Understanding the Molecular Basis of Toxin Promiscuity: The

Oct 22, 2014 - †Institute for Molecular Bioscience, ‡School of Biomedical Sciences, and §Centre for Advanced Imaging, The University of Queenslan...
0 downloads 10 Views 3MB Size
Download PDF
Article pubs.acs.org/jmc

Understanding the Molecular Basis of Toxin Promiscuity: The Analgesic Sea Anemone Peptide APETx2 Interacts with Acid-Sensing Ion Channel 3 and hERG Channels via Overlapping Pharmacophores Jonas E. Jensen,†,∥,# Ben Cristofori-Armstrong,†,# Raveendra Anangi,†,# K. Johan Rosengren,‡ Carus H. Y. Lau,† Mehdi Mobli,§ Andreas Brust,† Paul F. Alewood,† Glenn F. King,*,† and Lachlan D. Rash*,† †

Institute for Molecular Bioscience, ‡School of Biomedical Sciences, and §Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD 4072, Australia S Supporting Information *

ABSTRACT: The sea anemone peptide APETx2 is a potent and selective blocker of acid-sensing ion channel 3 (ASIC3). APETx2 is analgesic in a variety of rodent pain models, but the lack of knowledge of its pharmacophore and binding site on ASIC3 has impeded development of improved analogues. Here we present a detailed structure−activity relationship study of APETx2. Determination of a high-resolution structure of APETx2 combined with scanning mutagenesis revealed a cluster of aromatic and basic residues that mediate its interaction with ASIC3. We show that APETx2 also inhibits the off-target hERG channel by reducing the maximal current amplitude and shifting the voltage dependence of activation to more positive potentials. Electrophysiological screening of selected APETx2 mutants revealed partial overlap between the surfaces on APETx2 that mediate its interaction with ASIC3 and hERG. Characterization of the molecular basis of these interactions is an important first step toward the rational design of more selective APETx2 analogues.

■

INTRODUCTION Acid-sensing ion channels (ASICs) are proton-gated cation channels that are abundantly expressed throughout the central and peripheral nervous system, where they are primary acid sensors in the nociceptive pathway.1 Alternative splicing of five ASIC-encoding genes leads to the expression of seven subunits (ASIC1a and -1b, ASIC2a and -2b, and ASIC3−ASIC5) that combine to form hetero- or homotrimeric channels that differ in their pH sensitivity, kinetics, and tissue distribution. The ASIC3 subtype is an important therapeutic target as it has been shown to play an important role in inflammatory pain,2 arthritis,3 postoperative pain,4 migraine,5 and cardiac pain.6 ASIC3 has also been implicated in anxiety and insulin resistance.7 Despite its therapeutic relevance, there is currently a lack of selective and potent inhibitors of ASIC3 that could be used for the treatment of ASIC3-related diseases. The most potent and selective inhibitor of ASIC3 described to date is the 42-residue sea anemone peptide APETx2, which inhibits rat (r) ASIC3containing channels with IC50 values of 63 nM for homomeric channels and 0.1−2 μM for heteromeric channels.8 Peripheral administration of APETx2 was instrumental in defining the role of ASIC3 in peripheral pain, and APETx2 was shown to be analgesic in a variety of rodent pain models.2,5,9,10 These studies not only validated ASIC3 as a novel target for peripherally acting analgesics but also highlighted the value of APETx2 as a © 2014 American Chemical Society

research tool and potential therapeutic lead. Interestingly, APETx2 also inhibits the voltage-gated sodium (NaV) channel NaV1.8 (IC50 values between 55 nM and 2.6 μM depending on the study), which is also involved in peripheral pain processing.11,12 The structure of APETx2 is comprised of a four-stranded βsheet cross-braced by three disulfide bonds.13 This fold places APETx2 into the so-called defensin-like family of sea anemone peptides that includes APETx1 and several sodium and potassium channel-modulating peptides.13 The sequence of APETx2 is 64% identical with that of APETx1, a potent and selective inhibitor of the hERG channel (IC50 = 34 nM).14 The recently discovered paralog APETx3 differs by only one residue from APETx1, but it was reported to be inactive on hERG at concentrations of ≤50 μM.12 On the basis of the differences in sequence between APETx1 and APETx2, and the electrostatic surface of APETx2, it was proposed that APETx2 interacts with ASIC3 via a cluster of residues centered around Tyr16, Arg17, and Arg31.13 This hypothesis is consistent with the limited structure−activity relationship (SAR) data available for APETx2, which has revealed a critical functional role for Phe15, Arg17, and the N-terminus of the peptide.15−17 Received: September 11, 2014 Published: October 22, 2014 9195

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

Figure 1. (A) Stereoview of the ensemble of 20 APETx2 structures. The structures were overlaid over all backbone atoms. The four inter-cystine loops and the N- and C-termini are labeled. (B) Ensemble of APETx2 structures with the peptide backbone colored blue and the heavy atoms of cysteine and non-cysteine side chains colored yellow and green, respectively. (C) Overlay of the ensemble of APETx2 structures determined in this study (PDB entry 2MUB, blue) and the previously published ensemble (PDB entry 1WXN, red).13 The new structure is more precisely defined, and there are differences in the conformation of some of the loops. The significant difference in conformation of the C-terminal region is due to our determination that the Thr39−Pro40 peptide bond adopts a cis conformation rather than a trans conformation as reported previously.13 The N- and C-termini are labeled.

Table 1. Statistical Analysis of APETx2 Structures 2MUB experimental restraints interproton distance restraints intraresidue (i − j = 0) sequential (i − j = 1) medium-range (i − j = 2−4) long-range (i − j ≥ 5) hydrogen bond restraints disulfide bond restraints dihedral angle restraints rmsd from mean coordinate structure (Å) backbone atoms (residues 1−42) all heavy atoms (residues 1−42) stereochemical qualityb residues in favored Ramachandran region (%) Ramachandran outliers (%) unfavorable side chain rotamers (%) Clashscore, all atomsc overall MolProbity score

1WXN

156a 155 72 186 26 9 42 (31 backbone)

369 128 41 93 38 9 28

0.27 ± 0.07 1.00 ± 0.17

0.84 ± 0.16 1.54 ± 0.21

92.5 0 2.94 1.68 1.74 (72nd percentile)

90 2.5 0 16.69 2.27 (60th percentile)

a

Only structurally relevant (nonredundant) restraints, as defined by CYANA, are included. bAccording to MolProbity (http://molprobity.biochem. duke.edu). cDefined as the number of >0.4 Å steric overlaps per 1000 atoms.

