A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ ...channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.
Structure−activity relationships were investigated on the tricyclic dihydropyridine (DHP) KATP openers 9-(3-bromo-4-fluorophenyl)-5,9-dihydro-3H,4H-2,6-dioxa-4-azacyclopentabnaphthalene-1,8-dione (6) ...and 10-(3-bromo-4-fluorophenyl)-9,10-dihydro-1H,8H-2,7-dioxa-9-azaanthracene-4,5-dione (65). Substitution off the core of the DHP, absolute stereochemistry, and aromatic substitution were evaluated for KATP channel activity using Ltk− cells stably transfected with the Kir6.2/SUR2B exon 17− splice variant and in an electrically stimulated pig bladder strip assay. A select group of compounds was evaluated for in vitro inhibition of spontaneous bladder contractions. Several compounds were found to have the unique characteristic of partial efficacy in both the cell-based and electrically stimulated bladder strip assays but full efficacy in inhibiting spontaneous bladder strip contractions. For compound 23b, this profile was mirrored in vivo where it was fully efficacious in inhibiting spontaneous myogenic bladder contractions but only partially able to reduce neurogenically mediated reflex bladder contractions.
ATP-sensitive K+ (KATP) channels play an important role in the regulation of smooth muscle membrane potential. To investigate the properties of KATP channels in guinea pig urinary bladder smooth ...muscle cells, fluorescence-based assays were carried out with the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol DiBAC4(3). The prototypical channel openers, including pinacidil, (-)-cromakalim, and diazoxide, elicited concentration-dependent decreases in membrane potential that were attenuated by glyburide. Similar responses were evoked by a reduction in intracellular ATP levels by metabolic inhibition. The observed rank order potency (EC50) for evoking membrane potential changes by potassium channel openers, P1075 (53 nM) approximately Bay X 9228 > (-)-cromakalim approximately ZD6169 approximately pinacidil > Bay X 9227 approximately ZM244085 > diazoxide (59 microM), showed a good correlation with that of bladder smooth muscle relaxation, as assessed by isolated tissue bath studies. The maximal efficacies of (-)-cromakalim, pinacidil, Bay X 9228, and ZD6169 were comparable with the response achieved by the reference activator P1075. Whole cell currents in bladder smooth muscle cells were increased in both inward and outward directions by P1075 and were reversed by glyburide to control levels. The molecular composition assessed by reverse transcriptase-polymerase chain reaction analysis using subunit-specific primers revealed the presence of mRNA for inward rectifying potassium channel (KIR6.2) and sulfonylurea receptors (SUR)2B and SUR1. The subunit profile together with pharmacological properties suggests that the KATP channel in bladder smooth muscle cells could be composed of SUR2B associated with a single inward rectifier, KIR6.2. In summary, these studies have characterized the pharmacological profile using fluorescent imaging plate reader-based membrane potential techniques and provide evidence for the molecular identity of KATP channels expressed in guinea pig bladder smooth muscle cells.
Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ...ATP-sensitive K(+) (K(ATP)) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno3,2-bquinolin-8(4H)-one 1,1-dioxide (A-278637), a novel 1,4-dihydropyridine K(ATP) channel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated K(ATP) channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC(50) = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC(50) = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002) J Pharmacol Exp Ther 303:387-394, A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 (R)-4-3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino-3-ethyl-benzonitrile and ZD6169 (S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective K(ATP) channel openers for the treatment of overactive bladder via myogenic etiology.
Structure−activity relationships were investigated on a novel series of tricyclic dihydropyridine-containing KATP openers. This diverse group of analogues, comprising a variety of heterocyclic rings ...fused to the dihydropyridine nucleus, was designed to determine the influence on activity of hydrogen-bond-donating and -accepting groups and their stereochemical disposition. Compounds were evaluated for KATP activity in guinea pig bladder cells using a fluorescence-based membrane potential assay and in a pig bladder strip assay. The inhibition of spontaneous bladder contractions in vitro was also examined for a subset of compounds. All compounds studied showed greater potency to inhibit spontaneous bladder contractions relative to their potencies to inhibit contractions elicited by electrical stimulation.
