Article pubs.acs.org/jmc
Studies Examining the Relationship between the Chemical Structure of Protoxin II and Its Activity on Voltage Gated Sodium Channels Jae H. Park,* Kevin P. Carlin, Gang Wu, Victor I. Ilyin, Laszlo L. Musza, Paul R. Blake, and Donald J. Kyle Discovery Research, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, New Jersey 08512, United States S Supporting Information *
ABSTRACT: The aqueous solution structure of protoxin II (ProTx II) indicated that the toxin comprises a well-defined inhibitor cystine knot (ICK) backbone region and a flexible C-terminal tail region, similar to previously described NaSpTx III tarantula toxins. In the present study we sought to explore the structure−activity relationship of the two regions of the ProTx II molecule. As a first step, chimeric toxins of ProTx II and PaTx I were synthesized and their biological activities on Nav1.7 and Nav1.2 channels were investigated. Other tail region modifications to this chimera explored the effects of tail length and tertiary structure on sodium channel activity. In addition, the activity of various C-terminal modifications of the native ProTx II was assayed and resulted in the identification of protoxin IINHCH3, a molecule with greater potency against Nav1.7 channels (IC50 = 42 pM) than the original ProTx II.
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INTRODUCTION Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues, as well as tight coordination of distant functions. In vertebrates and invertebrates electrical signals play a fundamental role in the control of physiological processes. Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for action potential initiation and propagation in excitable cells, including nerve, muscle, and neuroendocrine cell types.1 VGSCs are composed of an α-subunit that forms the pore of the channel and one or two β-subunits that modulate the activity of the α-subunit and affect its expression in the plasma membrane. The α-subunit contains four domains (D1−D4) with each domain containing six transmembrane segments (S1−S6). Thus far, nine different α-subunit isoforms have been identified (Nav1.1− Nav1.9). Even though most excitable tissues contain more than one sodium channel isoform, there exist tissue specific expression patterns. For example, the Nav1.4 isoform is mainly expressed in skeletal muscle while the Nav1.5 isoform is mainly localized to cardiac tissue. The Nav1.7, Nav1.8, and Nav1.9 isoforms are predominantly expressed in the peripheral nervous system,2 while the remaining isoforms are expressed in neurons of both the central and peripheral nervous systems. Data from both animal studies and human subjects indicate that the Nav1.7 channel isoform plays an important role in pain signaling. Mice that had the Nav1.7 channels knocked out in a subset of nociceptors had an impaired ability to sense pain.3 In humans, the presence of nonsense mutations in the gene encoding the Nav1.7 channel has been associated with the absence of pain perception.4 Moreover, gain-of-function mutations in the human SCN9A gene that cause excessive channel activity have been associated with pain syndromes. © 2014 American Chemical Society
Mutations that limit the ability of the Nav1.7 channel to inactivate are associated with a hereditary condition known as paroxysmal extreme pain disorder. Gain-of-function mutations that cause the Nav1.7 channels to activate at lower membrane potentials result in a second pathophysiological condition known as primary erythromelalgia. Patients with this latter condition present complaints of severe burning pain in the extremities.2 Taken together, these data suggest that the Nav1.7 channel is a promising new drug target for modulating human pain conditions such as neuropathic pain, a condition with significant unmet clinical need. In addition, the finding that patients with nonfunctioning Nav1.7 channels showed no substantial deficits other than a reduction in the sense of smell4 suggests that a Nav1.7 channel-selective antagonist would be able to alleviate pain without eliciting the adverse side effects that are common in existing therapies. The challenge of designing a highly selective Nav1.7 channel blocker has proven to be quite difficult, and there has only been limited success reported thus far.5−7 The high sequence homology among the channel isoforms likely underpins highly similar steric and electrostatic binding sites on the various channel isoforms, ultimately making it difficult for small, druglike molecules to achieve Nav1.7 specificity. Multiple toxin-binding sites have been described for Nav channels (sites 1−7) that enhance or inhibit the flow of sodium ions across cell membranes. Venom peptides derived from a number of species, including spider, have been reported and generally bind at sites 1, 3, 4, and 6 of the VGSC. Some spider toxins, for example protoxin II (ProTx II, Thrixopelma pruriens) and phrixotoxin I (Phrixotrichus auratus), are gating modifiers Received: May 2, 2014 Published: July 15, 2014 6623
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that trap voltage-gated channels in nonconducting state(s).8 ProTx II is a 30 amino acid, three-disulfide bond inhibitor cystine knot (ICK) peptide that traps the voltage sensor and preferentially inhibits Nav1.7.8−11 Phrixotoxin I is one amino acid shorter than protoxin II but contains a similar ICK motif. Phrixotoxin I has been shown to act primarily upon potassium channels, but more recently an inhibitory function at certain sodium channels has also been described.12 Our present goal was to better understand the nature of ProTx II’s high affinity for Nav1.7 channels and selectivity over other Nav channel isoforms, as we hoped to uncover critical elements that may subsequently be applied to our design of small molecules. In this report, we describe the solution structure of ProTx II and its comparison to the previously reported solution conformation of NaSpTx3 family tarantula toxins (phrixotoxin I13 and GsMTx II14). We also describe the synthesis and in vitro measurement of Nav1.7 activity of several novel toxin peptides derived from protoxin II and phrixotoxin I. In the course of our work we made synthetic modifications to these peptides in order to explore the relationship(s) between sequence, structure, and biological activity, particularly on Nav1.7 channels.
