Side Chain Flexibilities in the Human Ether-a-go-go Related Gene

4266 J. Med. Chem. 2009, 52, 4266–4276 DOI: 10.1021/jm900002x

Side Chain Flexibilities in the Human Ether-a-go-go Related Gene Potassium Channel (hERG) Together with Matched-Pair Binding Studies Suggest a New Binding Mode for Channel Blockers Ulrich Zachariae,*,† Fabrizio Giordanetto,‡ and Andrew G. Leach† †

Physical and Computational Chemistry, AstraZeneca, Mereside, Alderley Park, Macclesfield SK11 4TG, U.K., and ‡Lead Generation, AstraZeneca, Pepparedsleden 1, 43183 Molndal, Sweden Received January 2, 2009

The cardiac hERG K+channel constitutes a long-standing and expensive antitarget for the drug industry. From a study of the flexibility of hERG around its internal binding cavity, we have developed a new structural model of drug binding to hERG, which involves binding orthogonal to the pore channel and therefore can exploit the up to 4-fold symmetry of the tetrameric channel. This binding site has a base formed by four tyrosine side chains that complement reported ligand-based pharmacophores. The model is able to rationalize reduced hERG potency in matched molecular pair studies and suggests design guidelines to optimize against hERG not relying simply on lipophilicity reduction. The binding model also suggests a molecular mechanism for the link between high-affinity hERG binding and C-type inactivation.

Introduction hERG is the gene coding the R-subunit of a K channel in cardiac muscle that controls the length of the cardiac action potential and hence the heart rate.1-3 Undesired drug blockade of K+ ion flux in hERG can lead to long QT syndrome, eventually inducing fibrillation and arrhythmia.2,3 hERG blockade is a significant problem experienced during the course of many drug discovery programs. Much effort has been made to provide tools to help rationally improve molecules to increase their chances of being safe medicines, including computational approaches.4,5 Notably, crystal structures of related channels and homology models of hERG based upon them have been reported, although the physiology of these other channels usually differs from that of hERG, which readily undergoes C-type inactivation upon membrane depolarization.2,6 C-Type inactivation is a process that leads to inhibition of ion flux in opened K+ channels and is believed to occur near the extracellular channel entrance.7,8 It is thought that the channel-forming section of hERG resembles that of structurally known K+ channels, which are formed by a tetramer of channel subunits consisting of a K+ ion selectivity filter, the pore helix, and two transmembrane helices.2,3,6 The tetramer forms a large internal, water-filled cavity. Previous studies have shown that this internal cavity is likely to be the key drug interaction site in hERG.2,3,9 More specifically, drug binding has proven to be most sensitive to alanine mutation of aromatic residues Tyr652 and Phe656,3,9 a

+

*To whom correspondence should be addressed. Phone: +44-1625518531; Fax: +44-1625-232693. E-mail: U.Zachariae@googlemail. com. a Abbreviations: hERG, human ether-a-go-go related gene potassium channel; TM, transmembrane; QT, time interval between Q and T spikes in electrocardiograms/cardiac repolarization phase; KcsA, potassium channel from the prokaryote Streptomyces lividans; TEA, tetraethylammonium; TBA, tetrabutylammonium; MD, molecular dynamics; CCR-5, chemokine (C-C motif) receptor 5; 5-HT1A, 5-hydroxytryptamine receptor 1A.

pubs.acs.org/jmc

Published on Web 06/17/2009

which are assumed to line the internal cavity. Mutations of polar residues presumed to be at the bottom of the K+ selectivity filter (see Figures 1 and 2) which forms another face of the cavity were reported to exert similarly disruptive effects on high-affinity drug blockade as those of Tyr652 and Phe656.3,9 Mutants in the same region that abolish C-type inactivation are incapable of binding drugs with high affinity.10 Present structural models of hERG blockade cannot readily explain either observation. Since all published structural studies of hERG block are based on static homology models of the channel, we intended to examine whether conformational flexibility can be found around the hERG cavity, with potential implications for drug binding modes to hERG. Literature reports of medicinal chemistry approaches that have been successful at reducing or eliminating hERG potency provide a source of inspiration for medicinal chemistry programs,4,11 and pharmacophore models provide some coherent view of the molecular features that lead to potency and some hints at how to reduce it.5,12,13 We have obtained a structural model of the binding site of hERG inspired by molecular dynamics simulations that we believe unifies these strands of work and that can be used to design molecules with reduced hERG potency. Results and Discussion Construction of the hERG Model. A number of crystal structures of K+ channels have been published, representing various degrees of channel openness or fully closed states. We selected the closed form of KcsA,14-16 solved as complexes with the channel blockers tetraethylammonium (TEA) or tetrabutylammonium (TBA), as a structural template for construction of a hERG homology model. The observation that methanesulfonanilides can be trapped in the internal cavity of hERG upon closure suggests that there is sufficient room for bound drugs in the closed state and that r 2009 American Chemical Society