9196

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

Figure 2. Concentration effect curves (left) for inhibition of rASIC3 by WT APETx2 and its analogues. The middle panel shows a comparison of the 15 N chemical shifts of individual residues for WT APETx2 and each of the mutants, which reveals that none of the mutations perturb the global fold of APETx2. In the right panel are shown the structures of APETx2 (PDB entry 2MUB) with residues mutated for SAR studies shown as sticks. Structural data were extracted from SOFAST-HMQC and HSQC spectra for the synthetic and recombinant peptide, respectively. Data are grouped according to location of the mutations in (A) loop 2, (B) loop 4, and (C) elsewhere. Data points for concentration effect curves represent the mean ± SEM (n ≥ 5).

involvement in hydrogen bonds. There was no evidence of line broadening or additional spin systems that would suggest conformational heterogeneity, indicating that APETx2 adopts a single, well-defined conformation in solution. In addition, heteronuclear 15N and 13C assignments were achieved using HSQC experiments recorded at natural abundance. The 1H chemical shifts were very similar to those reported previously,13 with the exception of changes related to the use of a more physiological pH (6.0) in the current study compared to the acidic pH (3.0) used previously. The torsion angle dynamics program CYANA20 was used to calculate the structure of APETx2 based on 569 interproton distance restraints and 42 dihedral angle restraints. The 20 conformers with best MolProbity score21 were selected to represent the solution structure of APETx2 (Figure 1A). The structural statistics and restraints used to generate the ensemble of APETx2 structures are given in Table 1, along with a comparison to the previously reported structure.13 The new structure is more precise [i.e., lower backbone and heavy atom rmsds (see Figure 1C)], and it has significantly higher stereochemical quality (Table 1). This structure is ranked in the 72nd percentile of published structures according to MolProbity21 and can be classified as “high-resolution” based on measures of precision and stereochemical quality.19 As reported previously,13 APETx2 adopts a compact fold consisting of a four-stranded antiparallel β-sheet comprising residues 2−5 (β1), 10−14 (β2), 28−31 (β3), and 36−39 (β4). The β-sheet is stabilized by three disulfide bonds together with an extensive network of hydrogen bonds.

Herein we describe an in-depth SAR study of APETx2. We determined a high-resolution solution structure of APETx2 and used scanning mutagenesis to identify residues that are critical for its activity at ASIC3. Furthermore, we show that APETx2 inhibits hERG at micromolar concentrations and demonstrate partial overlap between the APETx2 pharmacophores for ASIC3 and hERG. Drug-induced inhibition of hERG results in QT interval prolongation, which can lead to potentially lethal arrhythmias, and consequently, the Food and Drug Administration mandates safety screening at this target for new drugs.18 Thus, understanding the molecular basis of the interaction of APETx2 with ASIC3 and hERG will facilitate the rational engineering of APETx2 analogues that are devoid of hERG activity and thus more suitable for therapeutic development.

■

RESULTS High-Resolution Solution Structure of APETx2. The published ensemble of APETx2 structures (Protein Data Bank entry 1WXN)13 has root-mean-square deviations (rmsds) for the backbone and heavy atoms of 0.84 and 1.54 Å, respectively, which is classified as “medium-resolution”.19 For developing an atomic-resolution understanding of how APETx2 interacts with ASIC3, it was desirable to improve the resolution of this structure, and therefore, we recorded new nuclear magnetic resonance (NMR) data at ultra-high field (900 MHz). Complete 1H resonance assignments were made using twodimensional (2D) 1H−1H COSY, TOCSY, and NOESY spectra, including the side chain hydroxyl protons of Thr36 and Thr39, which were NMR-visible because of their 9197

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

Figure 3. APETx2 activity at hERG. (A) Sequence alignment of Anthopleura elegantissima toxins. Conserved residues, conservative substitutions, and cysteine residues are highlighted with light blue, dark blue, and yellow backgrounds, respectively; the rectangle highlights the position of the single point mutation differentiating APETx1 and APETx3 at residue 3. (B) Concentration effect curve for inhibition of hERG by APETx2 (n ≥ 4). (C) Effect of 3 μM APETx2 on hERG activation (n ≥ 9). The tail current was plotted as a function of voltage and fit to a Boltzmann equation. V0.5 = −29 ± 2.10 mV for control (---) and −5.0 ± 1.33 mV for APETx2 (). The inset shows the current−voltage relationship for hERG currents under control conditions and in the presence of 3 μM APETx2. Peak currents were measured at the end of a 2 s depolarization pulse. (D) Effect of 3 μM APETx2 on steady-state inactivation. Data are plotted as normalized peak current after inactivation at 50 mV against test potential and fit to a Boltzmann equation. V0.5 = −62.24 ± 1.29 mV for control and −60.57 ± 2.61 mV for APETx2 (n ≥ 5). Protocols for panel D were performed on the same oocyte as in panel C. All data points are normalized to control values and represent the mean ± SEM.

spectra of mutants produced recombinantly, which in either case allowed a direct comparison of the spectrum of each mutant with that of wild-type (WT) APETx2. This measure of structural integrity allows a distinction to be made between functionally deleterious mutations that arise from removal of key channel−peptide interactions and those that arise secondarily due to perturbations of the peptide structure. For most analogues, the 1H and 15N amide chemical shifts were very similar to those observed for the WT peptide, indicating that the mutations did not perturb the global fold of APETx2. Small local chemical perturbations were observed, as expected, for the residue that was mutated, and sometimes for spatially adjacent residues (Figure 2). However, for two analogues, Y13A and W14A, major differences were observed throughout the sequence, suggesting that these peptides were misfolded (Figure S1 of the Supporting Information). These residues are largely buried, particularly Tyr13 that is involved in aromatic ring stacking with Phe15 and Tyr32. Thus, we conclude that Tyr13 and Trp14 play an important role in the folding and structural integrity of APETx2, and therefore, the Y13A and W14A mutants were excluded from functional analyses. The activity of WT APETx2 and those of the remaining mutants were tested on Xenopus laevis oocytes expressing rASIC3. Concentration effect curves are shown for each mutant in Figure 2. The IC50 calculated for WT APETx2 was 61 nM, which corresponds well with that reported for the peptide isolated from the native source (IC50 = 63 nM).8 Mutation of Tyr16 to an Ala had a profound effect on the ability of APETx2 to inhibit rASIC3. Tyr16 is substantially more solvent exposed than Tyr13 or Trp14 and is positioned next to Phe33 and Leu34, which are important for activity (see below) and have perturbed chemical shifts in the HSQC spectrum of the Y16A mutant. To further examine the functional importance of Tyr16, we produced a more conservative Y16F mutant. The HSQC spectrum of the Y16F mutant overlays well with that of WT APETx2, with only a minor perturbation of the 15N chemical shift of residue 16,

