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A high percentage of drugs and drug candidates has been found to cause cardiotoxicity by reducing potassium conductance, more commonly known as QT prolongation. However, some compounds do not show direct block of ionic flow, suggesting that other mechanisms may also lead to reduction of potassium currents. Using the functional recovery after chemobleaching (FRAC) assay, we have examined a collection of drugs and drug-like compounds for potential perturbation of cardiac potassium channel trafficking. Here we report that a significant number of inhibitory compounds displayed effects on channel expression on the cell surface. Further investigation of celastrol (3-hydroxy-24-nor-2-oxo-1 (10Sanguinetti M.C. Mitcheson J.S. Trends Pharmacol. Sci. 2005; 26: 119-124Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar),3,5,7-friedelatetraen-29-oic acid), a cell-permeable dienonephenolic triterpene compound, revealed its potent inhibitory activity on both Kir2.1 and hERG potassium channels, causal to QT prolongation. In addition to acute block of ion conduction, celastrol also alters the rate of ion channel transport and causes a reduction of channel density on the cell surface. In contrast, celastrol has no effects on trafficking of either CD4 or CD8 membrane proteins. Furthermore, the potency for reducing surface expression is ∼5-10-fold more effective than that for either direct acute inhibition or reported cytoprotectivity via activation of the heat shock transcription factor 1. Because the reduction of potassium channel activity is a common form of druginduced cardiotoxicity, the potent inhibition of cell surface expression by celastrol underscores a need to evaluate drug candidates for their chronic effects on biogenesis of potassium channels. Our results suggest that chronic exposure to certain drugs may be an important aspect of acquired QT prolongation. A high percentage of drugs and drug candidates has been found to cause cardiotoxicity by reducing potassium conductance, more commonly known as QT prolongation. However, some compounds do not show direct block of ionic flow, suggesting that other mechanisms may also lead to reduction of potassium currents. Using the functional recovery after chemobleaching (FRAC) assay, we have examined a collection of drugs and drug-like compounds for potential perturbation of cardiac potassium channel trafficking. Here we report that a significant number of inhibitory compounds displayed effects on channel expression on the cell surface. Further investigation of celastrol (3-hydroxy-24-nor-2-oxo-1 (10Sanguinetti M.C. Mitcheson J.S. Trends Pharmacol. Sci. 2005; 26: 119-124Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar),3,5,7-friedelatetraen-29-oic acid), a cell-permeable dienonephenolic triterpene compound, revealed its potent inhibitory activity on both Kir2.1 and hERG potassium channels, causal to QT prolongation. In addition to acute block of ion conduction, celastrol also alters the rate of ion channel transport and causes a reduction of channel density on the cell surface. In contrast, celastrol has no effects on trafficking of either CD4 or CD8 membrane proteins. Furthermore, the potency for reducing surface expression is ∼5-10-fold more effective than that for either direct acute inhibition or reported cytoprotectivity via activation of the heat shock transcription factor 1. Because the reduction of potassium channel activity is a common form of druginduced cardiotoxicity, the potent inhibition of cell surface expression by celastrol underscores a need to evaluate drug candidates for their chronic effects on biogenesis of potassium channels. Our results suggest that chronic exposure to certain drugs may be an important aspect of acquired QT prolongation. Potassium channels play important roles in a variety of biological processes ranging from neuronal excitability to tumorgenesis (1Jan L.Y. Jan Y.N. Annu. Rev. Physiol. 1992; 54: 537-555Crossref PubMed Scopus (213) Google Scholar, 2Jan L.Y. Jan Y.N. Annu. Rev. Neurosci. 1997; 20: 91-123Crossref PubMed Scopus (462) Google Scholar, 3Mu D. Chen L. Zhang X. See L.H. Koch C.M. Yen C. Tong J.J. Spiegel L. Nguyen K.C. Servoss A. Peng Y. Pei L. Marks J.R. Lowe S. Hoey T. Jan L.Y. McCombie W.R. Wigler M.H. Powers S. Cancer Cell. 2003; 3: 297-302Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Of the more than 400 ion channel genes identified within the human genome, at least 167 are annotated to encode potassium channels (4Xu J. Chen Y. Li M. Targets. 