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Figure 1. (A) Upward-facing conformation of the Tyr652 side chains often found in conventional hERG homology models that use KcsA (Phe103) as a structural template. SF denotes the selectivity filter of the K+ channel, P denotes the pore helix, and S5 and S6 denote transmembrane helices that form the scaffold of the channel. (B) Down-facing state of the Tyr652 side chains derived from molecular dynamics simulations having implications for the size and shape of the internal cavity of hERG.

this state and states like it are likely to be relevant to hERG blockade by drug molecules.17 The sequence alignment that was employed (see Supporting Information) suggests that Tyr652 replaces Phe103 of KcsA, which lines the internal cavity, and the aromatic ring of Phe656 replaces the polar residue Thr107 of KcsA. Several multinanosecond simulations of a homology model of hERG, immersed in a membrane bilayer, were carried out, inspired by results obtained from simulations of the K+ channel KcsA (see Supporting Information). The side chain of Phe103 assumes an upright position in KcsA (cf. Figure S1), forming the boundary between the hydrophilic internal cavity and the membrane; common homology-modeling packages usually place Tyr652 also in an upward-facing conformation (Figure 1A). During the MD simulations, all four Tyr652 rings spontaneously adopted a “down” conformation (Figure 1B) in early stages of the trajectories, pointing into the hydrophilic, water-filled cavity. The rotation into the cavity can be ascribed to the side chains of Phe656 approximately one helical turn below Tyr652, which provide a nonpolar environment at the bottom of the channel cavity and can provide beneficial stacking interactions (Figure 2A). Also, tyrosine residues usually partition into lipid/aqueous interface regions rather than highly hydrophobic regions.18 The “down” conformations

Figure 2. (A) MD-derived hERG model. The central cavity is shown as a blue surface meshwork between the selectivity filter (SF), pore helix (P), and helix S6. EC denotes the extracellular side of the membrane protein. Orange broken lines highlight the H-bond between Asn629 and Ser620 (see text). Subunit 4 was omitted for clarity. (B) Docked binding mode of dofetilide (orange) in hERG showing the favorable fit of the molecule in the internal cavity (blue mesh), viewed perpendicular to the channel axis. (C) Binding mode of dofetilide viewed along the channel axis from the extracellular side showing the stacking of aromatic rings onto two Tyr652.

open small gateways into the lipid membrane. These gateways to the membrane (or transmembrane domains S1-S4) could rationalize why the hERG cavity can bind significantly larger drugs in its closed state than its size (as projected from KcsA) may suggest. The specific polarity and inherent flexibility of Tyr side chains lining the hERG cavity, as opposed

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to the more lipophilic Phe103 in KcsA or Ile470 in Shaker, could thus serve as an explanation for why hERG is so adaptable and promiscuous at binding large drugs. The side chains of Ser620 and Asn629 were restrained in the simulations so that they formed a direct hydrogen bond; this bond is a switch that permits hERG channels to undergo C-type inactivation.6-8 We assume that the channel’s ability to inactivate rapidly requires a direct interaction here, in accord with experimental data showing that a conservative mutation to a slightly bigger residue, Ser620Thr, suffices to interfere with inactivation of hERG.10 A direct link also agrees with structural data on KcsA mutants of the corresponding residues Glu71-Asp80, where a bridging water molecule, resolved in the X-ray structure, cannot completely recover C-type inactivation in the Glu71Ser mutant.19 The applied structural restraint led to a moderate bend of the pore helices, slightly increasing the lateral size of the cavity further. To investigate the effect the altered shape of the cavity might have on drug binding, a hERG structural model was constructed and used for subsequent docking calculations. It included the bend in the pore helices and “down”-facing Tyr652 side chains and thus “open” gateways toward the S1-S4 domain, although the Tyr652 side chains were modeled as rotamerically flexible in the docking runs. The original C4 symmetry of K+ channels was reimposed. The resulting hERG model and cavity shape are shown in Figure 2A. Relationship between Ligand Affinity and Lipophilicity. Affinity for the hERG channel is often highly dependent on the lipophilicity of the ligand.20 Many of the QSAR type approaches to modeling hERG potency single out log P, log D, or similar properties as the most influential.4,12,13,20,21 Most of the literature data sets that were investigated as potential probes of the new structural model being proposed here show this trend; clogP values for the large data sets found in Fletcher et al.,22 Owen et al.,23 and Bell et al.,25 plotted against hERG potency in the Supporting Information, clearly show a correlation between the two, while the data set of Rowley et al. shows population only of the two diagonal quadrants, indicating a key role for lipophilicity.26 The hERG potency of most compounds follows the rule that an increase in lipophilicity leads to more potent hERG binding. The macrocyclic compounds from Bell et al. exhibit a somewhat more complicated pattern, however.24 One reasonable deduction from this is that one good strategy for lowering hERG affinity is to lower the lipophilicity of a molecule.20 Such data sets in their totality are not very useful for validating the model; so long as the fairly hydrophobic cavity formed by the “down” conformation of the Tyr side chains is big enough to fit these ligands in them, the observed affinity can be viewed simply as a partitioning between the aqueous and hERG channel environments driven by nonspecific interactions. One aim of this study is to find specific changes in the molecular scaffold of druglike molecules that are capable of decreasing hERG liability independent of lipophilicity and test whether the protein structure is consistent with them. These are most likely to depend upon the details of interactions between the ligand and protein. Matched molecular pairs in which a small change in the molecular scaffold leads to a considerable lowering of hERG potency without a decrease in lipophilicity provide this kind of data set, and a number of them have been highlighted by Jamieson et al.4 The lead optimization of maraviroc by

