-based concentration-response assay, and selected compounds were subjected to secondary testing in an assay for endogenous KCa3.1 activity using human erythrocytes (red blood cell assay). Although the estimated potencies were slightly higher in the RBC assay, there was an overall good correlation across all clusters. The campaign led to the identification of several chemical series of KCa3.1 activators and inhibitors, comprising already known pharmacophores and new chemical series. One of these were the benzothiazinones that constitute a new class of highly potent KCa3.1 inhibitors, exemplified by 4-{[3-(trifluoromethyl)phenyl]methyl}-2H-1,4-benzothiazin-3(4H)-one (NS6180).
authors.affiliation: NeuroSearch A/S, Ballerup DK-2750, Denmark. susannejorgensen1@gmail.com
authors.forename: Susanne
authors.initials: S
authors.lastname: Jørgensen
authors.forename: Tino
authors.initials: T
authors.lastname: Dyhring
authors.forename: David T
authors.initials: DT
authors.lastname: Brown
authors.forename: Dorte
authors.initials: D
authors.lastname: Strøbæk
authors.forename: Palle
authors.initials: P
authors.lastname: Christophersen
authors.forename: Joachim
authors.initials: J
authors.lastname: Demnitz
mesh_terms: D000081:Acetamides / Q000138:chemical synthesis / Q000494:pharmacology; D000465:Algorithms; D003627:Data Interpretation, Statistical; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000188:drug therapy; D026902:Potassium Channel Blockers / Q000737:chemistry / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000819:agonists* / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000138:chemical synthesis / Q000494:pharmacology; D054852:Small Molecule Libraries; D013793:Thallium / Q000737:chemistry / Q000493:pharmacokinetics; D013843:Thiazines / Q000494:pharmacology; D014320:Triphenylmethyl Compounds / Q000138:chemical synthesis / Q000494:pharmacology
chemical_list: C581707:4-((3-(trifluoromethyl)phenyl)methyl)-2H-1,4-benzothiazin-3(4H)-one; D000081:Acetamides; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D011720:Pyrazoles; D054852:Small Molecule Libraries; C411671:TRAM 34; D013843:Thiazines; D014320:Triphenylmethyl Compounds; D013793:Thallium; C472774:senicapoc