2004; 3: 32-38Google Scholar). Hence, they represent a major repertoire of ion channel proteins. Mutations of potassium channels are causal to a number of human diseases including ataxia, epilepsy, long-QT syndrome, Bartter's syndrome, and familial persistent hyperinsulinaemic hypoglycemia of infancy (5Ashcroft F.M. Ion Channels and Diseases. Academic Press, London2000: 67-312Google Scholar). In addition to being targeted by therapeutics for treating diabetes, epilepsy, and neuropathic pain, unintended inhibition of potassium channel activities has become an important concern, especially for the cardiovascular system (6Curran M.E. Curr. Opin. Biotechnol. 1998; 9: 565-572Crossref PubMed Scopus (41) Google Scholar, 7Ford J.W. Stevens E.B. Treherne J.M. Packer J. Bushfield M. Prog. Drug Res. 2002; 58: 133-168Crossref PubMed Scopus (25) Google Scholar). In human cardiac myocytes, repolarization during an action potential is mediated by a number of potassium channels including Kv4.3, Kv1.4, Kv1.5, Kv2.1, hERG, KvLQT1/mink, and Kir2.x (for reviews, see Refs. 8Roden D.M. Balser J.R. George Jr., A.L. Anderson M.E. Annu. Rev. Physiol. 2002; 64: 431-475Crossref PubMed Scopus (229) Google Scholar and 9Shah M. Akar F.G. Tomaselli G.F. Circulation. 2005; 112: 2517-2529Crossref PubMed Scopus (135) Google Scholar). Their activities are temporally involved in different phases of cardiac repolarization. Compoundcaused reduction of potassium currents, particularly those mediated by hERG, KvLQT1/mink, and Kir2.1, often slows down the rate of repolarization and hence prolongs the duration of action potential (10Sanguinetti M.C. Mitcheson J.S. Trends Pharmacol. Sci. 2005; 26: 119-124Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This is also evident in human patients with long-QT syndrome who have genetic mutations in these genes. Both experimental evidence and computational simulation suggest that a small decrease of potassium conductance could significantly prolong the duration of cardiac action potential (11Nichols C.G. Lederer W.J. Am. J. Physiol. 1991; 261: H1675-H1686PubMed Google Scholar, 12Clancy C.E. Rudy Y. Cardiovasc. Res. 2001; 50: 301-313Crossref PubMed Scopus (151) Google Scholar). Because an unusually high percentage of compounds causing QT prolongation is associated with the reduction of cardiac potassium currents (13De Ponti F. Poluzzi E. Montanaro N. Eur. J. Clin. Pharmacol. 2000; 56: 1-18Crossref PubMed Scopus (237) Google Scholar), it is important to understand the mechanisms by which these compounds exert the inhibition. A considerable number of compounds are inhibitory to potassium channel current, but their potency of inhibition may not always be explained by acute inhibition of channel conductivity (14Redfern W.S. Carlsson L. Davis A.S. Lynch W.G. MacKenzie I. Palethorpe S. Siegl P.K. Strang I. Sullivan A.T. Wallis R. Camm A.J. Hammond T.G. Cardiovasc. Res. 2003; 58: 32-45Crossref PubMed Scopus (1324) Google Scholar). These compounds may exert their effects by alternative mechanisms including chronic mechanism of action. In general, reduction of potassium current on the cell surface could take place at two levels, ion conduction and channel density. The effect on channel conductivity can be measured by established techniques such as electrophysiological methods (4Xu J. Chen Y. Li M. Targets. 2004; 3: 32-38Google Scholar). However, changes of channel density are not readily assayed by existing high throughput technologies. Thus, it is not known to what extent the acquired QT prolongation is contributed to by chronic effects independent from direct block among those compounds that have been clinically associated with cardiotoxicity. A critical first step to address this question is to identify compounds that affect the surface expression of cardiac potassium channels. Recently, we reported the functional recovery after chemobleaching (FRAC) 2The abbreviations used are: FRAC, functional recovery after chemobleaching; hERG, human ether-a-go-go-related gene; MTSET, 2-(trimethylammonium)ethyl-methanethiosulfonate; BFA, brefeldin A; PBS, phosphate-buffered saline; HA, hemagglutinin; HBSS, Hanks' balanced salts solution. assay, which measures the rate of protein trafficking with effective throughput (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). This assay system enables an investigation of many compounds in parallel for their roles in regulation of potassium channels, particularly whether and to what extent they affect protein expression on the cell surface. Using this approach, we have screened a compound library of 2000 chemicals including human drugs, natural compounds, and drug-like chemicals, for either acute or chronic effects on cardiac potassium channels. Intriguingly, comparable numbers of compounds have been found to cause acute block or chronic inhibition. One