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Char-Chang Shieh

Publications and source records attributed to Char-Chang Shieh.

12 recordsLinked to original sources
PubMed2004

In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892.

1. Openers of ATP-sensitive K(+) channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. 2. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (-log EC(50)=7.63) in guinea-pig bladder smooth muscle cells. 3. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [(125)I]A-312110. A-151892 displaced [(125)I]A-312110 binding to bladder membranes with a -log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. 4. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with -log IC(50) values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (-log IC(50)=7.81) and portal vein (-log IC(50)=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. 5. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED(35%) values of 8.05 and 7.43, respectively. 6. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel K(ATP) channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms.

Acetamides↗
PubMed2004

Differential action potentials and firing patterns in injured and uninjured small dorsal root ganglion neurons after nerve injury.

The profile of tetrodotoxin sensitive (TTX-S) and resistant (TTX-R) Na(+) channels and their contribution to action potentials and firing patterns were studied in isolated small dorsal root ganglion (DRG) neurons after L5/L6 spinal nerve ligation (SNL). Total TTX-R Na(+) currents and Na(v) 1.8 mRNA were reduced in injured L5 DRG neurons 14 days after SNL. In contrast, TTX-R Na(+)currents and Na(v) 1.8 mRNA were upregulated in uninjured L4 DRG neurons after SNL. Voltage-dependent inactivation of TTX-R Na(+) channels in these neurons was shifted to hyperpolarized potentials by 4 mV. Two types of neurons were identified in injured L5 DRG neurons after SNL. Type I neurons (57%) had significantly lower threshold but exhibited normal resting membrane potential (RMP) and action potential amplitude. Type II neurons (43%) had significantly smaller action potential amplitude but retained similar RMP and threshold to those from sham rats. None of the injured neurons could generate repetitive firing. In the presence of TTX, only 26% of injured neurons could generate action potentials that had smaller amplitude, higher threshold, and higher rheobase compared with sham rats. In contrast, action potentials and firing patterns in uninjured L4 DRG neurons after SNL, in the presence or absence of TTX, were not affected. These results suggest that TTX-R Na(+) channels play important roles in regulating action potentials and firing patterns in small DRG neurons and that downregulation in injured neurons and upregulation in uninjured neurons confer differential roles in shaping electrogenesis, and perhaps pain transmission, in these neurons.

Action Potentials↗
PubMed2004

Potassium channel subtypes as molecular targets for overactive bladder and other urological disorders.

Potassium channels have re-emerged as attractive targets for overactive bladder and other urological diseases in recent years, in part due to an enhanced understanding of their molecular heterogeneity, tissue distribution, functional roles and regulation in physiological and pathological states. Cloning and heterologous expression analysis, coupled with the advancement of improved high-throughput screening techniques, have enabled expeditious identification of selective small-molecule openers and blockers for ATP-sensitive K+ channels, Ca2+-activated K+ channels and voltage-dependent K+ channel-KQT-like subfamily (KCNQ) members, and has paved the way in the assessment of efficacy and adverse effects in preclinical models. This review focuses on the rationale for molecular targeting of K+ channels, the current status of target validation, including preclinical proof-of-concept studies, and provides perspectives on the limitations and hurdles to be overcome in realising the potential of these targets for diverse urological indications such as overactive bladder, erectile dysfunction and prostate diseases.

Aged↗
PubMed2003

Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation.

The effects of 1-ethyl-2-benzimidazolinone (1-EBIO) and riluzole on human prostate cancer cells, LNCaP and PC-3, were evaluated using rubidium (86Rb(+)) efflux and proliferation assays. 1-EBIO and riluzole evoked concentration-dependent increases in 86Rb(+) efflux from LNCaP and PC-3 cells that were sensitive to inhibition by intermediate-conductance Ca(2+)-activated K(+) channel (IK(Ca)) blockers clotrimazole and charybdotoxin. Blockers of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, iberiotoxin, or small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel, apamin or scyllatoxin, had no effect. Concurrently, both 1-EBIO and riluzole evoked concentration-dependent increases in proliferation from human prostate cancer cell lines (LNCaP and PC-3 cells). Clotrimazole and charybdotoxin, but not iberiotoxin, apamin or scyllatoxin, inhibited 1-EBIO- and riluzole-evoked increases in proliferation from LNCaP and PC-3 cells. N-(3-(trifluoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea (NS-1608) and 2-amino-5-(2-fluorophenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BK(Ca) channel openers had no effect on LNCaP and PC-3 proliferation. These results demonstrate that IK(Ca) channels play an important role in the regulation of human prostate cancer cell proliferation.

Benzimidazoles↗
PubMed2003

The discovery of a new class of large-conductance Ca2+-activated K+ channel opener targeted for overactive bladder: synthesis and structure-activity relationships of 2-amino-4-azaindoles.

2-Amino-4-azaindoles have been identified as a structurally novel class of BK(Ca) channel openers. Their synthesis from 2-chloro-3-nitropyridine is described together with their in vitro properties assessed by 86Rb(+) efflux and whole-cell patch-clamp assays using HEK293 cells stably transfected with the BK(Ca) alpha subunit. In vitro functional characterization of BK(Ca) channel opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig bladders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.

