Physicochemical Features of the hERG Channel Drug Binding Site

Blockade of hERG K+ channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-π interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K+ channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. Blockade of hERG K+ channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-π interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K+ channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. Long QT syndrome (LQTS) 1The abbreviations used are: LQTS, long QT syndrome; hERG, human ether-a-go-go related gene; Kv, voltage-gated K+; V½, voltage half-point for channel activation; VHSA, van der Waals hydrophobic surface area; WT, wild type; Mes, 2-N-morpholinoethanesulfonic acid; EAG, ether-a-go-go. is a disorder of ventricular repolarization that predisposes affected individuals to cardiac arrhythmia and sudden death. Inherited LQTS is caused by mutations in K+ or Na+ ion channel genes or ankyrin-B (1Keating M.T. Sanguinetti M.C. Cell. 2001; 104: 569-580Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 2Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. DuBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Marec H.L. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (839) Google Scholar). Acquired LQTS is more common and can be induced as an unintended and rare side effect of treatment with many structurally diverse medications. In the past few years, several commonly used drugs (e.g. terfenadine, cisapride, sertindole, thioridazine, grepafloxacin) were withdrawn from the market, or their approved use was severely restricted, when it was discovered that they caused arrhythmia or were associated with unexplained sudden death, albeit very infrequently (3Pearlstein R. Vaz R. Rampe D. J. Med. Chem. 2003; 46: 2017-2022Crossref PubMed Scopus (167) Google Scholar). The molecular basis of drug-induced LQTS is block of human ether-a-go-go related gene (hERG) channels that conduct IKr, the rapid delayed rectifier K+ current important for repolarization of cardiac action potentials (4Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2161) Google Scholar, 5Trudeau M. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1101) Google Scholar). A reduction in IKr prolongs the action potential duration of ventricular myocytes, lengthens the QT interval and increases dispersion as measured by ECG recordings, and increases the risk of torsades de pointes, a ventricular tachyarrhythmia that can degenerate into fibrillation and cause sudden death. In a laboratory setting, it is possible to induce arrhythmia in animals with drugs that block voltage-gated K+ (Kv) channels other than hERG. However, in clinical practice, drug-induced LQTS is always attributable to direct or indirect (via interference with metabolism of a co-administered medication) block of hERG channels (6Redfern 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 (1329) Google Scholar). This understanding has prompted intense efforts to quantify hERG channel activity of new chemical entities during an early stage of the drug development process (6Redfern 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 (1329) Google Scholar, 7Fermini B. Fossa A.A. Nat. Rev. Drug Discov. 2003; 2: 439-447Crossref PubMed Scopus (428) Google Scholar). A better understanding of the molecular basis of hERG channel block could facilitate computer-assisted drug design and enable presynthetic, virtual screening of compounds for hERG activity. Moreover, a description of the physicochemical features of the drug binding site would complement pharmacophore models (8Cavalli A. Poluzzi E. De Ponti F. Recanatini M. J. Med. Chem. 2002; 45: 3844-3853Crossref PubMed Scopus (381) Google Scholar, 9Ekins S. Crumb W.J. Sarazan R.D. Wikel J.H. Wrighton S.A. J. Pharmacol. Exp. Ther. 2002; 301: 427-434Crossref PubMed Scopus (270) Google Scholar) of drugs that block hERG channels and define the molecular basis for receptor fields predicted by these models. Toward this goal, we have used site-directed mutagenesis and voltage clamp analysis of mutant channels expressed in Xenopus oocytes to elucidate the molecular mechanisms of hERG channel block by structurally diverse drugs, including MK-499, cisapride, terfenadine, vesnarinone, chloroquine, and quinidine (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar, 11Kamiya K. Mitcheson J.S. Yasui K. Kodama I. Sanguinetti M.C. Mol. Pharmacol. 2001; 60: 244-253Crossref PubMed Scopus (132) Google Scholar, 12Sanchez-Chapula J.A. Navarro-Polanco R.A. Culberson C. Chen J. Sanguinetti M.C. J. Biol. Chem. 2002; 277: 23587-23595Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 13Sanchez-Chapula J.A. Ferrer T. Navarro-Polanco R.A. Sanguinetti M.C. Mol. Pharmacol. 