The APETx2 structure presented here is generally consistent with that reported previously,13 but there are several noticeable differences in loop conformations and one additional major difference. APETx2 contains three proline residues that were all previously reported to adopt a trans conformation.13 In the study presented here, we confirmed the trans conformation for the Arg17−Pro18 and Cys20−Pro21 peptide bonds based on strong Hα(i−1)−Hδ(i) NOEs. In contrast, the strong Hα(i− 1)−Hα(i) NOE observed for Pro40 is suggestive of a cis conformation. This was confirmed using 1H−13C HSQC data, which revealed a Cβ−Cγ chemical shift separation of ∼10 ppm for Pro40, consistent with a cis conformation,22 compared to a Cβ−Cγ separation of 95% purity as judged by mass spectrometry and analytical RP-HPLC. The structural integrity of the mutated analogues was determined using either selective optimized flip angle shorttransient heteronuclear multiple quantum coherence (SOFAST-HMQC) spectra of mutants made using SPPS or 2D 1 H−15N heteronuclear single-quantum coherence (HSQC) 9198

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

observed at −20 mV, while at 40 mV, there was no apparent inhibition of peak current by 3 μM APETx2 (Figure 3C, inset). The mean voltage of half-maximal activation (V0.5) for hERG tail currents (−29.0 ± 2.10 mV) was shifted 24 mV to more positive potentials in the presence of 3 μM APETx2 (V0.5 = −5.01 ± 1.33 mV; p < 0.001) (Figure 3C), an effect similar to that observed previously for APETx1.14,24 APETx1 also induces a small negative shift in steady-state inactivation (SSIN) of hERG.14 In contrast, 3 μM APETx2 did not affect SSIN (Figure 3D), nor did it affect the test pulse current in the I−V curve at 40 mV (Figure 3C, inset). Thus, APETx2 does not appear to modify hERG channel inactivation. Because mutation of a single residue in APETx1 abolishes its hERG activity,12 we hypothesized that mutation of the corresponding residue in APETx2 (Ala3) might also reduce or abolish hERG activity without significantly affecting activity on ASIC3. Thus, we produced an A3P mutant of APETx2 and found that, as predicted, it inhibited rASIC3 with a potency similar to that of the WT peptide (Figure 4A). Surprisingly,

indicating that the mutant is properly folded. This mutant peptide weakly inhibited rASIC3 at low concentrations (∼15% inhibition at 300 nM) and caused concentration-dependent agonism at concentrations of >1 μM. Thus, we conclude that the Tyr16 side chain hydroxyl group is important for mediating the interaction of APETx2 with rASIC3. The R17A mutation resulted in a 44-fold loss of activity, consistent with previous findings.15 A further decrease in activity was observed in the R17E charge reversal mutant, confirming that Arg17 plays a key role in APETx2 inhibition of rASIC3, most likely via an electrostatic interaction with the channel. Mutation of Pro18 to Ala caused an only 3-fold decrease in activity, suggesting that Pro18 plays an only minor role in the APETx2−ASIC3 interaction. Four residues in loop 4 were mutated. The F33A and L34A mutants both had dramatically reduced activity. In contrast, mutation of Arg31 and Tyr32 to Ala resulted in only minor decreases in activity. Tyr32 is partially buried, and its mutation to alanine results in substantially altered 15N chemical shifts for the functionally critical Phe15, Tyr16, Arg17, Phe33, and Leu34 residues. Thus, the minor deleterious effect of mutating Tyr32 to Ala most likely results from alterations in the orientation of the side chains of neighboring residues of functional importance, rather than removal of a key part of the peptide pharmacophore. We also mutated five residues that were predicted to be distal to the APETx2 pharmacophore. As expected, mutation of Ser5, Asn8, Lys10, and Asp23 to Ala resulted in peptides that had activity very similar to that of WT APETx2. In contrast, mutation of Arg24 to Ala resulted in a 26-fold reduction in activity, a surprising result given that it is far removed from loops 2 and 4. NMR data indicated that the structural integrity of loops 2 and 4 was maintained in the R24A mutant, but this substitution did cause moderate 15N chemical shift changes for residues 22−27, as well as for residues close to the N- and Ctermini. A similar loss of activity was observed for the R24Q mutant, but only minor chemical shifts in the C-terminus were observed. Taken together, these data suggest that Arg24 is important for APETx2 activity at rASIC3 or that mutation of this residue induces conformational changes in the spatially proximal C-terminal region, which was previously shown to be functionally important (i.e., deletion of the three C-terminal residues reduces activity by 18-fold17). At this stage, we cannot distinguish between these possibilities. Modulation of hERG Activity by Wild-Type and Mutant APETx2. The single Thr3 to Pro substitution of APETx3 compared to APETx1 was reported to render the peptide inactive at hERG (Figure 3A).12 APETx2 was previously reported to lack hERG activity; however, it was assessed only at 300 nM (i.e., ∼5-fold higher than the IC50 for inhibition of rASIC3).8 Because of the 64% identical sequences of APETx2 and APETx1, we hypothesized that higher concentrations of APETx2 might inhibit hERG, which would seriously limit its potential as an analgesic. Indeed, we found that APETx2 inhibits hERG with an IC50 of 1.21 ± 0.05 μM with a maximal inhibition of 53.6 ± 1.5% (Figure 3B). APETx2 inhibited both peak (recorded at 0 mV) and tail hERG (recorded upon repolarization to −70 mV) currents. Inhibition was rapid, reaching a plateau within 1 min, and fully reversible upon washout (data not shown). Current−voltage (I−V) curves recorded in the absence or presence of 3 μM APETx2 show a voltage dependence of peptide action on hERG. The greatest reduction in peak current (∼58%) was