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of crude linear protoxin II in water was added and stirred for 24−48 h at room temperature at a concentration of 0.1 mg/mL. Once the reaction was completed, the pH of the reaction mixture was adjusted to 3 and the mixture purified by preparative HPLC. Peptide Analysis. Crude and purified peptides were analyzed using an Agilent 1100 series LC or LC−MS, equipped with a SymmetryShield, RP18, 5 μm, 4.6 mm × 150 mm column (Waters). Two gradients were used: (i) 10−60% B over 30 min with a flow rate of 1 mL/min; (ii) 10−60% B over 15 min with a flow rate of 1 mL/ min. Buffer A was 0.1% TFA in water, and buffer B was 0.1% TFA in acetonitrile. The purity of each peptide is ≥95%, and it was calculated from the average purity between two methods. Detection was at 220 nm. Peptide mass was analyzed with low resolution electrospray ionization. NMR Spectroscopy and Structural Analysis. Toxin samples were dissolved in 200 μL of a 90% H2O−10% D2O mixture and transferred to 3 mm i.d. WILMAD 335-PP NMR tubes. NMR spectra were collected on a Bruker Avance III 500 MHz spectrometer equipped with a z-gradient TCI cryoprobe or a Bruker Avance III 600 MHz spectrometer equipped with a z-gradient TXI probe. The experiments were performed at 293, 298, 303, or 308 K to optimize resolution and minimize critical peak overlaps. Collection of 1H and NOESY experimental data at multiple temperatures also allowed the calculation of the temperature coefficients of the amide resonances. All spectra were processed and analyzed with Bruker’s Topspin 3.0 or 3.1 software. Solvent suppressed one-dimensional experiments were recorded with either a WATERGATE scheme utilizing a binomial pulse sandwich15 or a noesypresat16 sequence. Homonuclear correlation (COSY)17 experiments were collected using gradients for coherence selection18 and in the absolute value mode. Phase sensitive nuclear Overhauser spectroscopy (NOESY)19 was carried out with a WATERGATE20 sequence for water suppression and with mixing times of 100, 200, and 300 ms, with states-TPPI phase cycling.21 Total correlation spectroscopy (TOCSY)22 was carried out with a DIPSI223 spin-lock of 120 ms duration with states-TPPI phase cycling, applying WATERGATE solvent suppression. One-bond 1H−13C heteronuclear correlation (HMQC) spectroscopy24 was run in phase sensitive mode with states-TPPI phase cycling. Heteronuclear multiple bond correlation (HMBC) spectroscopy25 was acquired with a gradient enhanced pulse sequence26 in absolute value mode. Phase sensitive 1 H−15N heteronuclear single quantum coherence (HSQC) spectroscopy27 was carried out with WATERGATE suppression28 and statesTPPI phase cycling. Amino acid spin systems were identified from COSY and TOCSY data. Sequential assignments were obtained from NH−Hα NOESY correlations and from HMBC data when available. NOESY spectra were collected with 100, 200, and 300 ms mixing times, without observing any significant spin diffusion effects. NOE cross-peaks were classified into strong, medium, weak, and very weak, and upper bound distance restraints of 2.4, 3.4, 4.2, and 5.4 Å, respectively, were used in structure calculations. Molecular visualization and modeling were performed using the software package SYBYL on a Silicon Graphics Octane workstation. Modeling was based on molecular mechanics using the Tripos force field29 and the Gasteiger−Hückel method30 for calculation of charges as implemented in version 7.0 of the software. The starting point for structure calculations used a homology model generated (Quanta 98, Accelrys) using the published coordinates of PaTx I13 (1v7f.pdb, model 1) obtained from the Protein Data Bank. Structural graphics were generated using UCFS Chimera version 1.8.1 (build 39231) running on Mac OS 10.8.4. Hydrophobic surface areas generated in UCFS Chimera rely on the hydrophobicity scale of Kyte and Doolittle.31 Electrophysiology. Cells. The Nav1.2 cell line was engineered inhouse, while the Nav1.7 cell line was subcloned from an in-licensed cell line. Both the human Nav1.7 and the rat Nav1.2 isoforms were expressed in HEK-293 cells. Cells were plated on 35 mm culture dishes precoated with poly-D-lysine in standard DMEM culture medium and incubated in a 5% CO2 incubator at 37 °C.