2003; 63: 1051-1058Crossref PubMed Scopus (120) Google Scholar). These studies identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain and predicted to face the central cavity of the channel (Fig. 1a) that are critical for high affinity binding of these drugs. Kv channels of the Kv1–4 families have an Ile or Val residue in the positions equivalent to Tyr-652 or Phe-656 of hERG (Fig. 1b). This suggests a plausible explanation for why hERG and not Kv1–4 channels are readily blocked by structurally diverse drugs: aromatic residues in S6 are required for high affinity binding. In addition, Kv1–4 channel α-subunits have a Pro-Val(Ile)-Pro motif in the S6 domain that was suggested to cause a bend in the α-helix and alter the shape of the central cavity as compared with that predicted for KcsA, a K+ channel that lacks these Pro residues. The S6 domain of hERG also lacks the Pro-Val-Pro motif and instead has the residues Ile-Phe-Gly in the equivalent positions. Thus, we previously proposed that aromatic residues (2 on each subunit, 8 per channel) that face the inner cavity, and the lack of the Pro-Val-Pro motif, were important determinants of the high affinity hERG channel binding site (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar). EAG channels also lack a Pro-Val-Pro motif and have a Tyr and Phe in the S6 domain but are relatively insensitive to drugs that block hERG. However, EAG channels were made sensitive to block by cisapride by moving the Tyr residue by one position in the S6 domain (14Chen J. Seebohm G. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12461-12466Crossref PubMed Scopus (177) Google Scholar). Pharmacophore models predict that important features of potent hERG channel blockers are 1) a basic nitrogen that is usually protonated at physiological pH and 2) three hydrophobic centers of mass (centroids) arranged in a specific spatial pattern around the centrally located nitrogen (8Cavalli A. Poluzzi E. De Ponti F. Recanatini M. J. Med. Chem. 2002; 45: 3844-3853Crossref PubMed Scopus (381) Google Scholar, 9Ekins S. Crumb W.J. Sarazan R.D. Wikel J.H. Wrighton S.A. J. Pharmacol. Exp. Ther. 2002; 301: 427-434Crossref PubMed Scopus (270) Google Scholar). For many potent hERG blockers, these centroids are aromatic groups. We and others have postulated that π -stacking between aromatic groups of the drug and Phe-656 and Tyr-652 of hERG are required for high affinity binding and channel block (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar, 15Lees-Miller J.P. Duan Y. Teng G.Q. Duff H.J. Mol. Pharmacol. 2000; 57: 367-374PubMed Google Scholar). In addition, because most hERG blockers contain a basic nitrogen, it has been suggested that cation-π interactions with Tyr-652 or Phe-656 might also be required for high affinity binding to the channel. In this study, we systematically investigated these hypotheses by mutating Tyr-652 and Phe-656 to several other amino acids and determined the sensitivity of the resulting mutant channels to block by cisapride, MK-499, and terfenadine. A quantitative comparison between the IC50 values for the mutant channels and the physicochemical properties of the side chain of the mutant residues was used to provide further insights into the molecular features of the hERG binding site. Molecular Biology—Point mutations of hERG subcloned into the pSP64 plasmid expression vector (Promega, Madison, WI) were made as described (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar). Constructs were confirmed with restriction mapping and DNA sequencing. Complementary RNAs for injection into oocytes were with of the expression with were with of hERG 2 use in voltage clamp of and of Xenopus oocytes and were as described M.C. J. Scopus Google Scholar). were at with a voltage clamp PubMed Scopus Google Scholar). were in a 2 2 to pH with were determined with to from to at a of and from a potential of were measured at quantify drug-induced block of hERG, were with to at from a potential of block of current was by the at the of the to a potential that the of or and were as in and in the use to of the of current and were of The of hERG were with a to the half-point and for the voltage of channel effect were to the to determine the drug required for and the are as of and between groups were the were at and physicochemical were for each of the amino acids at Phe-656 the Molecular is a of two-dimensional not the of a when the properties of of of these and and shape and and two-dimensional van der of surface and the values for the amino acids The for of was a J. Chem. Sci. 1995; Scopus Google Scholar) in the M. Drug D. Scholar, A.J. J. Chem. Sci. 2: Scopus Google Scholar). is an that has use in the area of in The to of that with the of In many the direct of to the is The or is identified as that the between the predicted and in values and the and the were on an 2 other analysis and was the of Phe-656 hERG The residue equivalent to Phe-656 in hERG is in (Fig. 1b). However, in most other Kv this amino acid is a Val or Ile and is the residue of a Pro-Val(Ile)-Pro motif that has been proposed to a bend in the S6 domain the site of D. M. Y. G. Nature. 