Figure 4. Effect of APETx2 analogues on hERG. (A and B) Concentration effect curves for inhibition of rASIC3 [A (n ≥ 6)] and hERG [B (n ≥ 4)] by WT APETx2 and the A3P mutant. (C) Summary of the degree of shift in V0.5 of the tail current induced by 3 μM peptide (normalized to the maximal current amplitude of each respective treatment condition) (n ≥ 6). Via one-way ANOVA, *p < 0.05 and **p < 0.001 compared to WT.

however, the A3P mutant was almost equipotent with WT APETx2 on hERG (Figure 4B); the mutation caused no change in the effect of the peptide on channel activation (Figure S2 of the Supporting Information) or inactivation kinetics (data not shown). SAR studies have not been conducted to elucidate the molecular epitopes on APETx1 that mediate its interaction with hERG. On the basis of the distance between functionally important hERG channel residues at the APETx1 binding site and the distances among residues Lys18, Tyr32, Phe33, and Leu34 of APETx1, these residues were hypothesized to comprise the hERG pharmacophore.24 The latter three residues are conserved in APETx2, which suggests that Lys18 (Pro in APETx2) is the most likely contributor to the difference in selectivity and potency between APETx1 and APETx2. Zhang and colleagues hypothesized that the side chain of Lys18 in APETx1 makes a salt bridge interaction with Glu518 in the S3b loop of hERG.24 9199

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

To explore the hERG pharmacophore of APETx2, we examined the activity of several APETx2 mutants at a single concentration of 3 μM and compared the degree of peptideinduced shift in the V0.5 of hERG activation to that of WT APETx2 (Figures 4C). Because the A3P mutant had an efficacy similar to that of WT APETx2, we examined the activity of a truncation mutant missing two N-terminal residues (3−42). The 3−42 deletion mutant showed an intermediate shift in the V0.5 of activation, between that of the control and WT APETx2. Three loop 4 mutants were also tested against hERG: the Y32A mutant was equipotent with WT APETx2, whereas the F33A and L34A mutations significantly altered activity at hERG. Interestingly, although the F33A mutant did not affect the peak current, it significantly dampened the tail current (Figure S2 of the Supporting Information), possibly indicating a mechanism of action or binding mode different from that of WT APETx2. We next tested the activity of mutants N8A and P18A; mutations at these positions, which are both Lys in APETx1, have only minor effects on the activity of APETx2 against ASIC3. The P18A mutant had significantly lower activity against hERG, whereas the N8A mutant was similar to WT APETx2.

■

DISCUSSION APETx2 Pharmacophore: ASIC3 versus hERG. Although APETx2 was discovered nearly a decade ago, it is still the most potent and selective inhibitor of ASIC3 known. It has proven to be a valuable research tool as well as a promising therapeutic lead with the potential for treating peripheral pain. In conjunction with previous work highlighting the importance of Phe1516 and the N-terminus of APETx223 for inhibition of rASIC3, it is clear that modification to four hydrophobic residues (Phe15, Tyr16, Phe33, and Leu34) as well as Arg17 and Thr2 has a profound effect on the potency of APETx2. These residues form a contiguous surface spread across loops 2 and 4 and the N-terminus of APETx2; we conclude that this surface constitutes the primary ASIC3 pharmacophore (Figure 5A). In contrast to the published report that APETx2 has no activity against hERG,8 we found that APETx2 inhibits hERG with a mechanism of action similar to that of its structural relative, the potent hERG inhibitor APETx1. Although a relatively weak inhibitor, the IC50 of APETx2 for hERG is only 7-fold higher than the IC50 for human ASIC3. Furthermore, we established the critical importance of Pro18, Phe33, and Leu34 and a minor role of the N-terminus for hERG inhibition (Figure 5B), thereby revealing partial overlap in the peptide’s pharmacophore for ASIC3 and hERG (Figure 5C). A large focus was placed on residues in loops 2 and 4, and the Nterminus, which have previously been suggested to be important for the APETx2−ASIC313 and APETx1−hERG24,25 interactions. Analysis of the primary structure of APETx1 and APETx2 shows a high level of sequence identity (64%) with only a few differences in loops 2 and 4 (Figure 3A). Tyr16 and Arg17 make up a crucial part of the APETx2 pharmacophore for interaction with ASIC3 and differ markedly in APETx1 (Gly16 and Thr17). On the other hand, Pro18 was identified as being important for hERG activity but does not appear to contribute substantially to ASIC3 activity. The remaining important residues for ASIC3 activity (Thr2, Phe15, Arg24, Tyr32, Phe33, and Leu34) are the same in both toxins. This strongly suggests that the difference in ion channel selectivity between

Figure 5. Molecular surface representations of APETx2 showing the major sites of interaction with ASIC3 and hERG. APETx2 residues that, when mutated to alanine, have a major or minor impact on the peptide’s ability to inhibit ASIC3 are colored red or orange, respectively (A), while those that affect hERG inhibition (B) are colored purple or blue, respectively. The left and right views are related by a 180° rotation. (C) APETx2 pharmacophores for interaction with ASIC3 and hERG mapped onto a single structure. The two views are related by a 90° rotation. Residues that are important only for interaction with ASIC3 or hERG are colored red or purple, respectively, whereas residues colored green are crucial for interaction with both channels.

APETx1 and APETx2 is primarily a result of three residues: Tyr16, Arg17, and Pro18. Pro18 in APETx2 is replaced with a positively charged lysine in APETx1, which was suggested to be crucial for hERG activity.24 Thus, we predict that mutation of Pro18 in APETx2 to a negatively charged Asp or Glu residue will further weaken its interaction with hERG, while retaining substantial ASIC3 activity. In addition to Pro18, Asn8 (also a lysine in APETx1) was more important for activity at hERG than ASIC3, and it therefore represents another potential site for engineering more ASIC3-selective APETx2 analogues. In addition to APETx1−3, peptidomic26 and transcriptom27,28 ic studies have revealed the existence of a diverse family of APETx-like peptides abundant in the Actiniidae family of sea anemones. However, the molecular targets of these peptides remain largely unknown. Two other sea anemone peptides, BDS-I and BDS-II, also belong to this structural family and have been shown to modulate KV and NaV channels.29,30 Consistent with these reports, APETx2 has also been shown to interact with NaV channels. Most notably, it inhibits the nociceptor-selective subtype NaV1.8.11,12 This confounds its use as a “selective” tool for studying the role of ASIC3, particularly 9200