of the identified inhibitors is celastrol, a bioactive compound from the celastraceae plant, also called the Chinese Thunder God vine (16Ngassapa O. Soejarto D.D. Pezzuto J.M. Farnsworth N.R. J. Nat. Prod. 1994; 57: 1-8Crossref PubMed Scopus (58) Google Scholar, 17Zhou B.N. Mem. Inst. Oswaldo. Cruz. 1991; 86: 219-226Crossref PubMed Scopus (29) Google Scholar). We investigated the effects of celastrol on both channel conduction activity by electrophysiological recording and channel protein trafficking by the FRAC assay (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). Celastrol has an unusual pharmacological profile including both acute and chronic inhibition for Kir2.1 and hERG potassium channels but not for the homologous Kv2.1 potassium channel. Cell lines are established by standard protocols using pcDNA3.1. HEK 293 stable cell lines expressing Kir2.1, hERG, and Kv2.1 channel and human neuroblastoma cell line SH-SY5Y were maintained in Dulbecco’s modified Eagle’s medium/F-12 50/50 medium supplied with 10% fetal bovine serum, penicillin/streptomycin, l-glutamine. The HEK 293 cell lines are supplemented with 500 μg/ml G418 (complete medium). The compound library (Spectrum Collection, 2000 compounds, supplied as 10 mm Me2SO solution) used in this study was purchased from MicroSource Discovery Inc. (Groton, CT). This library consists of mostly human therapeutic drugs or drug-like compounds and natural products. The compounds were diluted with deionized water to reach a concentration of 100 μm with 5% Me2SO. This was used as the 10× compound solution to be introduced to the cell culture. HEK 293 cells stably expressing Kir2.1 were plated at 4 × 104/100 μl complete medium per well in poly-l-Lysine coated 96-well plates. Three hours later, 50 μl of complete medium containing 15 mm sodium butyrate was added to each well and at with 5% of complete medium containing mm was added to the and for Cell and were using a compound and the assay were and on a system after of mm solution was added to the cell and at for each cell was and with complete medium containing mm on an of the 10× compound solution was added to of each cell assay μl of 5% Me2SO solution was added to each well in the first of each cell and μl of 10 μm brefeldin A in 5% Me2SO was added to each well in the of each cell with the compound these as and Me2SO concentration in the cell medium was at or for cell to its the compounds, the cells were at with 5% for each cell was and with complete medium and at for 15 the was to a 96-well The cells were with in in both and cell were measured by an Ion stable cell line was plated at × in with mm were first with complete medium containing mm for of the were with mm for and cells were with medium containing mm of celastrol were added to the cells using in Me2SO and cells were with the compound for the cells were with medium and in medium for 15 at were to plates. were with in in both and were measured on an Ion and was as (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). was the from different for celastrol were by assay to the of hERG hERG stable cell line was plated at × in a One after different of celastrol were added to the cell from Me2SO Me2SO and were used as and at medium were to medium containing the of compounds and mm with the compounds, cells were with and was by medium containing 50 mm the were to a were with in in both and were measured on an Ion and rate was as was the from two different was used to the the of containing 10 to with was maintained by a system were from with the solution containing to with the was which was by currents were using an at and by and by were at and to different or for of potassium channels. current for each of channel were to evaluate the inhibition by the Kir2.1 channels, membrane was maintained at and a of ranging from to currents at each step were hERG channels, the of a of and a step of either or to currents. currents were by an and the were measured by the to the of the Kv2.1 channels, currents were measured were from to at a were using and in and are as was using the HEK 293 cells were with and Kir2.1 or CD8 by using after cells were and in at 4 × were added to each well from or Me2SO and an of Me2SO was added to well used as a were at with compounds for were with and by with mm in for at were with balanced salts solution mm and fetal bovine and with on for were with medium and with for 15 on the cells were with and the surface was measured by using Kir2.1 and hERG stable cell lines were plated in at the density of × The cells were with compounds with Me2SO HEK 293 cells were plated at × and the with CD4 or CD8 were added after of with compounds, cells were with and with mm mm at 4 