2000; PubMed Scopus Google Scholar). We determined the and of mutating Phe-656 to other residues, including Ile and of the mutant by of Phe-656 to or relatively by to a voltage from a potential of are in The for these mutant channels were and at a voltage when were with (Fig. These properties are to hERG and because the of the from rapid they that the mutant channels a relatively voltage of The voltage of channel of hERG a half-point of and a of of the mutations caused relatively in the for or in the for the of at was the that by of the mutations induced in the of but K+ was not altered the lack of on potential of the current was between the of or and physicochemical as hydrophobicity, surface or of the side group for the amino acid for Phe not of Phe-656 to or reduced expression and of channels (Fig. and the voltage and of were not determined for these mutant of mutant hERG with determined with determined in a new of of hERG sensitivity of Phe-656 mutant channels to drugs was determined by the in current by to of block of and mutant Phe-656 hERG channels with or are in for for these channels are in is important for drug of Phe-656 to the other aromatic amino acids or Tyr altered block by by a of and In the IC50 was by of when Phe-656 was mutated to or mutations that also severely channel (Fig. the IC50 for was when Phe-656 was mutated to the residues or For these residues, the IC50 was by a of and We also determined the sensitivity of all mutant channels to block by cisapride (Fig. and (Fig. to MK-499, the IC50 values for these drugs were when Phe-656 was mutated to Tyr or and the most by to or of Phe-656 to the hydrophobic residues or Ile or effect on channel sensitivity to block by cisapride and of IC50 values and determined from for MK-499, cisapride, and block of Phe-656 and Tyr-652 mutant hERG of IC50 values and determined from for MK-499, cisapride, and block of Phe-656 and Tyr-652 mutant hERG channels of hERG by drugs (e.g. quinidine and is J.A. Navarro-Polanco R.A. Culberson C. Chen J. Sanguinetti M.C. J. Biol. Chem. 2002; 277: 23587-23595Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 13Sanchez-Chapula J.A. Ferrer T. Navarro-Polanco R.A. Sanguinetti M.C. Mol. Pharmacol. 2003; 63: 1051-1058Crossref PubMed Scopus (120) Google and the potential to determine IC50 can be However, Curran M.E. Keating M.T. Sanguinetti M.C. Res. PubMed Scopus Google Scholar) and R. Res. Scopus Google Scholar) not block hERG in a and cisapride voltage F. T. Escande D. I. J. Pharmacol. Exp. Ther. 2001; Google Scholar). The Phe-656 that altered the voltage of was the IC50 values for this mutant channel were not We also for voltage of block for and was for mutant channels by all three drugs for each not Thus, it is to block of hERG by these drugs at to determine IC50 The for the in IC50 to for each mutant channel was as a of a of and properties of the amino acids for The for analysis were three the side chain surface of from to and residue surface and the two-dimensional from For all the was for and for terfenadine. The that was correlated with in for all three was the two-dimensional van der Waals hydrophobic surface area is by the van der Waals surface of hydrophobic in for the of the of and was in to the surface area not this into the for the of physicochemical in a of the predicted in for the three drugs on the values as described values for residues can be determined by the for the residue into each of the three in the of of for between physicochemical properties and drug sensitivity of Phe-656 mutant chain surface surface in a new The between and values were further the for residues that altered the channel were from the The for and and are in related to high as including the S.A. J. Chem. Sci. Scopus (839) Google a from in and a of the of hydrophobic and from in that they for all in each than the hydrophobic In to the of two-dimensional van der Waals surface of and were the more correlated but not as correlated as the the all compounds and all amino was the most correlated and the most and were always the and most correlated a that is a the The was used to for models and identified the of and the of nitrogen as a of in for all three In this is a for residues that contain more than one nitrogen and For for the of predicted IC50 values was when was the but the to the quantitative activity to for MK-499. The for the for cisapride and were as 1) 2) The values from these are in A better could also be by from the analysis because this residue the basic nitrogen of the drug and binding affinity more than on hydrophobic surface area of the side Together, these that the important physicochemical of Phe-656 is hydrophobic not per at for hERG residue equivalent to Tyr-652 in hERG is in in Kv1–4 this amino acid is Ile (Fig. 1b). Tyr-652 was mutated to 8 other residues to determine the effect of the and of the side group on drug of Tyr-652 were well and the mutant channels K+ and relatively properties (Fig. and might by to the group or cation-π or π -stacking with the of of Tyr-652 to Phe or reduced or not the sensitivity to (Fig. a and and hERG, block of current was not were for cisapride (Fig. and (Fig. that an aromatic group be a side chain is not an essential of this residue for drug hERG was insensitive to (Fig. and reduced sensitivity to cisapride and (Fig. and for the lack of importance of the group of of Tyr-652 to other or hydrophobic residues also reduced drug block (Fig. For the IC50 for was for and for The effect of Tyr-652 mutations on IC50 values for all three drugs are in The the voltage of by but block by cisapride was For the reduction of current between and at potentials from to of and hERG channels by all three drugs were also of voltage this of potentials not was a between hydrophobic of residue and sensitivity to drugs. For the for the of and in for was The for this was for cisapride and However, the of was and was between in drug when the aromatic residues were from the the between and in for the Phe-656 mutant channels and for MK-499, cisapride, and terfenadine, We that an aromatic side group for residue is required to high potency block by these three drugs. Introduction of Pro-Val-Pro into S6 of hERG and Drug Kv channels have a Pro-Val-Pro motif in the S6 domain (Fig. 1b). This is Ile-Phe-Gly in hERG, Phe is The of 2 Pro residues in the S6 domain induce a bend in the domain D. M. Y. G. Nature. 