EXPERIMENTAL SECTION
Materials and Methods. Materials. The side chain protecting groups for amino acids were as follows: tBu for aspartic acid, glutamic acid, serine, threonine, and tyrosine; Trt for cysteine and glutamine; Pbf for arginine; Boc for lysine and tryptophan. All orthogonally protected amino acids were purchased from Protein Technologies, Inc. (Tucson, AZ), Anaspec (Fremont, CA), and Chem Impex (Wooddale, IL). HCTU was purchased from Protein Technologies, Inc. (Tucson, AZ) and Anaspec (Fremont, CA). H-Trp(Boc)-2-Cl-Trt resin (0.42 mmol/g) and Fmoc-Rink amide resin (0.47 mmol/g) were purchased from Bachem (Torrence, CA). Fmoc-Ile-Wang LL resin (0.29 mmol/ g), Fmoc-Leu-Wang LL resin (0.24 mmol/g), Fmoc-Gly-Wang LL resin (0.35 mmol/g), and H-Met-2-Cl-Trt resin (0.64 mmol/g) were purchased from Novabiochem (Gibbstown, NJ). NMM, DIPEA, piperidine, DMF, DCM, 2,4,6-collidine, TFA, TIS, 3,6-dioxa-1,8octanedithiol, thioanisole, phenol, 0.1% TFA in acetonitrile, 0.1% TFA in water, aniline, and methanol were purchased from Sigma-Aldrich (St. Louis, MO) Linear Peptide Synthesis. The linear peptides were synthesized on a Pioneer peptide synthesizer (Applied Biosystems), and amino acids were assembled stepwise on a 0.05−0.1 mmol scale using 6- to 8fold excess of Fmoc amino acids. Prior to each coupling step, the Fmoc protecting groups were removed using 20% piperidine in DMF. Couplings were accomplished using Fmoc protected amino acids using HCTU and 2,4,6-collidine in DMF/DCM (1:1). Following synthesis, linear peptides were treated with TFA/TIS/3,6-dioxa-1,8-octanedithiol/thioanisole/phenol/H2O (81.5:1:2.5:5:5:5 by volume) for 2−3 h at room temperature to cleave from the resin. Cleaved peptides were treated with cold diethyl ether to precipitate the peptides, and then the precipitated peptides were washed with diethyl ether 3 times. Crude linear peptides were purified by reversed phase preparative HPLC, and white solids were obtained. C-Terminal Modification. The mixture of orthogonally protected protoxin II (0.13 g, 18 μmol, 1.0 equiv), HATU (67 mg, 176 μmol, 10 equiv), and collidine (47 μL, 352 μmol, 20 equiv) in DMF (10 mL) was stirred for 10 min. A derivatizing agent, RNH2 or ROH (176 μmol, 10 equiv), was added to the mixture and stirred for 3 h. Following removal of DMF, the resulting oil was run through a preparative HPLC instument to purify the linear protoxin II derivatives. Amidated linear protoxin II was treated with a TFA cocktail to remove the side chain protecting groups. Air Oxidation. GSH (0.15 mM) and GSSG (0.3 mM) were added to a mixture of urea (2 M) and Tris-HCl (0.1 M) in milli-Q water. pH was then adjusted to 8 using saturated aqueous NaHCO3. A solution 6624
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Electrophysiological recordings were made from the cultured cells approximately 12−48 h after plating. Electrophysiology. On the day of experimentation, the 35 mm dish was placed on the stage of an inverted microscope and continuously perfused with fresh extracellular solution. A gravity-driven superfusion system was used to apply compounds directly to the cell under evaluation. This system consists of a linear array of glass pipettes connected to a motorized horizontal translator. The outlet of this superfusion system was positioned approximately 100 μm from the cell of interest. Sodium currents were recorded under voltage clamp in the wholecell configuration using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), 1322A A/D converter (Molecular Devices), and pClamp software (version 8, Molecular Devices). Borosilicate glass pipettes had resistance values between 1.5 and 3.0 MΩ when filled with pipet solution. Series resistance (3 μM.