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

2.1 mm × 50 mm, particle size of 5 μm, flow rate of 0.25 mL/min, gradient from 10 to 50% solvent B over 30 min, with solvent as described above) and obtained with purities of >95%. The HPLC k′ value [(peptide retention time − solvent retention time)/solvent retention time] for each peptide is as follows: WT APETx2, 13.54; 3− 42, 11.38; A3P, 9.73; S5A, 10.65; N8A, 12.45; K10A, 13.11; Y16A, 9.34; Y16F, 14.46; R17A, 9.14; R17E, 13.16; P18A, 11.00; D23A, 12.93; R24A, 13.37; R24Q, 15.91; R31A, 13.42; Y32A, 9.54; F33A, 10.15; L34A, 11.34. Peptide identity was confirmed using matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) MS using a model 4700 Proteomics Bioanalyzer (Applied Biosciences). RP-HPLC fractions were mixed in a 1/1 (v/v) ratio with α-cyano-4hydroxycinnamic acid matrix (5 mg/mL in a 50/50 acetonitrile/H2O mixture and 0.1% TFA), and MALDI-TOF mass spectra were collected in positive reflector mode. The observed and theoretical monoisotopic masses (M+H+) for each peptide are as follows: WT APETx2, 4558.927 (4558.915); 3−42, 4400.761 (4400.846); A3P, 4584.792 (4584.931); S5A, 4542.625 (4542.920); N8A, 4515.699 (4515.910); K10A, 4501.492 (4501.857); Y16A, 4466.794 (4466.889); Y16F, 4542.657 (4542.920); R17A, 4473.620 (4473.851); R17E, 4531.582 (4531.857); P18A, 4532.839 (4532.900); D23A, 4514.631 (4514.926); R24A, 4473.573 (4473.851); R24Q, 4530.518 (4530.873); R31A, 4473.508 (4473.851); Y32A, 4466.712 (4466.889); F33A, 4482.662 (4482.884); L34A, 4516.510 (4516.868). Concentration Determination. The concentration of a stock solution of synthetic WT APETx2 was determined by amino acid analysis at the Australian Proteome Analysis Facility (Sydney, Australia). The area under the RP-HPLC peak (at 214 nm) for all analogues was then compared to the area from a known amount of the standard WT APETx2, which allowed accurate determination of the concentration of each analogue. NMR Spectroscopy. Lyophilized APETx2 was reconstituted at a final concentration of 300 μM in 20 mM phosphate buffer and 5% D2O (pH 6.0) then filtered by centrifugation at 14000 rpm for 10 min in a low-protein binding Ultrafree-MC centrifugal filter [0.22 μm pore size (Millipore, Billerica, MA)]. The eluate was transferred to a 5 mm outer diameter susceptibility-matched NMR cell (Shigemi). NMR data were acquired at 25 °C using a Bruker Avance II 900 MHz spectrometer equipped with a cryogenically cooled probe (Bruker BioSpin GmbH, Rheinstetten, Germany). A series of 2D homonuclear 1H NMR spectra were collected to determine the structure of WT APETx2, including total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and double-quantum-filtered correlation spectroscopy (DQF-COSY) spectra. TOCSY spectra were acquired using an MLEV isotropic mixing period of 80 ms, while a 200 ms mixing time was used to collect NOESY data. The 2D data sets were generally recorded with 4096 data points in the direct dimension and 512 increments in the indirect dimension over a sweep width of 12 ppm. Watergate or excitation sculpting was used for solvent suppression, and all data sets were referenced to the solvent signal, 4.773 ppm at 298 K. In addition, a natural abundance 1H−13C 2D HSQC data set was recorded using 32 scans and 400 increments over a sweep width of 80 ppm. SOFAST-HMQC spectra were recorded for WT APETx2 and all mutants. These were recorded using a repetition time of 0.1 s and 4096 transients per increment (with a total of 30−40 increments). All data were processed using the Rowland NMR toolkit or Topspin (Bruker Biospin). Free induction decays were multiplied by a Gaussian window function, and the indirect dimension was zero filled and linearly predicted as appropriate prior to Fourier transformation. Medium- and long-range NOESY cross-peaks were assigned both manually and in an automatic fashion using CYANA.35 Data sets recorded on a lyophilized sample dissolved in 100% D2O were used to identify slowly exchanging amide protons involved in hydrogen bonds. 1 H−15N and 1H−13C SOFAST-HMQC data were subsequently assigned using the 1H chemical shifts determined from the homonuclear data. The complete set of 1H, 15N, and 13C chemical shift assignments has been deposited in BioMagResBank (accession number 25205). Structures were calculated using interproton distances based on NOESY peak intensities, backbone dihedral angles derived

in in vivo models of peripheral pain. However, this activity may prove to be highly beneficial and fortuitous for its development as an analgesic lead compound. It remains to be seen if the hERG activity of APETx2 can be separated from its NaV1.8 activity. Given the promiscuous pharmacology of the APETxlike peptides that have been characterized to date,12,29 further structure−function studies will be critical for ascertaining the role of specific residues in targeting each class of channels. This knowledge will aid in the development of peptide analogues with low levels of, or negligible, hERG channel blockade at concentrations relevant for ASIC3 inhibition (0.5−10 μM) and thus greater potential for therapeutic development. In summary, we demonstrated that APETx2 has promiscuous pharmacology, and we elucidated the molecular basis of its interaction with ASIC3 and hERG. The data presented here open up the possibility of rationally engineering APETx2 variants with improved potency and selectivity toward ASIC3, and thus greater therapeutic value.

