jm501765v (2)

16
Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Na V 1.7 Sodium Channel Justin K. Murray, Joseph Ligutti, Dong Liu, Anruo Zou, Leszek Poppe, Hongyan Li, § Kristin L. Andrews, Bryan D. Moyer, Stefan I. McDonough, Philippe Favreau, # Reto Stö cklin, # and Les P. Miranda* ,Departments of Therapeutic Discovery, Neuroscience, and § Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States Therapeutic Discovery and Neuroscience, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States # Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland * S Supporting Information ABSTRACT: Na V 1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identication of a 34-residue peptide, termed GpTx-1, with potent activity on Na V 1.7 (IC 50 = 10 nM) and promising selectivity against key Na V subtypes (20× and 1000× over Na V 1.4 and Na V 1.5, respectively). NMR structural analysis of the chemically synthesized three disulde peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp 29 , Lys 31 , and Phe 34 near the C-terminus are critical for potent Na V 1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against Na V 1.4. A structure-guided campaign aorded additive improvements in potency and Na V subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a Na V 1.7 IC 50 value of 1.6 nM and >1000× selectivity against Na V 1.4 and Na V 1.5. INTRODUCTION Voltage-gated sodium channels (VGSCs or Na V s) initiate and propagate action potentials in excitable cells such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells. 1 Structurally, they consist of an approximately 260 kDa α-subunit and associated smaller β- subunits. 2 The α-subunit has four domains (IIV), each domain containing six transmembrane helices (S1S6). The S5S6 domains govern the main aspects of ion permeation, and domains including most prominently xed charge within the S4 transmembrane α-helix transduce depolarizing voltages into physical opening of the channel. The family of VGSCs consists of nine known subtypes (Na V 1.1Na V 1.9). These subtypes show tissue specic localization and functional dierences with Na V 1.1, Na V 1.2, and Na V 1.3 found principally in the central nervous system, Na V 1.6 located both centrally and peripherally, and Na V 1.7, Na V 1.8, and Na V 1.9 expressed primarily in the peripheral nervous system. 3 Na V 1.4 is present in skeletal muscle, and Na V 1.5 is found predominantly in cardiac muscle. 4 Three VGSCs (Na V 1.5, Na V 1.8, and Na V 1.9) are resistant to blockade by the sodium channel blocker tetrodotoxin (TTX), 5 demonstrating subtype specicity within this gene family. A role for the Na V 1.7 channel in pain perception was established by clinical gene-linkage analyses that revealed gain- of-function mutations in the SCN9A gene that encodes the α- subunit of Na V 1.7 channels as the etiological basis of inherited pain syndromes such as inherited erythromelalgia and paroxysmal extreme pain disorder. 6 Loss-of-function mutations result in the complete inability to sense any form of pain. 7 Global deletion of SCN9A in mice abolishes perception of thermal, mechanical, inammatory, and chemical pain, 8 and cell-specic deletion reduces responsiveness to several forms of pain. 9 On the basis of such evidence, decreasing Na V 1.7 channel activity in peripheral sensory neurons has been proposed as an eective pain treatment. 10 A role for Na V 1.7 in itch also is suggested by clinical genetics. 11 Broad Na V antagonists, such as TTX, lidocaine, bupivacaine, phenytoin, lamotrigine, and carbamazepine, have been shown to be useful for attenuating Received: November 13, 2014 Published: February 6, 2015 Article pubs.acs.org/jmc © 2015 American Chemical Society 2299 DOI: 10.1021/jm501765v J. Med. Chem. 2015, 58, 22992314

Upload: justin-murray

Post on 16-Feb-2017

165 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: jm501765v (2)

Engineering Potent and Selective Analogues of GpTx-1, a TarantulaVenom Peptide Antagonist of the NaV1.7 Sodium ChannelJustin K. Murray,† Joseph Ligutti,‡ Dong Liu,‡ Anruo Zou,‡ Leszek Poppe,† Hongyan Li,§