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Price et al. and that of N-{3-[4-(4-cyclohexylmethanesulfonylaminobutyl)piperazin-1-yl]phenyl}acetamide (PRX-00023) by Becker et al. provide further sets.11,27 The concept of matched molecular pair analysis has been described elsewhere.28 Nonetheless, some of the lipophilicity dependent data sets provide a check on the model and test its robustness with regard to a more diverse set of chemical space. Thus, a set of antipsychotics from Ekins et al.29,30 and compounds from Bell et al.,24,25 Fletcher et al.,22 and Rowley et al.26 were also investigated as described below for matched molecular pairs.28 The results are summarized in the Supporting Information. All compounds exhibiting potency toward hERG fit into the cavity in perpendicular binding modes (see below) and obtain reasonable to high scores. They interact strongly with the array of Tyr652 side chains and the selectivity filter. They therefore provide support for the model if only by not being inconsistent with it. Matched Molecular Pair Study of hERG Affinities. Docking was used to investigate the SAR of matched pair binding to our hERG model, employing the program GOLD.31 The conformational states of the side chains of all four Tyr652 residues were taken from rotamer libraries; the side chains could each adopt one of four energetically favorable rotameric states.32 The simulations had shown that, in particular, up and down conformations are accessible. The binding site can therefore access five different combinations of up and down conformations of the Tyr side chains. This is in addition to the all four up binding site seen in the KcsA crystal structure to which binding is presumably weaker because of the reduced enclosure. Each of the conformations presents a differently sized and shaped (mostly lipophilic) pocket, the sort of pocket that is consistent with the observed trend for hERG binding to track with lipophilicity.4,12,13,20 The GOLDscore scoring function was used to rank the docked poses. It is optimized for finding poses very close to crystallographically determined ligand binding modes. As stated explicitly in the GOLD manual, GOLDscore is not designed to reproduce affinity trends among larger sets of molecules.33 It has been widely recognized that the scoring functions commonly used in drug design are capable of generating meaningful poses but fail at predicting ligand affinity.34,35 Therefore, scoring functions alone cannot be used to validate or invalidate a homology model. The crystallographic conformation of ligands is usually found among the better-scoring poses but does not necessarily correspond to the highest-ranking pose.34 In the context of very similar molecules, such as the matched pairs described below,28 the scoring problem of docking two molecules has conceptually been reduced to the slightly perturbed scoring problem of a single molecule. The correct pose can be found among the higher-ranking poses of most molecules such that an analysis of a large number of exhaustive dockings on matched pairs may be able to approximately rank the differential affinity of the two molecules for the proposed protein structure. In most of the cases described, this approach reproduced differential experimental affinities in a qualitative way. The averaged GOLDscores for the best few (usually 20) poses in most instances identified the more hERG-active molecule in a pair. However, we emphasize that GOLDscore is not inherently designed to reproduce target (or antitarget) affinities of large data sets correctly33 and can therefore (if at all) be used only in the context of closely related matched molecular pairs and not in QSAR-like studies across larger sets of molecules.

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We also emphasize that the analysis of the docked poses and their molecular interactions, as opposed to the scores, should serve as the key source for understanding the SAR in all cases and should reemphasize the complicating effect that changes in bulk lipophilicity of the ligand can bring by driving

Figure 3. (A) Schematic drawing of our MD-derived hERG model showing a ligand (cisapride) bound perpendicular to the channel axis. The ligand is shown in orange: EC, extracellular side; IC, intracellular side. (B) Schematic model of drug binding in the closed state of hERG as used in previous structural studies.3,6,27 The channel axis is denoted as z.