Although ATP-sensitive K + channels continue to be explored for their therapeutic potential, developments in high-affinity radioligands to investigate
native and recombinant K ATP channels have been ...less forthcoming. This study reports the identification and pharmacological characterization of a novel
iodinated 1,4-dihydropyridine K ATP channel opener, 125 IA-312110 (9R)-9-(4-fluoro-3- 125 iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano3,4- b thieno2,3- e pyridin-8(7H)-one-1,1-dioxide. Binding of 125 IA-312110 to guinea pig cardiac ( K D = 5.8 nM) and urinary bladder ( K D = 4.9 nM) membranes were of high affinity, saturable, and to a single set of binding sites. Displacement of 125 IA-312110 by structurally diverse potassium channel openers (KCOs) indicated a similar rank order of potency in both guinea
pig cardiac and bladder membranes ( K i , heart): A-312110 (4.3 nM) > N -cyano- N â²-(1,1-dimethylpropyl)- N â³-3-pyridylguanidine (P1075) > (-)- N -(2-ethoxyphenyl)- N â²-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine (Bay X 9228) > pinacidil > (-)-cromakalim > N -(4-benzoyl phenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropionamine (ZD6169) > 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione
(ZM244085) â« diazoxide (16.7 μM). Displacement by K ATP channel blockers, the sulfonylurea glyburide, and the cyanoguanidine N -1-(3-chlorophenyl)cyclobutyl- N â²-cyano- N â³-3-pyridinyl-guanidine (PNU-99963) were biphasic in the heart but monophasic in bladder with about a 100- to 500-fold difference
in K i values between high- and low-affinity sites. Good correlations were observed between cardiac or bladder-binding affinities
of KCOs with functional activation as assessed by their respective potencies to either suppress action potential duration
(APD) in Purkinje fibers or to relax electrical field-stimulated bladder contractions. Collectively, these results demonstrate
that 125 IA-312110 binds with high affinity and has an improved activity profile compared with other radiolabeled KCOs. 125 IA-312110 is a useful tool for investigation of the molecular and functional properties of the K ATP channel complex and for the identification, in a high throughput manner, of both novel channel blockers and openers that
interact with cardiac/smooth muscle-type K ATP channels.
The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K(ATP) channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon ...17- mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk(-)cells (L-cells) with K(IR) 6.2 were determined by measuring changes in membrane potential responses evoked by K(+) channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) fluorescence. The rank order potency of a variety of structurally distinct K(+) channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17- cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of 125IA-312110 binding with the same K(+) channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K(ATP) channels containing SUR2B exon 17- represent a major splice variant expressed in urinary bladder smooth muscle.
A series of novel cyanoguanidine derivatives was designed and synthesized. Condensation of N-(1-benzotriazol-1-yl-2,2-dichloropropyl)-substituted benzamides with N-(substituted-pyridin-3-yl)-N ...‘-cyanoguanidines furnished N-{2,2-dichloro-1-N ‘-(substituted-pyridin-3-yl)-N ‘ ‘-cyanoguanidinopropyl}-substituted benzamide derivatives. These agents were glyburide-reversible potassium channel openers and hyperpolarized human bladder cells as assessed by the FLIPR membrane potential dye (KATP-FMP). These compounds were also potent full agonists in relaxing electrically stimulated pig bladder strips, an in vitro model of overactive bladder. The most active compound 9 was evaluated for in vivo efficacy and selectivity in a pig model of bladder instability. Preliminary pharmacokinetic studies in dog demonstrated excellent oral bioavailability and a t 1/2 of 15 h. The synthesis, SAR studies, and biological properties of these agents are discussed.
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![[125I]A-312110, a Novel Hig...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0026-895X/small "[125I]A-312110, a Novel High-Affinity 1,4-Dihydropyridine ATP-sensitive K+ Channel Opener: Characterization and Pharmacology of Binding")