Kristin L. Andrews,∥ Bryan D. Moyer,‡ Stefan I. McDonough,⊥ Philippe Favreau,# Reto Stocklin,#

and Les P. Miranda*,†

†Departments of Therapeutic Discovery, ‡Neuroscience, and §Pharmacokinetics & Drug Metabolism, Amgen Inc., One AmgenCenter Drive, Thousand Oaks, California 91320, United States∥Therapeutic Discovery and ⊥Neuroscience, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States#Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland

*S Supporting Information

ABSTRACT: NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for thetreatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residuepeptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20×and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptidewas consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp29, Lys31, and Phe34

near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-foldselectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity,culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity againstNaV1.4 and NaV1.5.

■ INTRODUCTION

Voltage-gated sodium channels (VGSCs or NaVs) initiate andpropagate action potentials in excitable cells such as central andperipheral neurons, cardiac and skeletal muscle myocytes, andneuroendocrine cells.1 Structurally, they consist of anapproximately 260 kDa α-subunit and associated smaller β-subunits.2 The α-subunit has four domains (I−IV), eachdomain containing six transmembrane helices (S1−S6). TheS5−S6 domains govern the main aspects of ion permeation,and domains including most prominently fixed charge withinthe S4 transmembrane α-helix transduce depolarizing voltagesinto physical opening of the channel. The family of VGSCsconsists of nine known subtypes (NaV1.1−NaV1.9). Thesesubtypes show tissue specific localization and functionaldifferences with NaV1.1, NaV1.2, and NaV1.3 found principallyin the central nervous system, NaV1.6 located both centrallyand peripherally, and NaV1.7, NaV1.8, and NaV1.9 expressedprimarily in the peripheral nervous system.3 NaV1.4 is presentin skeletal muscle, and NaV1.5 is found predominantly incardiac muscle.4 Three VGSCs (NaV1.5, NaV1.8, and NaV1.9)are resistant to blockade by the sodium channel blocker

tetrodotoxin (TTX),5 demonstrating subtype specificity withinthis gene family.A role for the NaV1.7 channel in pain perception was

established by clinical gene-linkage analyses that revealed gain-of-function mutations in the SCN9A gene that encodes the α-subunit of NaV1.7 channels as the etiological basis of inheritedpain syndromes such as inherited erythromelalgia andparoxysmal extreme pain disorder.6 Loss-of-function mutationsresult in the complete inability to sense any form of pain.7

Global deletion of SCN9A in mice abolishes perception ofthermal, mechanical, inflammatory, and chemical pain,8 andcell-specific deletion reduces responsiveness to several forms ofpain.9 On the basis of such evidence, decreasing NaV1.7 channelactivity in peripheral sensory neurons has been proposed as aneffective pain treatment.10 A role for NaV1.7 in itch also issuggested by clinical genetics.11 Broad NaV antagonists, such asTTX, lidocaine, bupivacaine, phenytoin, lamotrigine, andcarbamazepine, have been shown to be useful for attenuating

Received: November 13, 2014Published: February 6, 2015

Article

pubs.acs.org/jmc

© 2015 American Chemical Society 2299 DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

Page 2: jm501765v (2)

pain in humans and animal models but have a variety of sideeffects due to a lack of isoform specificity.12 A primarychallenge in the development of a NaV1.7 antagonist as atherapeutic is attaining sufficient selectivity against NaV1.5,expressed in cardiac tissue, and NaV1.4, in skeletal muscle, so asnot to impair normal cardiac and skeletal muscle function.13

Spider venoms contain many peptide toxins that targetvoltage-gated ion channels, including KV, CaV, and NaVchannels, and have been useful tools to study channel structureand function.14 Two well-characterized examples of NaV1.7inhibitory peptides that display different NaV selectivity profilesand promiscuities toward other voltage-gated ion channelfamilies are Huwentoxin-IV (HWTX-IV) from the venom ofthe Chinese bird spider Selenocosmia huwena15 and Protoxin-II(ProTxII), isolated from the tarantula Thrixopelma pruriens.16