the terminal tryptophan was substituted with isoleucine in addition to either the α- or N-methylated Leu at the L29 position (compounds 13 and 15). When potencies were compared, compound 12 (α-methylated) and compound 14
(N-methylated) demonstrated a very slight decrease in Nav1.7 potency. The effect of restricting the potential for α helix formation was much more apparent when the terminal tryptophan was substituted with isoleucine. In this case, the 6628
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α-methylated peptide retained its Nav1.7 potency while the Nmethylated version was more than 53-fold less potent. These results supported the hypothesis that even though the ProTx II tail region was flexible, certain conformations of the tail were preferred for potency against the Nav1.7 channel isoform. Moreover, the Trp30 residue may bind to the Nav1.7 channel pocket that has a proton acceptor site. This hydrogen bonding may further override the conformational effect and give compound 14 relatively high potency against Nav1.7. After observing the indication of the potential role of the Cterminal tryptophan residue in the interaction with the Nav1.7 channel, we decided to investigate if the functionalization of this residue would lead to significant changes. Three ProTx II compounds were synthesized with the C-terminal carboxylic acid modified to amidated functional groups (compounds 16, 17, 19). When the carboxylic acid functional group was modified to a primary amide (compound 16) or phenylamide (compound 19), the activity at the Nav1.7 channel did not change significantly. Potency against Nav1.2 channels was affected, being increased approximately 10- to 30-fold relative to the parent ProTx II. Interestingly, when the carboxylic acid was modified to a methylamide (compound 17), the potency at Nav1.7 was increased into the picomolar range (IC50 = 42 pM; Figure 4B). Activity on Nav1.2 channels also increased with this compound, but the overall selectivity (83-fold) was comparable to the parent ProTx II molecule (105 fold). Since the CONH2 modification of ProTx II (compound 16) significantly improved potency against Nav1.2 channels over ProTx II’s (COOH) and the terminal methylated compound 17 (CONHCH3) showed the highest potency against Nav channels, we were interested to see if a methyl ester group (COOCH3, compound 18) would increase the selectivity by decreasing potency against Nav1.2. Unfortunately, compound 18 showed similar nonselective activity to compound 16 and compound 19. When we arrange the order of potency of C-terminal modified peptides against Nav1.7, compound 17 is the most potent peptide and compound 1 and compound 19 are the least potent peptide. This resulted in potency order of NHCH3 > NH2 > OCH3 > NHPh ∼ OH). Given that the C-terminus containing OCH3 and OH can only exist in a transconformation and they are the least potent, then we believe that it is likely that potent NHCH3 containing C-terminus predominantly exist in the cis-amide conformation. Furthermore, compound 19 (NHPh) had relatively lower potency, possibly due to its sterically hindered substituent that would favor the trans-amide conformation at the C-terminal and therefore reduce the potency against Nav1.7 channels. After improving the potency of PaTx I at Nav1.7 by substituting its tail sequence to that of ProTx II, we tested whether the potency of other natural toxins could be increased by simply substituting the ProTx II tail. On the basis of our previous study,12 we chose two from NaSpTx3 family, VsTx II and JzTx XII, as these are very weak Nav1.7 antagonists (VsTx II, IC50 ≈ 9.3 μM; JzTx XII, IC50 ≈ 1.5 μM). Compounds 20 and 21 were synthesized consisting of VsTx II and JzTx XII with ProTx II tails, respectively. Compound 20 showed an approximately 30-fold increase in potency compared to the original toxin (IC50 = 293 nM vs IC50 = 9621 nM); however, its potency against the Nav1.2 isoform remained unchanged. This resulted in a selectivity improvement from 6.2-fold to 43-fold. When the sequences of compound 20 and compound 4 (ProTx II (M19L)) are compared, it can be seen that there exists only a
