Homology Modeling of Human Etheragogo Related Gene

At least two groups33'34 have reported hERG homology models using available atomic resolution structures of bacterial K+ channels KcsA35 (closed) and MthK36 (open). These channels contain only two transmembrane domains (equivalent to helices S5/S6 in Figure 3), and the models therefore only cover the predicted structure of the hERG pore. The basic architecture of hERG channel is expected to be similar to that of other voltage-gated K+ channels, such as KvAP (Figure 3).37 The channel pore domain is formed by tetramerization from helices S5 and S6, as well as the pore helix P and the selectivity filter loop. The selectivity filter lies on the extracellular side of the membrane. The movement of S6 helices with respect to each other in a crossover fashion renders the channel closed, with the water-filled cavity isolated from cytosol. The voltage-sensing paddles formed by helices S3b and S4 are responsible for the voltage dependence exhibited by KvAP. The debate about the exact position of the S4 segment and the details of voltage sensing at the molecular level is ongoing.38,39 While the structure and location of the paddles with respect to the membrane may be different in hERG, the basic structure of the pore is likely to be reasonably conserved.

Two bands of aromatic residues are predicted to line the cavity, with each monomer contributing Phe656 and Tyr652 (Figure 4).15,33,34 These residues are both located on the S6 helix, with the tetrad of Phe656 situated closer to the mouth of the channel, and the four Tyr652 residues further toward the pore helix. The homology model is corroborated by earlier mutagenesis data. Sanguinetti and co-workers used alanine scanning to identify key residues responsible for hERG blockade by potent inhibitors terfenadine, cisapride, and MK-499.33 Phe656 and Tyr652 appeared to be the primary interaction points. The current consensus implicates Phe656 in p-stacking interactions with the ligands, while Tyr652 is thought to participate in a cation-p interaction with the protonated basic nitrogen present in most of the reported hERG blockers. Recently, the potency for hERG blockade by these three drugs was shown by systematic mutagenesis to correlate well with measures of hydrophobicity of residue 656, such as its side chain van der Waals

Figure 3 Structural model of hERG channels. Key elements of hERG channel topology illustrated using the x-ray structure of KvAP37 Two of the four subunits comprising the tetrameric channel are shown. (Reproduced with permission from Aronov, A. M. Drug Disc. Today 2005, 10, 149-155 © Elsevier.)

Figure 4 Model of the pore portion of hERG channel. The P-S6 fragment is shown for a dimer. Aromatic residues Phe656 and Tyr652 are critical for hERG block by most known small molecule ligands. Polar residues Thr623 and Ser624 modulate the binding potency for a number of reported hERG blockers. An hERG blocker is represented schematically based on published evidence. One or two hydrophobes interact with Phe656, a positive charge is stabilized by cation-n interaction with Tyr652, and the generally hydrophobic tail contains an acceptor able to interact with the polar residues on the loop that connects the pore helix to the selectivity filter. (Reproduced with permission from Aronov, A. M. Drug Disc. Today 2005, 10, 149-155 © Elsevier.)

hERG blocker

Figure 4 Model of the pore portion of hERG channel. The P-S6 fragment is shown for a dimer. Aromatic residues Phe656 and Tyr652 are critical for hERG block by most known small molecule ligands. Polar residues Thr623 and Ser624 modulate the binding potency for a number of reported hERG blockers. An hERG blocker is represented schematically based on published evidence. One or two hydrophobes interact with Phe656, a positive charge is stabilized by cation-n interaction with Tyr652, and the generally hydrophobic tail contains an acceptor able to interact with the polar residues on the loop that connects the pore helix to the selectivity filter. (Reproduced with permission from Aronov, A. M. Drug Disc. Today 2005, 10, 149-155 © Elsevier.)

Figure 5 Structure of ibutilide.

hydrophobic surface area.24 In the case of residue 652, the presence of an aromatic residue in this position is required for high-affinity hERG blockade, consistent with the importance of the cation-n interaction predicted by ligand-based models.