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potency through nonspecific partitioning. Our study here therefore focuses on the analysis of binding modes. For comparison, the obtained scores are given in the Supporting Information. In Figure 2, panels B and C, is shown the binding mode of the hERG blocker dofetilide.10 It fundamentally differs from previous poses in that it was found to be perpendicular to the channel axis so that the 4-fold symmetry of the channel interacts synergistically with the roughly 2-fold symmetry of the ligands (Figure 3). The central charge binds close to an original K+ binding site on the axis, an electrostatically favored place, while the aromatic side groups stack onto the aromatic side chains of two oppositely located Tyr652. The polar center of the molecule additionally interacts with the lower part of the selectivity filter through hydrogen bonds (residues Thr623, Ser624, Val625; see Figure 2B). In the case of terfenadine by contrast, a slightly less potent binder, the adjacent phenyl rings on the same side of the molecule stack on two neighboring Tyr652 side chains rather than opposite ones (Supporting Information Figure S2), reemphasizing that the cavity varies in size and shape through the flexibility of Tyr652. The binding position of cisapride is shown in Figure S3 and further corroborates the model. The model is consistent with effective binding of the more potent known hERG blockers, all of which yield well scored poses by GOLD (see below). The first of the matched pairs type of molecules studied is taken from the development of maraviroc (4), Pfizer’s CCR5 antagonist. Price et al. describe how structural changes to an initial lead permitted increases in potency against CCR5 and the elimination of binding to the hERG channel.11 Focusing on the final fixes to hERG, the process started from compound 1 with a cyclobutyl left-hand side. A binding mode was proposed by Price et al. for an earlier lead compound, but it is not clear that this model provided the insight that the transformation from 1 to 2 to 3 to 4 (Figure 4) might be beneficial.11 Indeed, from the picture of the binding mode presented, it is likely that the region of the ligand that is varied in this series is in a fairly open part of the pore such that potency might be expected to follow increases in lipophilicity. The docking poses for 1- 4 in our homology model (see Figure 5) suggest that the two ends of the molecule protrude toward the cavity edges into the narrow gateways at either side of the channel. The drop in hERG potency that is observed experimentally, most notably for maraviroc itself (4), corresponds to increasing clashing with the edges of the channel for 2 and 4 and to the need to adopt a more compact but higher energy gauche conformation for 3. Maraviroc (4) itself also has to change conformation in order to fit into the pocket at all. The binding mode of these fairly rigid compounds suggests that bulky, nonplanar substituents at either end of the molecule might be beneficial and that having them at both ends ought to produce a cooperative loss of potency.

Figure 4. Compounds taken from the optimization away from hERG of the CCR5 antagonist maraviroc (4).11 The log D values were taken from this reference.


Figure 5. (A) Binding mode of 1 in the hERG homology model (peach), viewed perpendicular to and along the channel axis, respectively. (B) Binding mode of 2 (peach), viewed perpendicular to and along the channel axis. (C) Binding mode of 3 (peach), viewed perpendicular to and along the pore axis of hERG. (D) Pose of 4 (peach) binding to hERG, viewed perpendicular to and along the pore axis.