Like many other spider toxins, these two peptides conform tothe inhibitory cystine knot (ICK) peptide structural motif17 andinhibit channel activation by binding to the voltage sensor andlocking the channel in a closed conformation. HWTX-IV,ProTxII, and two other reported NaV1.7 inhibitory peptides, μ-conotoxin KIIIA18 from cone snail venom and centipede toxinpeptide μ-SLPTX-Ssm6a,19 have been prepared and charac-terized in our lab for comparison of their biologic activities.Herein we report our identification and characterization of

GpTx-1, a known antagonist of TTX-sensitive sodiumchannels,20 from the venom of the tarantula spider

Grammostola porteri.21 GpTx-1 was first reported as a CaVchannel blocker after isolation from the venom of the closelyrelated Chilean tarantula Grammostola rosea and named GTx1-15 (UniproKB: accession no. P0DJA9).22 It was later identifiedin the venom of Paraphysa scrofa (Phrixotrichus auratus).23 Onthe basis of its potency and desirable NaV subtype selectivityprofile, we selected GpTx-1 as a lead in our effort to developtherapeutically useful NaV1.7 peptides. We describe a significantpeptide medicinal chemistry effort to investigate the GpTx-1structure−activity relationships and engineer analogues withimproved levels of NaV1.7 potency and selectivity against theimportant off-target NaV isoforms NaV1.4 and NaV1.5.

■ RESULTS AND DISCUSSION

High-Throughput Screening of Venom Fractions. Toidentify a novel peptide inhibitor with NaV potency, 84 venomfractions from the tarantula Grammostola porteri (AtherisLaboratories, Switzerland, Melusine ref. MLU-020007) werescreened for activity against NaV1.7 (Figure 1). A 384-wellIonWorks Quattro (IWQ) platform, which evaluates receptorinhibition with a population patch clamp, was utilized for itshigh-throughput screening capability. Several venom fractionswith significant (>80% inhibition of peak current) NaV1.7inhibitory activity were identified, the first of which was fraction31. A second aliquot of this fraction was tested in the NaV1.7and NaV1.5 IWQ assays to confirm the activity of the hit and

Figure 1. (A) Reversed phase (RP) HPLC fractionation of crude venom extracted from Grammostola porteri. The tick marks along the x-axisrepresent time slices of fractionation. (B) Activity of the isolated venom fraction in the NaV1.7 IonWorks Quattro (IWQ) assay. Fraction 31(indicated with rectangular box) contained a major peak in the RP-HPLC chromatogram that exhibited >80% inhibition of peak current in the ionchannel assay and was later identified as GpTx-1.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2300

Page 3: jm501765v (2)

evaluate selectivity. All samples were tested for potency onsodium channels with electrophysiology to give a directmeasure of receptor inhibition. The validated hit fraction wasthen analyzed by high-resolution electrospray ionization (ESI)and matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectrometry (MS), which indicated thatthe fraction was a mixture of at least four distinct peptidespecies (Figures 2 and 3, respectively). The active fraction wasthen separated by reversed phase (RP) HPLC, and thecorresponding subfractions were screened for activity in theNaV1.7 and NaV1.5 IWQ assays. Subfraction 11 was the majorpeak in the RP-HPLC chromatogram and showed >90%inhibition of NaV1.7 activity (Figure 4). Deconvolution ofsubfraction 11 by Edman degradation and MS/MS sequencingrevealed the primary peptide sequence of GpTx-1 (1, Figure 5).GpTx-1 is a 34 residue, C-terminally amidated polypeptidecontaining six cysteine residues engaged in three disulfidebonds and is a putative member of NaSpTx family 1.24 Toconfirm its identity and activity, synthetic GpTx-1 waschemically synthesized using Fmoc solid-phase peptide syn-thesis (SPPS) to generate the linear peptide sequence, whichwas then oxidatively folded, purified by RP-HPLC to producethe final product (Figure 6), and tested.25 A coelution of thesynthetic and native products (1:1) was observed, confirmingthe authenticity of the synthetic versus native product (seeSupporting Information).Results of Electrophysiology Studies. Chemically