■

EXPERIMENTAL SECTION

Peptide Synthesis of APETx2 and Its Mutants. WT APETx2 and several mutants (Y16A, R17A, P18A, R24A, R31A, Y32A, and F33A) were synthesized using SPPS in which an N-terminal Gly1− Ser19 thioester fragment was ligated with a Cys20−Asp42 peptide amide using conditions described previously.23 Single-residue mutations were introduced during fragment synthesis23 in which peptides were assembled via SPPS using a stepwise in situ neutralization protocol for Boc chemistry31 followed by HF cleavage.23 Peptide fragments were purified by preparative HPLC [Vydac C18 column, 250 mm × 21 mm, flow rate of 15 mL/min, gradient of 5 to 45% solvent B (0.043% TFA in 90% acetonitrile) in solvent A (0.05% TFA in water) over 40 min] to purities of >95% (confirmed by analytical HPLC) before they were subjected to native chemical ligation as described previously.23 The ligation product mixture was loaded onto a preparative RP-HPLC column (Vydac C18 column, 21 mm × 250 mm), and full-length, reduced synthetic APETx2 was eluted with a linear gradient from 0 to 30% solvent B over 10 min followed by 30 to 50% solvent B over 40 min, at a flow rate of 15 mL/min. Fractions were analyzed using electrospray ionization mass spectrometry (ESIMS) and analytical HPLC before being pooled and lyophilized. Oxidative folding was performed under the conditions described previously for chemically synthesized APETx2.23 Folding was monitored using analytical RP-HPLC. Oxidized APETx2 was purified on a Vydac C18 column (250 mm × 10 mm) with a linear gradient from 5 to 30% solvent B over 10 min followed by 30 to 50% solvent B over 40 min using a flow rate of 4 mL/min. Fractions were analyzed using HPLC−ESI-MS, and pure fractions with HPLC purities of >95% were pooled and lyophilized. Recombinant Production of APETx2 and Its Mutants. APETx2 mutants were produced as previously described.16 Briefly, genes encoding His6-tagged MBP-APETx2 fusion proteins, with a TEV protease cleavage site inserted between the MBP and APETx2 coding regions, were synthesized by GeneArt (Regensburg, Germany) and subcloned into a variant of the pLic-MBP vector.32−34 Plasmids were transformed into E. coli BL21(λDE3) cells, and fusion protein expression was induced with 1 mM IPTG. Cells were harvested by centrifugation and lysed using a high-pressure cell disruptor at 26 kPa (TS Series Bench-top model, Constant Systems Ltd.). The His6-MBPAPETx2 fusion proteins were captured by passing the soluble cell extract [buffered in 20 mM Tris and 200 mM NaCl (pH 8.0)] over an Ni-NTA Superflow resin (Qiagen, Valencia, CA). The His6-MBP tag was removed by TEV protease, and then the recombinant APETx2 was purified using RP-HPLC on a Zorbax 300SB C18 column (250 mm × 4.6 mm) using a flow rate of 1 mL/min and a gradient from 20 to 40% solvent B over 40 min. Purity and Integrity of Peptides. Purified mature peptides were analyzed using narrow-bore RP-HPLC (Thermo Aquasil C18 column, 9201

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

from TALOS+ analysis of chemical shifts,36 side chain χ1 dihedral angles derived from 3J(HαHβ) coupling constants in combination with intraresidue Hα−Hβ and HN−Hβ NOE intensities, and information about hydrogen bonds derived from the D2O exchange experiment. A set of 100 structures was calculated using the torsion angle dynamics program CYANA. The 30 structures with the lowest target function were analyzed using MolProbity.21 A set of 20 structures with the best MolProbity scores (i.e., highest stereochemical quality) were chosen to represent the solution structure of APETx2. Atomic coordinates for APETx2 and all restraint files used for the structure calculations have been deposited in the Protein Data Bank (entry 2MUB). Electrophysiological Measurements. X. laevis oocytes were defolliculated by being treated with collagenase (Sigma type I). rASIC3 and hERG cRNA was synthesized using an mMessage mMachine cRNA transcription kit and healthy stage V−VI oocytes injected with 4 ng of cRNA (40 nL of a 100 ng/μL solution). Oocytes were kept at 17 °C in ND96 solution [96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES (pH 7.45)] supplemented with 5 mM pyruvic acid, 50 μg/mL gentamicin, and 2.5% horse serum. Membrane currents were recorded 1−6 days after cRNA injection under voltage clamp (Axoclamp 900A amplifier, Molecular Devices, Sunnyvale, CA) using two standard glass microelectrodes with resistances of 0.5−2 MΩ when filled with a 3 M KCl solution. Data acquisition and analysis were performed using pCLAMP software (version 10, Molecular Devices). All experiments were performed at room temperature (18−21 °C) in an ND96 solution containing 0.1% fatty acid-free bovine serum albumin to minimize adsorption of APETx2 to plastic tubing. ASIC3 recordings were performed on oocytes clamped at −60 mV with data sampled at 2000 Hz and filtered at 0.01 Hz. Changes in extracellular pH were induced using a microperfusion system that allowed rapid changes of solutions (∼1.5 mL/min). HEPES was replaced by MES to buffer the pH 6 stimulus solution. Serial dilutions of test peptides (from 0.3 nM to 30 μM) were administered after stimulation at pH 7.45, and oocytes were bathed in the toxin solution until the next round of stimulation (55 s exposure). hERG-expressing oocytes were clamped to a holding potential of −80 mV, and then 1 s pulses to a test potential of 0 mV were applied every 10 s until currents stabilized. The current−voltage (I−V) relationship for hERG was measured by applying 2 s pulses to test potentials ranging from −70 to 40 mV in 10 mV increments and then by a repolarizing step to −70 mV for 1 s.37 SSIN was assayed using a triple-pulse voltage protocol. Cells were depolarized to 50 mV for 300 ms (P1), and then short P2 pulses from −120 to 70 mV in 10 mV increments were applied every 10 s to allow channels to relax to a steady state of inactivation. Currents were elicited by a P3 pulse to 50 mV (for 50 ms) and peak tail currents plotted as a function of the previous voltage step.14 Deactivation at more negative voltages was not corrected for. All hERG data were sampled at 10 kHz and filtered at 1 kHz. Data were normalized to the maximal current amplitude and fit with the Boltzmann equation: G = Gmax/[1 + exp(V0.5 − V)/k], where V0.5 is the voltage corresponding to the half-maximal effect. Data Analysis. The Hill equation was fit to concentration− response data to obtain the half-maximal response (IC50) and Hill slope. Data are presented as the mean ± SEM (n is the number of individual oocytes taken from at least three different frogs). Statistical analyses comparing differences between WT and mutant peptides or controls were performed by either an unpaired t test or analysis of variance followed by a Dunnett post hoc test, with p values indicated in the figure legends or text.

■

Accession Codes

PDB and BMRB accession codes for APETx2 are 2MUB and 25205, respectively.