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With only one end providing a steric block to protrusion through the gateways, the molecule might slide across the channel and push the other end further out; this is prevented by adding bulk at both ends, as achieved with the i-Pr triazole and the difluorocyclohexyl groups of maraviroc. The drop in potency in this set occurs despite an increase in lipophilicity. An additional block to maraviroc binding might arise from the perturbation of the axial-equatorial preference for the amide substituent on the difluorinated cyclohexane. Whereas on a cyclohexane ring with CH2 at the 4 position B3LYP/6-31G* calculations (see Experimental Section) suggest a preference for the amide to be equatorial by ∼1.7 kcal/ mol, this is reduced to 0.3 kcal/mol in the CF2 case. The axial conformation fits less well into the hERG model, and hence, an entropic penalty to binding might be expected to supplement the penalty incurred by a poor fit to the cavity. The second set of compounds was highlighted in a review by Jamieson et al. of examples of small structural changes that modulate hERG potency.4,24,25,36 Of particular interest to us, they identify a set of small changes that are either lipophilicity neutral or involve a small increase in lipophilicity. We selected three of these changes, the conversion of 5 to 6, 7 to 8, and 12 to 13, for further analysis. The change from 5 to 6 represents a subtle change to a small flexible molecule, from 7 to 8 a change in a large rigid molecule, and from 12 to 13 the rearrangement of molecular features. Two of these changes (5-6, 7-8) represent cases in which a considerable rise in lipophilicity is recorded, and in two of the three sets, optimization against hERG is achieved by increasing the size of the molecular scaffold. Examination of the binding modes of the more potent hERG inhibitors in each matched molecular pair would suggest the transformation that proved successful in each case, as well as a number of alternatives, some of which are described below. These binding modes are shown in Figures 7, 9, and 12. Compounds 5 and 6 come from the work of Blum et al. on a series of neuropeptide Y Y5-receptor antagonists.36 The binding mode of 5 (Figure 6) shown in Figure 7 shows that the Tyr652 side chains interact favorably with the imidazole ring through aromatic-aromatic interactions. The ethanolamine side chain of 5 trails out of the binding pocket through the entrance of one of the gateways at the side. This is a congested region of the pocket, and adding bulk at the position adjacent to the amide will not be well tolerated, as is observed; poses that retain most of 5 and 6 in almost identical positions differ in GOLDscore by over 10 units, emphasizing the penalty of introducing bulk at this position. The binding mode also suggests that dimethylation next to the alcohol might also reduce potency, although the tertiary alcohol may be unstable. The authors of this work rationalize this change as rigidifying the chain; this may play a part in the potency modulation but probably a smaller one than the size effect described.36 A set of compounds (7-9) shown in Figure 8 from the work of Bell et al. on farnesyltransferase inhibitors have also

Figure 6. Compounds taken from the optimization of a set of neuropeptide Y Y5-receptor antagonists.36 The clogP values were taken from Jamieson et al.4

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Figure 7. Binding mode of compound 5 (A) and the modified compound 6 (B), which is a less potent hERG binder.

Figure 8. Compounds taken from the optimization of a set of farnesyltransferase inhibitors.24 The clogP values were taken from Jamieson et al.4

been studied.24 The binding mode of the macrocycle 7 is shown in Figure 9 and highlights that this has a very snug fit to the channel, in a binding mode in which only one of the four tyrosine side chains is flipped down. The rigid shape of 7 is able to make good aromatic-aromatic interactions with two of the Tyr side chains in their up conformation. Expansion of the cyclopentyl ring in 7 makes the molecule too big to fit as well, despite the increase in lipophilicity as indicated by the increase in content of short contacts identified by the GOLD pose viewer which increase both in number (from 15 to 18) and in severity. The authors note that adding an O instead of a CH2 to generate 9 has a minimal effect on hERG potency.24 This cyclic ether benefits from the O atom being able to interact favorably with the side chain of Ser624 as is shown in Figure 9. This permits the larger size of the molecule to be accommodated and enhances potency (compared to 8) despite the decrease in lipophilicity. The model is not able to differentiate the difference between the corresponding epimeric pair that is also reported in the same paper, but the experimental difference in that case is much smaller than for 7 and 8 (the two may be within experimental error of one another in fact). One feature of a protein based model differing from physical descriptor based modeling is that particular stereochemical arrangements can be distinguished. In the publication from which 7-9 come, there are three sets of compounds that are epimeric pairs with one stereocenter defined, the other unknown. The protein structure shows a clear preference for one of the epimers in each case, and we are thus able to predict that for compounds 10a and 10b (compounds 26 and 27 in the original paper) and 11 (compound 36 in the original paper) the more potent hERG binders have the stereochemistry indicated in Figure 10. A pair of compounds taken from optimization of a different series of farnesyltransferase inhibitors have also been studied and are detailed in Figure 11.25 The binding mode of 12 shown in Figure 12 has all four Tyr side chains flipped down and positions all three of the ligand aromatic rings where they can interact in a π-π fashion with three of

Figure 9. Binding modes for compounds 7 (A), 8 (B), and 9 (C) in the hERG channel. A beneficial interaction that may be experienced by 9 with Ser624 is indicated with a dashed red line.

them. At the same time, the carbonyl in the ring is able to interact favorably with Ser624. The carbonyl transposition