Additionally, residues Thr623 and Val625, located near the pore helix, were implicated in hERG binding to MK-499, but the effect was moderate in the cases of terfenadine and cisapride.15 Both Thr623 and neighboring Ser624 (Figure 4) have been shown to have pronounced effects on hERG block by vesnarinone, clofilium, and ibutilide (Figure 5). These polar residues may be able to interact with the polar tails present in many of the potent hERG blockers.15'17'34 A recent study of the structural determinants of hERG channel block by clofilium and ibutilide provided further evidence that residues at the base of the pore helices, which also face into the central cavity, can form interactions with drugs that may facilitate high-affinity binding.40 Mutation of T623A and S624A resulted in approximately 90- and 60-fold increases in ibutilide IC50 values, respectively. It is the specific interaction with Ser624 that appears to be responsible for the slow-off rates that characterize clofilium block of both wild-type and mutant D540 K hERG channels. Based on the docking model of clofilium within the inner cavity of the hERG homology model, Perry and co-workers invoke polar interactions between the side chain hydroxyl of Ser624 and the chlorine atom in clofilium as the interaction that contributes to stabilization of clofilium binding.40 The proposed orientation of both clofilium and ibutilide within the hERG channel includes aryl groups pointing toward the selectivity filter, a cation-p interaction of Tyr652 with the positively charged amine, and a hydrophobic contact of the fatty side chains of the drugs with Phe656. The role of Val625 in interactions with hERG blockers is less well understood, primarily because according to K+ channel crystal structures,35'36 the corresponding valine side chain is buried in the hydrophobic core surrounding the selectivity filter, and does not point into the inner cavity of the channel. Thus, the reason for the sensitivity of hERG block to the V625A mutation could lie in the allosteric effect of this residue on the conformation of neighboring side chains of Thr623 and Ser624.

A combination of structural features is thought to be responsible for the 'binding promiscuity' of hERG relative to other K+ channels. The aromatic residues critical for binding to structurally diverse drugs are missing in channels of the Kv1-4 families, replaced by Ile or Val in positions equivalent to Tyr652 and Phe656.15,17,24 The exact positioning of the aromatic residues is also of critical importance for binding. Structurally related EAG channels were made sensitive to the hERG blocker cisapride by moving the Tyr residue by one position along the S6 helix.41 Finally, the hERG channel lacks the Pro-Val(Ile)-Pro motif on the S6 helix, which is present in Kv1-4 channels and is thought to decrease the size of the inner cavity by inserting a kink in the inner helices.15,24

The only published example of the use of homology models for quantitative prediction of hERG blockade is the study by Rajamani and colleagues.42 The method described uses a two-state homology model to represent the flexibility of the channel. Two hERG homology models were built based on the crystal structure of the closed state of KcsA.43 The lower resolution structure of MthK in the open state was used as a guide providing insight into the movement of the S6 helix in going from the open to the closed states of the channel. As a result, two models of hERG channel were built - the partially open state (10° translation away from the KscA reference state) and the fully open state (19° translation, closely corresponding to MthK). IC50 values for 32 ligands assembled by Cavalli and co-workers44 (hERG pIC50 ranging between 4 and 9) were used to derive the linear interaction energy (LIE) correlation. The ligand set was docked to the homology models, the best poses (one for each ligand) were energy minimized, and used to derive the van der Waals and electrostatic contributions to the binding energy. Models derived for the two homology models separately resulted in poor fit (e.g., r2 = 0.24 for the partially open state). However, when a preference of each compound for a particular state was established by comparing the estimated interaction energy for each ligand in both states, separate better fits were obtained, with 21 ligands preferring the open state and 11 ligands the partially closed state of the channel. The final combined model:

produced r2 = 0.82 (rmse = 0.56) for 27 ligands, with five compounds identified as potential outliers.42 The study highlighted the difficulty of using hERG homology models for quantitative predictions of hERG blockade and the need for modeling techniques that can capture the flexibility of the K+ channel.

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