single amino acid mutation at the 11th position (Ser to Glu). This single amino acid change caused significant potency reductions at both Nav1.7 (∼172-fold) and Nav1.2 (∼285fold) isoforms. This indicates that the serine residue at the 11th position plays an important role in determining the affinity for Nav1.7 and Nav1.2 channels. The finding that Ser11 is important for Nav channels activity is consistent with the finding of Priest et al.,34 who identified this residue as important for Nav1.5 channel activity. The fact that the mutation of this residue affects activity on multiple Nav isoform indicates that it does not play a role in the Nav1.7 selectivity of ProTx II. More generally, this finding indicates that the tail portion is not the only important determinant of potency but amino acid residues in the body portion of the tertiary structure play a role in how the toxins interact with sodium channels. This conclusion is also consistent with the alanine scan experiments performed by Priest et al.34 When the ProTx II tail was substituted for the native JzTx XII tail (compound 21), there was no change in potency against either Nav1.7 or Nav1.2 channels as seen with the tail substitution of VsTx II. This is further evidence that the tail region is not the sole determinant of potency. When the sequence of compound 21 was compared with ProTx II, it can be seen that there are two amino acid differences in the body region: Met to Tyr at the 19th position and Arg to Glu at the 22nd position. Given that these same two residues (Met19 and Arg22) individually were identified as important for maintaining potency against Nav1.5 channels,10 it appears that these amino acids are critical for the fundamental interaction between the peptide and the channel. This comparison further demonstrates that the body portion of these toxins especially front view in Figure 3A is playing some complementary role in determining potency against Nav channels.
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CONCLUSION ProTx II and several peptide analogues were synthesized inhouse, and the aqueous solution structure was investigated by NMR spectroscopy. Not surprisingly, the structure exhibited the well described ICK folding pattern with a flexible Cterminal tail region, similar to other toxins in the NaSpTx3 family. Despite the high sequence homology and similar structural folding, it was previously noted that ProTx II and PaTx I showed profound differences in activity on voltage-gated sodium channels.12 Activity of chimeric toxins of ProTx II and the PaTx I, synthesized by switching tail regions, indicated that the flexible tail region plays an important role in Nav channel potency and to some degree isoform selectivity. Amino acid substitution experiments demonstrated that amino acids in the body region especially those on the surface shown in Figure 3A on left side, such as Ser11, Glu12, Tyr19, and Arg22, can also contribute to Nav potency and selectivity. When the PaTx I tail (KKII) was substituted for the ProTx II tail (KKKLW), PaTx I potency was improved 423 times at Nav1.7 channel and 208 times at Nav1.2 channel. Experiments designed to constrain the flexible tail region indicated that the α-helical conformation is favored over a β-sheet conformation. The potency of ProTx II was improved 24-fold when the C-terminal functional group was changed from a carboxylic acid to a methylamide. Our current SAR effort was able to identify a number of structural aspects of the ProTx II peptide that are important determinants of Nav channel potency and to a lesser extent Nav isoform selectivity. This effort led to compound 17, which to our knowledge is the most potent Nav1.7 channel inhibitor 6629
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voltage-gated sodium channels. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E2724−E2732. (6) Kers, I.; Csjernyik, G.; Macsari, I.; Nylöf, M.; Sandberg, L.; Skogholm, K.; Bueters, T.; Eriksson, A. B.; Oerther, S.; Lund, P. E.; Venyike, E.; Nyström, J. E.; Besidski, Y. Structure and activity relationship in the (S)-N-chroman-3-ylcarboxamide series of voltagegated sodium channel blockers. Bioorg. Med. Chem. Lett. 2012, 22, 5618−5624. (7) Kers, I.; Macsari, I.; Csjernyik, G.; Nylöf, M.; Skogholm, K.; Sandberg, L.; Minidis, A.; Bueters, T.; Malmborg, J.; Eriksson, A. B.; Lund, P. E.; Venyike, E.; Luo, L.; Nyström, J. E.; Besidski, Y. Phenethyl nicotinamides, a novel class of Nav1.7 channel blockers: structure and activity relationship. Bioorg. Med. Chem. Lett. 2012, 22, 6108−6115. (8) Sokolov, S.; Kraus, R. L.; Scheuer, T.; Catterall, W. A. Inhibition of sodium channel gating by trapping the domain II voltage sensor with protoxin II. Mol. Pharmacol. 