in 13 causes a change of shape; notably the meta-chlorophenyl group is twisted with regard to the carbonyl, which in turn is coplanar with the two ring bonds attached to the nitrogen of the amide. This effectively blocks one face of the aromatic ring, preventing it from forming either such optimal π-π interactions as 12 or a hydrogen bond to Ser624. In both of the latter two cases, 7 and 12, the authors of the original work note the general correspondence between hERG potency and in vivo cardiotoxicology findings but other than those transformations that reduce lipophilicity give no indication of how the beneficial changes were designed.24,25 Finally, a recent example of hERG optimization by design is given by Becker et al.,27 where hERG optimization of a 5-HT1A agonist is described. Selected compounds are detailed in Figure 13. The docked binding modes of compounds 15 and 16 are shown in Figure 14. Compound 15 was obtained from compound 14 through optimization of target potency but also introduced a raised potency for hERG despite a reduced lipophilicity. Our model clearly ranks 15 as the most potent compound in the set (not just the two compounds highlighted here), in agreement with observed potency. The docked pose for 15 suggests π-π interactions for the p-tolyl group, and hydrogen bonds to the two acceptors of the sulfonamide make it a good pose (Figure 14A). The model does distinguish 14 and 15 in terms of scoring, although the differences are difficult to account for and presumably represent a less favorable balance between the internal strain and binding to the protein when the moderately twist-inducing o-OMe substituent is in place in 14. The model very clearly explains the decreased hERG affinity for 16 (compared to 15), as it places the cyclohexyl group in contact with one of the Tyr side chains (Figure 14B). This hydrophobic-π interaction is less effective at stabilizing the bound ligand than the π-π interaction present in 14 and 15. In this section, a number of examples of hERG binders and close structural analogues have been used to demonstrate that our model is consistent with much of the observed SAR. Conventional models of hERG blockade (either lipophilicity dominated QSAR models or structural models based on an unmodified closed state) are not able to so

Figure 10. Predicted stereochemistry of the more potent epimers of 10a,b and 11 taken from Bell et al.’s optimization of farnesyltransferase inhibitors.24

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readily explain the success of those transforms that rely on structural modifications that raise lipophilicity or leave it unchanged. Most of the matched pairs presented here represent optimization by modifications at the extremities of the molecule or by increasing the general size of the molecule. It is much harder to understand why these changes led to a loss in hERG potency when a longitudinal binding mode is assumed. In that mode, adding groups to the peripheral sections of the compounds would either enhance interaction with hERG in the region of the cavity or interact strongly with residues at the intracellular end of S6 (cf. Figure 3), and thus, it is likely that this would interfere with channel closing, such as seen recently with hERG activators.37 In contrast, those residues observed to interact strongly with hERG blockers are located in the center of S6 and at the bottom of the selectivity filter.2,3,9,10 The general architecture of potent hERG blockers, as captured by previously published pharmacophore models, is much more compatible with binding perpendicular to the pore axis, as it naturally reflects many features found in the pharmacophore models such as their inherent symmetry. The agreement is outlined in the following section. Comparison with Pharmacophore Models. The binding modes and binding site can provide an interpretation for the origin of a number of published observations and pharmacophores concerning hERG potency. The base of the binding site as formed by all four Tyr652 side chains in the flipped down conformation is shown in 2D cartoon fashion in Figure 15A with some key distances indicated. The corresponding distances for the most potent compound used to derive most of the pharmacophore models, astemizole, in its best scored docked pose are shown in Figure 15B. These are compared (in Figure 15C) with some ligand derived geometrical parameters (pharmacophores) that were found to correspond to potent hERG binders. The pharmacophores are docked into our hERG model in Figure 15D.

Figure 12. Binding modes of compounds 12 (A) and 13 (B) in the hERG channel.

Figure 11. Compounds taken from the optimization of a different series of farnesyltransferase inhibitors.25 The clogP values were taken from Jamieson et al.4

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Figure 13. Examples taken from a lead optimization program for 5-HT1A agonists by Becker et al.27 The clogP was computed using the AstraZeneca c-lab platform (see Supporting Information).

Figure 14. Docked poses of compounds 15 (A) and 16 (B).

These are limited, for the sake of illustration, to the interactions likely to be with the Tyr side chains (hydrophobe and aromatic) and exclude a number of acceptor features that we believe would be consistent with interaction with residues in the lower part of the selectivity filter. This part of our structural model is defined with less confidence than the base formed by the tyrosine side chains. A few hydrophobic points are also excluded because they fall some way from the plane defined by the features highlighted. These may also involve interaction with the bottom of the selectivity filter or less optimal interaction with the array of Tyr side chains. In each case these other features are indicated in Figure 15. Pharmacophore features may also be present because of the limited structural variation in the training set used to derive them. Our interpretation of the pharmacophore models is described briefly below. (i) In their 1992 paper on class III electrophysiological agents, Morgan and Sullivan identify a generic substructure Ar-L-NR2 where Ar is an electron poor aromatic (phenyl para substituted with nitro, cyano, NHSO2Me, and imidazol-1-yl), L is a one to four atom spacer, and NR2 is a basic amine.38 The most potent compounds have a second L-Ar substituent on