■

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +61 7 3346 2982. Fax: +61 7 3346 2090. E-mail: l. [email protected]. *Telephone: +61 7 3346 2025. Fax: +61 7 3346 2101. E-mail: [email protected]. Present Address ∥

J.E.J.: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark. Author Contributions #

J.E.J., B.C.-A., and R.A. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Prof. John Wood (University College London, London, U.K.) and Prof. Jamie Vandenberg (Victor Chang Cardiac Research Institute, Sydney, Australia) for rASIC3 and hERG clones, respectively. We acknowledge financial support from the Australian National Health & Medical Research Council (Principal Research Fellowship to G.F.K. and Project Grant APP1012338 to G.F.K. and L.D.R.), the Australian Research Council (Future Fellowship to K.J.R. and M.M.), and the Institute for Molecular Bioscience, The University of Queensland (PhD Scholarship to J.E.J.).

■

ABBREVIATIONS USED ASIC, acid-sensing ion channel; hERG, human ether-a-go-go related gene; HF, hydrogen fluoride; SPPS, solid phase peptide synthesis; MBP, maltose binding protein; SAR, structure− activity relationship; TEV, tobacco etch virus; PDB, Protein Data Bank; SEM, standard error of the mean

■

REFERENCES

(1) Diochot, S.; Baron, A.; Salinas, M.; Douguet, D.; Scarzello, S.; Dabert-Gay, A. S.; Debayle, D.; Friend, V.; Alloui, A.; Lazdunski, M.; Lingueglia, E. Black Mamba Venom Peptides Target Acid-Sensing Ion Channels To Abolish Pain. Nature 2012, 490, 552−555. (2) Deval, E.; Noël, J.; Lay, N.; Alloui, A.; Diochot, S.; Friend, V.; Jodar, M.; Lazdunski, M.; Lingueglia, E. ASIC3, a Sensor of Acidic and Primary Inflammatory Pain. EMBO J. 2008, 27, 3047−3055. (3) Izumi, M.; Ikeuchi, M.; Ji, Q.; Tani, T. Local ASIC3 Modulates Pain and Disease Progression in a Rat Model of Osteoarthritis. J. Biomed. Sci. 2012, 19, 77−84. (4) Deval, E.; Noël, J.; Gasull, X.; Delaunay, A.; Alloui, A.; Friend, V.; Eschalier, A.; Lazdunski, M.; Lingueglia, E. Acid-Sensing Ion Channels in Postoperative Pain. J. Neurosci. 2011, 31, 6059−6066. (5) Yan, J.; Wei, X.; Bischoff, C.; Edelmayer, R. M.; Dussor, G. pHEvoked Dural Afferent Signaling is Mediated by ASIC3 and is Sensitized by Mast Cell Mediators. Headache 2013, 53, 1250−1261. (6) Immke, D. C.; McCleskey, E. W. ASIC3: A Lactic Acid Sensor for Cardiac Pain. Sci. World J. 2001, 1, 510−512. (7) Wu, W. L.; Cheng, C. F.; Sun, W. H.; Wong, C. W.; Chen, C. C. Targeting ASIC3 for Pain, Anxiety, and Insulin Resistance. Pharmacol. Ther. 2012, 134, 127−138. (8) Diochot, S.; Baron, A.; Rash, L. D.; Deval, E.; Escoubas, P.; Scarzello, S.; Salinas, M.; Lazdunski, M. A new sea anemone peptide, APETx2, Inhibits ASIC3, a Major Acid-Sensitive Channel in Sensory Neurons. EMBO J. 2004, 23, 1516−1525.

ASSOCIATED CONTENT

S Supporting Information *

Overlaid SOFAST-HMQC spectra of WT APETx2 and a Y13A mutant (Figure S1) and additional details of the electrophysiology experiments. This material is available free of charge via the Internet at http://pubs.acs.org. 9202

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203

Journal of Medicinal Chemistry

Article

(27) Kozlov, S.; Grishin, E. The Mining of Toxin-Like Polypeptides from EST Database by Single Residue Distribution Analysis. BMC Genomics 2011, 12, 88−99. (28) Rodríguez, A. A.; Cassoli, J. S.; Sa, F.; Dong, Z. Q.; de Freitas, J. C.; Pimenta, A. M.; de Lima, M. E.; Konno, K.; Lee, S. M.; Garateix, A.; Zaharenko, A. J. Peptide Fingerprinting of the Neurotoxic Fractions Isolated from the Secretions of Sea Anemones Stichodactyla helianthus and Bunodosoma granulifera. New Members of the APETxLike Family Identified by a 454 Pyrosequencing Approach. Peptides 2012, 34, 26−38. (29) Liu, P.; Jo, S.; Bean, B. P. Modulation of Neuronal Sodium Channels by the Sea Anemone Peptide BDS-I. J. Neurophysiol. 2012, 107, 3155−3167. (30) Diochot, S.; Schweitz, H.; Béress, L.; Lazdunski, M. Sea Anemone Peptides with a Specific Blocking Activity Against the Fast Inactivating Potassium Channel Kv3.4. J. Biol. Chem. 1998, 273, 6744− 6749. (31) Schnölzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. In Situ Neutralization in Boc-Chemistry Solid Phase Peptide Synthesis. Rapid, High Yield Assembly of Difficult Sequences. Int. J. Pept. Protein Res. 1992, 40, 180−193. (32) Cabrita, L. D.; Dai, W.; Bottomley, S. P. A Family of E. coli Expression Vectors for Laboratory Scale and High Throughput Soluble Protein Production. BMC Biotechnol. 2006, 6, 12−19. (33) Saez, N. J.; Mobli, M.; Bieri, M.; Chassagnon, I. R.; Malde, A. K.; Gamsjaeger, R.; Mark, A. E.; Gooley, P. R.; Rash, L. D.; King, G. F. A Dynamic Pharmacophore Drives the Interaction Between Psalmotoxin-1 and the Putative Drug Target Acid-Sensing Ion Channel 1a. Mol. Pharmacol. 2011, 80, 796−808. (34) Klint, J. K.; Senff, S.; Saez, N. J.; Seshadri, R.; Lau, H. Y.; Bende, N. S.; Undheim, E. A.; Rash, L. D.; Mobli, M.; King, G. F. Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli. PLoS One 2013, 8, e63865. (35) Herrmann, T.; Güntert, P.; Wüthrich, K. Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA. J. Mol. Biol. 2002, 319, 209−227. (36) Shen, Y.; Delaglio, F.; Cornilescu, G.; Bax, A. TALOS+: A Hybrid Method for Predicting Protein Backbone Torsion Angles from NMR Chemical Shifts. J. Biomol. NMR 2009, 44, 213−223. (37) Twiner, M. J.; Doucette, G. J.; Rasky, A.; Huang, X. P.; Roth, B. L.; Sanguinetti, M. C. Marine Algal Toxin Azaspiracid is an Open-State Blocker of hERG Potassium Channels. Chem. Res. Toxicol. 2012, 25, 1975−1984.