for the Kir2.1 stable cell were at at 4 for and the were with protein at 4 The were with of and in μl of for at for The were to 10% and to The membrane was with 5% in at 4 and with at with the membrane was with were using the on a the hERG stable cell line and the cell were with for at and on or The were with either or by the were as hERG surface expression were using by the density of the form and the to that of SH-SY5Y cells were plated in at × the cells were with compounds with Me2SO for were with and with μl of Cell were and at 4 100 μl of the was with at for and on a were with or by and the were by The density of the hERG form was and to that of as a identify small that may have acute or chronic effects on Kir2.1 potassium channel we screened a library of 2000 compounds using the FRAC assay (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). The assay of of potassium channels and of by Because the such as MTSET, to the of Kir2.1, which the channel conduction, we are to the rate of channel transport by the to after the channel functional recovery at different after (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). the compounds or after the can whether a compound or the activity by channel proteins. A compound may exert its effect on transport activity of channels. The compound collection used in this of human drugs, natural and drug-like A of the and of the compounds is in 1. The compounds were screened at 10 μm using a Kir2.1 channel stable cell The activity of Kir2.1 was by the of to that of by (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar, PubMed Scopus Google Scholar). compounds a more than of the concentration with that these compounds are to the cell for Hence, they were not in the for the of this compounds at least inhibition of Kir2.1 channel and were for compounds can be by different The compounds no significant of could be a of surface expression direct inhibition of ion conduction by the channels. These two mechanisms of action may be by the compound effects with or compounds with more than reduction in were for the two we the FRAC assay and the with the and the compounds a of inhibition in both the inhibitory effect is independent of the compounds more than significant inhibitory effect in cells but than 10% reduction in their effect is compounds inhibitory effect in both but with a inhibition in the the compound effects may affect both channel activity and surface These results suggest that a significant percentage of compounds affect channel surface expression in cell Celastrol was identified as of the natural inhibition on Kir2.1 channel Because of its therapeutic potential C. Chen J. J. 2005; PubMed Scopus Google Scholar and see celastrol was for more compounds in the screened library to celastrol celastrol and its inhibitory effects in FRAC the the no This that the effects on Kir2.1 channels were In FRAC celastrol inhibition with but 10% inhibition In contrast, a drug which has been reported to block hERG channels D. S. J. J. Pharmacol. 2003; PubMed Scopus Google Scholar), a of inhibition independent of the potency of the inhibitory effect of celastrol, we assay in the of different of celastrol with or The inhibition potency is μm with more than the measured These suggest that celastrol has effects on Kir2.1 both the channel activity and channel protein expression on the cell surface. more acute block of the Kir2.1 channel activity by celastrol, we measured the Kir2.1 currents and after the of μm celastrol using recording of μm celastrol, the cell Kir2.1 current was by This that celastrol can the Kir2.1 channel. whether the acute inhibition effect of celastrol is to cardiac potassium channels, we measured the compound effect on the hERG and Kv2.1 channels Celastrol at 10 μm the hERG current by 10% However, 10 μm celastrol no effect on Kv2.1 a potassium channel. these the and of celastrol in acute inhibition of cardiac Kir2.1 and hERG potassium channels. for at the protein the inhibitory effect of celastrol on Kir2.1 channel surface expression was by using to an Kir2.1 with an was in HEK 293 cells and with an S. Li M. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). of celastrol significantly Kir2.1 protein on the cell surface with cells with Me2SO as In contrast, which inhibition in not the Kir2.1 surface expression The surface expression of the Kir2.1 channel could be the of of transport or reduction in protein We celastrol for effect on the surface expression of CD8 with considerable with either celastrol in Me2SO or Me2SO not the surface expression of CD8 protein In contrast, of BFA, a that trafficking by of the J. J.S. Cell. 56: Full Text PDF PubMed Scopus Google Scholar), CD8 surface of surface expression by that celastrol surface expression of the Kir2.1 protein by with no effect on CD8 In contrast, the surface expression of both Kir2.1 and CD8 by Furthermore, and of Kir2.1 and CD8 that celastrol not affect the protein after for at These results the that celastrol transport of Kir2.1 channel to the cell surface. The acute inhibition by celastrol of both Kir2.1 and hERG potassium currents the question whether celastrol also the biogenesis of We examined whether celastrol could also the surface expression of hERG The hERG channel protein is as a form of and a form of is that the protein can on the cell surface R. A. A. J. Physiol. Scopus Google Scholar). A stable cell line expressing hERG potassium channel was used with celastrol significantly the of the protein but no effect on the protein the hERG protein celastrol could exert its effect by the no effect on of This was by the of CD4 in the and of Celastrol has no effect on the or the extent of of CD4 from we an assay using HEK 293 cells stably expressing hERG channel. This expression system has been to hERG with considerable to Q. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with celastrol at of either or 400 hERG channel by and of surface expression by a potent and at displayed reduction to celastrol at these not have an acute effect on hERG potassium current in not The inhibition of is in with reduction in of evaluate the effect of celastrol on biogenesis of hERG we used a human neuroblastoma cell line which hERG potassium current M. A. A. L. Pharmacol. 1998; 54: PubMed Scopus Google Scholar). The hERG are as a form by E. H. Eur. J. Pharmacol. 2003; PubMed Scopus Google Scholar). of celastrol at both and 400 for a reduction in the form The extent of reduction was by the of of hERG to that of a of and 400 celastrol and with the of the cells with Me2SO of the in both and E. Sci. 1998; PubMed Scopus Google Scholar). from many both and density of membrane are critical for of membrane consists of of transport from the of in the to the functional such as the cell surface. small including drugs and may exert their effects by with their targeted membrane at or to the of However, is known whether and drugs may exert their effects in of of the of effective effects could take place at of protein In FRAC we have a duration for compound effect on The the of some of the other chronic effects including and transcription (15Sun H. Shikano S. Xiong Q. Li M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16964-16969Crossref PubMed Scopus (20) Google Scholar). In this is the of which of compounds that either or inhibition. Using this we have identified and compounds with more than inhibition of Kir2.1 at 10 μm of these also displayed acute inhibition of hERG activity of displayed effects on Kir2.1 in the of than that in the of These results that a considerable of compounds in this library are of effects on channel expression on the cell surface. that causes QT prolongation but has no acute effects on cardiac potassium channels including hERG, KvLQT1/mink, Kv4.3, or the cardiac sodium was to hERG protein expression on the cell surface J.S. Chen X. J. J. Pharmacol. 2005; PubMed Scopus Google Scholar, E. L. A.T. J. Chen D. J. Pharmacol. 2005; PubMed Scopus Google Scholar). these results suggest that chronic effects by certain compounds on channel expression are more for the need to evaluate compound effects on biogenesis of ion channels. Celastrol is a potent and compound from a to the R. C. 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J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). is that celastrol effects on heat shock and potassium channels two different Because of the celastrol has been investigated for therapeutic in treating C. Chen J. J. 2005; PubMed Scopus Google Scholar). is of to evaluate whether celastrol could have effects on cardiac in In addition to pharmacological action on many drugs of especially in Drug 1998; PubMed Scopus Google Scholar). In many of these drug causes of in In the of expression a mechanism that is with J. S. A. M. L. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). These that drug to targeted an important in surface either or Celastrol also acute inhibitory effects with the to with the channel proteins. potassium channels, a of is to potassium channels J. D. C. R. PubMed Scopus Google Scholar). the of celastrol to be investigated in of mechanism of that is a of chronic effects from drugs as a of biogenesis of potassium channels. Thus, chronic exposure to certain drugs may be an important aspect of acquired QT prolongation. We Jan and for Kir2.1 and hERG and of the Li for and on this
Sun et al. (Thu,) studied this question.