2000; PubMed Scopus Google Scholar) and the of the inner We mutated and to Pro and Phe-656 to Val to a Pro-Val-Pro motif in hERG. with Pro-Val-Pro not at potentials as as (Fig. and were relatively insensitive to (Fig. blocked Pro-Val-Pro mutant channels by of these channels was readily of because the channels could not and drug in the central cavity as with hERG J.S. Chen J. Sanguinetti M.C. J. 2000; PubMed Scopus Google Scholar). of Pro-Val-Pro reduced drug this be to an altered shape or of the inner cavity because the mutations also altered We previously that the most important determinants of the drug binding site of hERG were 2 aromatic residues located in the S6 domain (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar). we these findings to that hydrophobic of Phe-656 and of Tyr-652 determine the sensitivity of hERG to block by structurally diverse drugs to cause of Phe-656 to or Tyr caused in properties of the channel and IC50 for block by MK-499, cisapride, or terfenadine. In of Phe-656 to or channel and drug However, in drug sensitivity was not related to gating. For the and voltage of for and were drug was relatively insensitive of the amino acid side group was the of the altered potency of drug block induced by of The position of Phe-656 in the S6 domain of hERG why the of mutant channels was models of the hERG channel on the of J. R.A. A. R. Science. PubMed Scopus Google Scholar) or Y. A. M. R. Nature. 2002; PubMed Scopus Google Scholar) channels that Phe-656 residues from all face the inner cavity and one in the and of the channel. In the the Phe-656 residues the of a of Phe-656 to residues with side might the of the that when the channel is in the it is of K+ in the of the side chain by a group to Val or from to is to channel might why channels with Phe-656 mutated to the residues and not at Introduction of a Pro-Val-Pro motif into the S6 domain of hERG also reduced drug However, the of this is because the Pro-Val-Pro also altered properties of the channel. aromatic side group for residue was required to drug We J.A. Ferrer T. Navarro-Polanco R.A. Sanguinetti M.C. Mol. Pharmacol. 2003; 63: 1051-1058Crossref PubMed Scopus (120) Google Scholar) and others (8Cavalli A. Poluzzi E. De Ponti F. Recanatini M. J. Med. Chem. 2002; 45: 3844-3853Crossref PubMed Scopus (381) Google Scholar, R.A. Vaz J. Chen M. J. Rampe D. Med. Chem. 2003; PubMed Scopus Google Scholar) previously suggested that the basic nitrogen of hERG channel blockers could a cation-π interaction with (8Cavalli A. Poluzzi E. De Ponti F. Recanatini M. J. Med. Chem. 2002; 45: 3844-3853Crossref PubMed Scopus (381) Google Scholar) have a molecular analysis pharmacophore for hERG that a of between the basic nitrogen and a critical hydrophobic of mass for potent hERG channel a different R.A. Vaz J. Chen M. J. Rampe D. Med. Chem. 2003; PubMed Scopus Google Scholar) predicted a between the basic nitrogen and this These well with a predicted of between Tyr-652 and Phe-656 in of hERG on the (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar). Thus, Tyr-652 a cation-π interaction with the centrally located basic nitrogen of MK-499, cisapride, terfenadine, and many other potent hERG For drugs (e.g. π -stacking is the interaction with It is important to that not all hERG channel blockers to the receptor site. For we previously (10Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (861) Google Scholar) that block of hERG by was affected but it was affected at all by cisapride or with mutations of located at the of the or the residue proposed to be the for of the S6 domain during channel Y. A. M. R. Nature. 2002; PubMed Scopus Google Scholar). Thus, all three drugs with Tyr-652 and Phe-656, they with to interaction with residues located to the In addition, potency blockers of hERG not to as with the S6 aromatic residues. For block of hERG by is affected by or mutations A. H.J. J. Pharmacol. 2003; PubMed Scopus Google suggesting that other residues are more critical of the binding site for this drug J.S. J. Pharmacol. 2003; PubMed Scopus Google Scholar). In the potency for block of hERG channels by three structurally diverse drugs cisapride, was well correlated with the two-dimensional approximation of the van der Waals hydrophobic surface area for the side chain of residue and was not related to per In an aromatic side group at residue was essential for high affinity block, suggesting the importance of a cation-π interaction between Tyr-652 and the basic nitrogen of the drugs. Together, these findings provide a refined molecular understanding of the hERG binding site and specific residues to the receptor fields previously predicted by a pharmacophore on quantitative activity analysis of potent We and for