(9) Karczewski, J.; Spencer, R. H.; Garsky, V. M.; Liang, A.; Leitl, M. D.; Cato, M. J.; Cook, S. P.; Kane, S.; Urban, M. O. Reversal of AcidInduced and Inflammatory Pain by the Selective ASIC3 Inhibitor, APETx2. Br. J. Pharmacol. 2010, 161, 950−960. (10) Deval, E.; Gasull, X.; Noël, J.; Salinas, M.; Baron, A.; Diochot, S.; Lingueglia, E. Acid-Sensing Ion Channels (ASICs): Pharmacology and Implication in Pain. Pharmacol. Ther. 2010, 128, 549−558. (11) Blanchard, M. G.; Rash, L. D.; Kellenberger, S. Inhibition of Voltage-Gated Na+ Currents in Sensory Neurones by the Sea Anemone Toxin APETx2. Br. J. Pharmacol. 2012, 165, 2167−2177. (12) Peigneur, S.; Béress, L.; Möller, C.; Marí, F.; Forssmann, W. G.; Tytgat, J. A Natural Point Mutation Changes Both Target Selectivity and Mechanism of Action of Sea Anemone Toxins. FASEB J. 2012, 26, 5141−5151. (13) Chagot, B.; Escoubas, P.; Diochot, S.; Bernard, C.; Lazdunski, M.; Darbon, H. Solution Structure of APETx2, a Specific Peptide Inhibitor of ASIC3 Proton-Gated Channels. Protein Sci. 2005, 14, 2003−2010. (14) Diochot, S.; Loret, E.; Bruhn, T.; Béress, L.; Lazdunski, M. APETx1, a New Toxin From the Sea Anemone Anthopleura elegantissima, Blocks Voltage-Gated Human Ether-A-Go-Go-Related Gene Potassium Channels. Mol. Pharmacol. 2003, 64, 59−69. (15) Anangi, R.; Chen, C. C.; Lin, Y. W.; Cheng, Y. R.; Cheng, C. H.; Chen, Y. C.; Chu, Y. P.; Chuang, W. J. Expression in Pichia pastoris and Characterization of APETx2, a Specific Inhibitor of Acid Sensing Ion Channel 3. Toxicon 2010, 56, 1388−1397. (16) Anangi, R.; Rash, L. D.; Mobli, M.; King, G. F. Functional Expression in Escherichia coli of the Disulfide-Rich Sea Anemone Peptide APETx2, a Potent Blocker of Acid-Sensing Ion Channel 3. Mar. Drugs 2012, 10, 1605−1618. (17) Jensen, J. E.; Mobli, M.; Brust, A.; Alewood, P. F.; King, G. F.; Rash, L. D. Cyclisation Increases the Stability of the Sea Anemone Peptide APETx2 but Decreases its Activity at Acid-Sensing Ion Channel 3. Mar. Drugs 2012, 10, 1511−1527. (18) Vandenberg, J. I.; Perry, M. D.; Perrin, M. J.; Mann, S. A.; Ke, Y.; Hill, A. P. hERG K+ channels: Structure, Function, and Clinical Significance. Physiol. Rev. 2012, 92, 1393−1478. (19) Kwan, A. H.; Mobli, M.; Gooley, P. R.; King, G. F.; Mackay, J. P. Macromolecular NMR Spectroscopy for the Non-Spectroscopist. FEBS J. 2011, 278, 687−703. (20) Güntert, P. Automated NMR Structure Calculation with CYANA. Methods Mol. Biol. 2004, 278, 353−378. (21) Davis, I. W.; Leaver-Fay, A.; Chen, V. B.; Block, J. N.; Kapral, G. J.; Wang, X.; Murray, L. W.; Arendall, W. B., III; Snoeyink, J.; Richardson, J. S.; Richardson, D. C. MolProbity: All-Atom Contacts and Structure Validation for Proteins and Nucleic Acids. Nucleic Acids Res. 2007, 35, W375−W383. (22) Schubert, M.; Labudde, D.; Oschkinat, H.; Schmieder, P. A Software Tool for the Prediction of Xaa-Pro Peptide Bond Conformations in Proteins Based on 13C Chemical Shift Statistics. J. Biomol. NMR 2002, 24, 149−154. (23) Jensen, J. E.; Durek, T.; Alewood, P. F.; Adams, D. J.; King, G. F.; Rash, L. D. Chemical Synthesis and Folding of APETx2, a Potent and Selective Inhibitor of Acid Sensing Ion Channel 3. Toxicon 2009, 54, 56−61. (24) Zhang, M.; Liu, X. S.; Diochot, S.; Lazdunski, M.; Tseng, G. N. APETx1 from Sea Anemone Anthopleura elegantissima is a Gating Modifier Peptide Toxin of the Human Ether-A-Go-Go- Related Potassium Channel. Mol. Pharmacol. 2007, 72, 259−268. (25) Chagot, B.; Diochot, S.; Pimentel, C.; Lazdunski, M.; Darbon, H. Solution Structure of APETx1 from the Sea Anemone Anthopleura elegantissima: A New Fold for an HERG Toxin. Proteins 2005, 59, 380−386. (26) Oliveira, J. S.; Zaharenko, A. J.; Ferreira, W. A., Jr.; Konno, K.; Shida, C. S.; Richardson, M.; Lúcio, A. D.; Beirão, P. S.; de Freitas, J. C. BcIV, a New Paralyzing Peptide Obtained from the Venom of the Sea Anemone Bunodosoma caissarum. A Comparison with the Na+ Channel Toxin BcIII. Biochim. Biophys. Acta 2006, 1764, 1592−1600. 9203

dx.doi.org/10.1021/jm501400p | J. Med. Chem. 2014, 57, 9195−9203