2008, 73, 1020−1028. (9) Schmalhofer, W. A.; Calhoun, J.; Burrows, R.; Bailey, T.; Kohler, M. G.; Weinglass, A. B.; Kaczorowski, G. J.; Garcia, M. L.; Koltzenburg, M.; Priest, B. T. ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors. Mol. Pharmacol. 2008, 74, 1476−1484. (10) Smith, J. J.; Cummins, T. R.; Alphy, S.; Blumenthal, K. M. Molecular interactions of the gating modifier toxin ProTx-II with Nav1.5: implied existence of a novel toxin binding site coupled to activation. J. Biol. Chem. 2007, 282, 12687−12697. (11) Xiao, Y.; Blumenthal, K.; Jackson, J. O., 2nd; Liang, S.; Cummins, T. R. The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation. Mol. Pharmacol. 2010, 78, 1124− 1134. (12) Park, J. H.; Carlin, K. P.; Wu, G.; Ilyin, V. I.; Kyle, D. J. Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7-selective peptideprotoxin II. J. Pept. Sci. 2012, 18, 442−448. (13) Chagot, B.; Escoubas, P.; Villegas, E.; Bernard, C.; Ferrat, G.; Corzo, G.; Lazdunski, M.; Darbon, H. Solution structure of phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus. Protein Sci. 2004, 13, 1197−1208. (14) Oswald, R. E.; Suchyna, T. M.; McFeeters, R.; Gottlieb, P.; Sachs, F. Solution structure of peptide toxins that block mechanosensitive ion channels. J. Biol. Chem. 2002, 277, 34443− 34450. (15) Liu, M.; Mao, X.; Ye, C.; Huang, H.; Nicholson, J. K.; Lindon, J. C. Improved WATERGATE pulse sequence for solvent suppression in NMR spectroscopy. J. Magn. Reson. 1998, 132, 125−129. (16) Nicholson, J. K.; Foxall, P. J.; Spraul, M.; Farrant, R. D.; Lindon, J. C. 750 MHz 1H and 1H−13C NMR spectroscopy of human blood plasma. Anal. Chem. 1995, 67, 793−811. (17) Aue, W. P.; Bartholdi, E.; Ernst, R. R. Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 1976, 64, 2229−2246. (18) Hurd, R. E. Gradient-enhanced spectroscopy. J. Magn. Reson. 1990, 87, 422−428. (19) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. Investigation of exchange processes by two-dimensional NMR spectroscopy. J. Chem. Phys. 1979, 71, 4546−4563. (20) Piotto, M.; Saudek, V.; Sklenár, V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 1992, 2, 661−665. (21) Marion, D.; Ikura, M.; Tschudin, R.; Bax, A. Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J. Magn. Reson. 1989, 85, 393−399. (22) Braunschweiler, L.; Ernst, R. R. Coherence transfer by isotropic mixing: application to protein correlation spectroscopy. J. Magn. Reson. 1983, 53, 521−528. (23) Shaka, A. J.; Lee, C. J.; Pines, A. Iterative schemes for bilinear operatorsapplication to spin decoupling. J. Magn. Reson. 1988, 77, 274−293.
described to date. Moreover, this compound has maintained similar selectivity as ProTx II, which is the most isoform selective natural toxin for Nav1.7.
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ASSOCIATED CONTENT
S Supporting Information *
1
H, 13C, and 15N chemical shift assignments of selected compounds; selected NOESY and HSQC spectra of compound 1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 609-409-5120. E-mail: jaehyun.park@pharma. com. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED Boc, tert-butyloxycarbonyl; tBu, tert-butyl; COSY, correlation spectroscopy; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle medium; DMF, N,N-dimethylformamide; Fmoc, fluorenylmethyloxycarbonyl; EGTA, ethylene glycol tetraacetic acid; GSH, reduced Lglutathione; GsMTx II, Grammostola spatulata mechanotoxin II; GSSG, oxidized (−)-glutathione; HCTU, 2-(6-chloro-1Hbenzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); HMBC, heteronuclear multiple-bond correlation; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum correlation; ICK, inhibitor cystine knot; JzTx XII, jingzhaotoxin XII; NaSpTx III, sodium channel blocking spider toxin family III; NMM, Nmethylmorpholine; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PaTx I, phrixotoxin I; Pbf, pentamethyldihydrobenzofuran-5-sulfonyl; ProTx II, protoxin II; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TOCSY, total correlation spectroscopy; Tris-HCl, Trizma hydrochloride; Trt, triphenylmethyl; VGSC, voltage-gated sodium channel
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REFERENCES
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Journal of Medicinal Chemistry
Article
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dx.doi.org/10.1021/jm500687u | J. Med. Chem. 2014, 57, 6623−6631