the amine. This geometrical arrangement corresponds roughly to an amine spaced ∼2.5 to ∼9 A˚ away from an aromatic ring, which fits very well with the geometry we propose for the binding site. If the basic nitrogen sits along the central axis where the electrostatics are tuned to stabilize K+ cations, then the aromatic ring ought to be able to stack favorably onto one of the flipped down Tyr side chains. Furthermore, the potency benefit of a 2-fold symmetric or pseudosymmetric structure fits very well with the up to 4-fold symmetry of the binding site we have obtained. (ii) Ekins et al. derived a five-point pharmacophore including the basic center and four hydrophobic points.29 The general arrangement is consistent with hydrophobes that are distributed around a ring about the size of that formed by the four Tyr side chains. A three-point subset shown in part ii of Figure 15C is particularly well suited to interacting with two adjacent Tyr side chains. (iii) By contrast, the pharmacophore derived by Cavalli et al. consisting of three aromatic points and a basic nitrogen fits well with the three aromatic pharmacophore points stacking onto three adjacent Tyr side chains.13 This is well exemplified by the binding of astemizole in Figure 15B. (iv) In his 2008 paper, Aronov summarizes the published pharmacophores into one that resembles that proposed by Cavalli et al. but with three hydrophobes not limited to aromatics.12 A further pharmacophore point contributing potency is also noted, an acceptor. This may be consistent with an interaction with one of the side chains of residues in the selectivity filter, but this has not been investigated. (v) Related to the general pharmacophore of Aronov is one for the binding of uncharged compounds, also proposed by Aronov, in 2006.39 Although these compounds may bind to a different state or in a different way, it is interesting to note that the subset of hydrophobes (the pharmacophore also includes three acceptor points) fits reasonably well with binding to a site with three Tyr groups flipped down. A subset of the compounds studied by Ekins et al. detailed under item iii was used to derive a pharmacophore model also apparently containing no positive ionizable feature. In both these cases, the molecules used to derive the pharmacophores (notably the more potent examples) are likely to be protonated at physiological pH and the absence of a positive ionizable feature most likely reflects the inability to have these features overlap and not their absence. The observed binding pose for these compounds can recapitulate the derived pharmacophore if only the hydrophobic features are considered. (vi) Finally, we note that Aptula and Cronin found that log D and Dmax, the maximum diameter of a molecule, could


Figure 15. (A) A cartoon of the binding site obtained when the side chains of Tyr652 flip down with key distances indicated to the nearest A˚. (B) Astemizole (peach) docked to the hERG model. Distances between the aromatic rings and the charged central nitrogen are given in A˚. (C) Schematic representations of substructures that bind tightly to hERG or pharmacophores derived from tight binding ligands. These are described in the text. (D) Pharmacophores docked to our hERG model support the suggested binding site perpendicular to the pore axis.

be used to build a QSAR model of hERG potency. Potency increased with Dmax, and Dmax ranged from 12.4 to 22.0 A˚. One particularly striking feature of the first four of these motifs (parts i-iv of Figure 15C) is that the ligands that are

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most potent present a pseudo-2-fold pharmacophoric symmetry. It is easy to understand why this might be preferred if the binding site has the 4-fold symmetry we propose and binding is orthogonal to the axis along which K+ permeates (Figures 2A and 3A) but much less easy to rationalize in the type of binding mode along the axis that is usually proposed (Figure 3B) in protein structure led studies.2,3,6,27 Figure 15D illustrates the agreement between the features presented by our suggested hERG binding site and the pharmacophores docked into the hERG model. Molecular Interpretation. The binding mode of hERG blockers seen in our docking study largely corresponds to the crystallographically determined positions of intracellular TBA or TEA in KcsA.14-16 In an electrophysiological study on the Shaker K+ channel mutant Ile470Tyr (Ile470 is homologous to hERG Tyr652), Klement et al. suggested that the Tyr rings point into the cavity in the inactivated state, whereas they assume an upright position in the open, activated state.40 Similarly, Chen et al. had shown earlier that it is the position of aromatic residues in helix S6 that determines sensitivity of hERG toward cisapride.41 In line with this, we suggest that the side chain flexibility of Tyr652 couples C-type inactivation and high-affinity drug blockade in hERG. The binding modes found in our study require at least one Tyr652 side chain in its “down” state. Tight drug block would therefore depend on the channel’s ability to inactivate. This observation has in fact been made experimentally in a number of binding studies, but its structural basis has remained elusive so far.42,43 We suggest that the probability of “down”-facing Tyr652 rings is raised in the inactivated state and that it is therefore more compatible with drug binding.40 Furthermore, the lower section of the channel’s selectivity filter is strongly involved in drug interactions in our model, which explains why mutations in this region have a strong impact on drug blockade.3,9,10 Interestingly, another residue found to be an important determinant for drug binding, Gly648,3,9 is located precisely where our model suggests that the gateways toward the S1-S4 domain or membrane are formed. The binding mode seen in our study is not the only way in which molecules can block hERG. Molecules such as fluvoxamine (and perhaps also other psychoactive drugs such as doxepin) show a particularly fast onset of channel block that is independent of mutations around Tyr652 and Phe656.44,45 They are believed to bind near the extracellular channel entrance, like peptide blockers of potassium channels.44 An extracellular binding site is also seen additionally for TBA or TEA in KcsA.14-16 Molecules that bind near the extracellular entrance cannot be treated by the same design guidelines, and it is possible that some molecules considered in this study fall into this category. The conventional model of binding with a more longitudinal binding mode along the channel axis may also be adopted by a significant number of molecules. Literature reports of a functional difference between hERG blockade by extended and more compact molecules are acknowledged here, in which cases of hERG blockade by molecules of more compact conformation are reported to be largely independent of interaction with Tyr652 and Phe656.43 Binding of more compact molecules may be more compatible with the traditional binding model, which does not require “down” conformations of Tyr652 and thus entails a smaller cavity. Alternatively, the traditional model of hERG binding could be favored in lower-affinity interaction, which could also involve switching between