Amines↗
PubMed2003

Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry.

Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK(Ca) channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC(50) values ranging from 1 to 4 microM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BK(Ca) channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC(50) values of the inhibitors in AAS format were comparable to those derived from (86)Rb(+) efflux assays. The present studies show that the pharmacological profiles of BK(Ca) channels assessed by AAS compare well with those obtained using the (86)Rb(+) efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K(+) channel modulators.

Benzimidazoles↗
PubMed2003

Automated Parallel Oocyte Electrophysiology Test station (POETs): a screening platform for identification of ligand-gated ion channel modulators.

Ligand-gated ion channels (LGICs) play important roles in the regulation of cellular function and signaling and serve as excellent drug targets. However, fast desensitization of most LGICs limits the choice of reliable methods to identify agonists, antagonists, and/or modulators in a high throughput manner. In this study, automated Parallel Oocyte Electrophysiology Test station (POETs) was used to screen a directed compound library against a rapidly desensitizing LGIC and to characterize further the pharmacological properties of the hits. POETs allows up to six two-electrode voltage-clamp experiments to be performed in parallel by automatically loading of the oocytes into flowcells, assessing individual oocyte behavior prior to initiating experiments. Oocytes injected with cRNA were transferred from a chilled 96-well plate into flowcells by the instrument, where they were impaled under software control by two independent electrodes. Expression was tested by measuring current responses to rapid application of agonists. Compounds, prepared in a 96-well format, were tested for effects by coapplication with agonist at a single concentration of 30 microM over 2 s. After compound application, oocytes were washed for a minimum of 30 s, and used repeatedly if the test compounds had no significant effect on the control response. Typical throughput could reach approximately 14 plates/day depending on the protocol. Pilot library screening revealed a hit rate of 0.06%, with active compounds having IC(50) values of 4-40 microM. Hits were also confirmed in native neurons using patch-clamp techniques. We conclude that automated POETs serves as a suitable platform for screening and expedient identification of LGIC modulators.

Algorithms↗
PubMed2003

[125I]A-312110, a novel high-affinity 1,4-dihydropyridine ATP-sensitive K+ channel opener: characterization and pharmacology of binding.

Although ATP-sensitive K+ channels continue to be explored for their therapeutic potential, developments in high-affinity radioligands to investigate native and recombinant KATP channels have been less forthcoming. This study reports the identification and pharmacological characterization of a novel iodinated 1,4-dihydropyridine KATP channel opener, [125I]A-312110 [(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyridin-8(7H)-one-1,1-dioxide]. Binding of [125I]A-312110 to guinea pig cardiac (KD = 5.8 nM) and urinary bladder (KD = 4.9 nM) membranes were of high affinity, saturable, and to a single set of binding sites. Displacement of [125I]A-312110 by structurally diverse potassium channel openers (KCOs) indicated a similar rank order of potency in both guinea pig cardiac and bladder membranes (Ki, heart): A-312110 (4.3 nM) > N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) > (-)-N-(2-ethoxyphenyl)-N'-(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) >> diazoxide (16.7 microM). Displacement by KATP channel blockers, the sulfonylurea glyburide, and the cyanoguanidine N-[1-(3-chlorophenyl)cyclobutyl]-N'-cyano-N"-3-pyridinyl-guanidine (PNU-99963) were biphasic in the heart but monophasic in bladder with about a 100- to 500-fold difference in Ki 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 [125I]A-312110 binds with high affinity and has an improved activity profile compared with other radiolabeled KCOs. [125I]A-312110 is a useful tool for investigation of the molecular and functional properties of the KATP 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 KATP channels.

Adenosine Triphosphate↗
PubMed2003

High throughput electrophysiology using a fully automated, multiplexed recording system.

The drug discovery process centers around finding and optimizing novel compounds active at therapeutic targets. This process involves direct and indirect measures of how compounds affect the behavior of the target in question. The sheer number of compounds that must be tested poses problems for classes of ion channel targets for which direct functional measurements (e.g., traditional patch-clamping) are too cumbersome and indirect measurements (e.g., Ca(2+)-sensitive dyes) lack sufficient sensitivity or require unacceptable compromises. We present an optimized process for obtaining large numbers of direct electrophysiological measurements (two-electrode voltage-clamp) from Xenopus oocytes using a combination of automated oocyte handling, efficient and flexible liquid delivery, parallel operation, and powerful integrated data analysis. These improvements have had a marked impact, increasing the contribution electrophysiology makes in optimizing lead compound series and the discovery of new ones. The design of the system is detailed along with examples of data generated in support of lead optimization and discovery.

Animals↗
PubMed2002

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, alpha-dendrotoxin (alpha-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K(+) currents of the sensory neurons. alpha-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to -50 mV and little inactivation during 250 ms pulses. In current-clamp experiments alpha-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. alpha-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K(+) currents function in response to the loss of the alpha-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

Algorithms↗
PubMed2002

(-)-(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637): a novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions. I. In vitro characterization.

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-hexahydrothieno[3,2-b]quinolin-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.

ATP-Binding Cassette Transporters↗