Article

several states. Some reports indicate that lower-affinity hERG block may be independent of C-type inactivation.42 Binding perpendicular to the channel axis can explain a number of biophysical observations (especially in high affinity interaction with hERG) better than longitudinal binding modes. It is conceivable that larger molecules bind perpendicular to the channel axis and thus make full use of the possible gates toward the membrane. The affinity of the perpendicular binding modes is enhanced by synergistic use of the 4-fold symmetry of the channel, close and multiple π-π stacking interactions, and the polarity of the selectivity filter. In this case, our model suggests, in line with some published SAR, that once the diameter of a molecule is large enough to ensure that it protrudes into the gateways in its low energy conformations, introducing appropriate steric bulk at the periphery (preferably at both ends) can reduce hERG potency. This contrasts to the expected effect of increased bulk and lipophilicity which should increase hERG potency. It was outside the scope of this study to investigate binding to an open state model of hERG, which may be governed by an altered arrangement of aromatic residues relative to each other and to the selectivity filter. Furthermore, binding to hERG is not likely to be restricted to just three specific modes (perpendicular, longitudinal, or extracellular) but can rather be assumed to have contributions from at least both internal states and possibly more. We are aiming to carry out further studies, including molecular dynamics simulations of binding to both open and closed hERG states, mutant binding analyses, and further matched-pair studies to shed light on this and further questions around the promiscuity of hERG binding. Experimental Section The hERG model was built using structures of the closed conformation of KcsA as structural templates (PDB codes 2bob, 2hjf, 2hvk).14-16 These structures were solved as complexes with the channel blockers TBA and TEA, and thus enable a practical comparison of cation locations between TBA/TEA and other hERG ligands. Sequence to structure alignments were carried out with PSI-BLAST46 and manually refined. On the basis of the alignment, a standard MODELLER47 run was followed by progressive side chain and backbone optimization based on the templates’ geometric restraints. The alignment of the hERG amino acid sequence to structurally known K+ channels is problematic, since hERG has a 43-residue insertion in the linker between TM helix S5 and the pore helix (P), which renders the alignment of S5 residues uncertain; while the conserved K+ selectivity filter provides an unambiguous anchor for aligning P, SF, the pore loop, and TM helix S6 of hERG. Residues from S5 do not participate in drug binding, whereas S6 provides the main interaction sites for hERG blockers.2 We focused on a model of the hERG sections P-SF-S6, while the outer regions of S5 were kept for the sake of structural stability. Docking was carried out with GOLD 4.0 using the GOLDscore scoring function with all four hERG Tyr652 side chains taken from rotamer libraries.31-33 Density functional theory calculations employed Becke’s threeparameter exchange functional combined with the Lee-YangParr correlation functional.48,49 All calculations were performed in Gaussian 03 and all minima verified by having all positive vibrational frequencies.50 For details of MD simulations and docking runs, see Supporting Information.

Acknowledgment. We thank Martin Packer, Claire Gavaghan, and Chris Pollard for fruitful discussions. Al Rabow and Martin Packer are gratefully acknowledged for carefully reading the manuscript. We thank Huw D. Jones


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for software and computer support. U.Z. acknowledges support from Bert L. de Groot and Helmut Grubmuller. Supporting Information Available: Supplementary computational methods, Figures S1-S4, lipophilicity trends in the published data sets, QM optimized coordinates for maraviroc models, coordinates of the hERG model, and all docking scores. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Side Chain Flexibilities in the Human Ether-a-go-go Related Gene

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