synthesized GpTx-1 (1) was characterized in a manualelectrophysiology whole-cell patch clamp assay using human

clones of several NaV subtypes (Figure 7). To test for inhibitionor stabilization of as many channel gating states as possible,dose−response curves were measured with voltage clamped toholding potentials that imposed steady 20% fractionalinactivation. The IC50 values of NaV1.8, NaV1.7, NaV1.5,NaV1.4, and NaV1.3 inhibition for GpTx-1 were 12.2 ± 2.2,0.0044 ± 0.0020, 4.20 ± 0.09, 0.301 ± 0.041, and 0.0203 ±0.0069 μM, respectively, confirming that GpTx-1 is a potentpeptide inhibitor of NaV1.7 with moderate selectivity againstNaV1.4 and excellent selectivity against the TTX-resistant(TTX-R) channels NaV1.5 and NaV1.8. Manual patch clampelectrophysiology was also performed with GpTx-1 on sensoryneurons isolated from mouse dorsal root ganglia (DRG) toevaluate physiologic relevance. The TTX-S current in theseneurons includes a component attributable to NaV1.7.

8 TheIC50 for inhibition of this current in DRG by GpTx-1 was0.0063 μM.26 Taken together, these electrophysiology resultsconfirm GpTx-1 as a potent NaV1.7 inhibitory peptide with apromising NaV subtype selectivity profile, making it a suitablestarting point for further structure−activity relationship (SAR)investigation.

NMR Structural Analysis of GpTx-1. To investigate thedisulfide architecture of the folded peptide, the NMR solutionstructure of synthetic GpTx-1 was examined. The primarily β-type structure of the peptide (backbone RMSD of 0.1 Å) isstabilized by three disulfide bonds and 11 hydrogen bondsbetween backbone residues (see Figure 8 for the ensemble ofthe 10 lowest energy conformations). The secondary structuralmotifs are a Type II β-turn between Gly4 and Arg7, followed by

Figure 2. High resolution electrospray ionization-mass spectrometry (ESI-MS) analysis of fraction 31 from the initial fractionation of Grammostolaporteri venom. The labeled peaks indicate the m/z ratios observed for the different ionization states ([M + 4H+]4+ = 1018.93, [M + 5H+]5+ = 815.35,and [M + 6H+]6+ = 679.64) of a peptide with a monoisotopic molecular weight of 4071.7 Da that was eventually identifed as GpTx-1. Additionalpeaks in the mass spectrum indicate that the venom fraction is a mixture of at least four distinct peptides.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2301

Page 4: jm501765v (2)

a β-strand between Arg7 and Ile10, then a type I β-turn betweenIle10 and Asn13, with an α-turn between Cys17 and Leu21, andfinally a β-hairpin between Val22 and Lys31. The NMR analysiswas consistent with the assumed disulfide connectivity of the sixcysteine residues as Cys2Cys17, Cys9Cys23, and Cys16Cys30 or a C1C4, C2C5, C3C6 pattern,27 revealing thatGpTx-1 contains an inhibitory cystine knot (ICK) motif.Comparison to Other Nav1.7 Inhibitory Peptides. The

peptide sequence, NaV1.7 potency, and NaV subtype selectivityof synthetic GpTx-1 (1) was compared to previously reportedNaV1.7 inhibitory peptides. Two voltage gating modifierpeptides from spider venom, HWTX-IV (2) and ProTxII (3),and the pore-blocking μ-conotoxin KIIIA18 (4) were chemicallysynthesized according to literature procedures and tested side-by-side with GpTx-1 against human clones of NaV1.7, NaV1.5,and NaV1.4 with the PatchXpress (PX) planar patch clampautomated electrophysiology system (Table 1). In our hands,ProTxII was the most potent peptide antagonist of NaV1.7(IC50 = 0.003 μM) but showed the least selectivity against theother NaV isoforms. HWTX-IV had moderate potency againstNaV1.7 (IC50 = 0.033 μM) with good selectivity against NaV1.5(IC50 = 25 μM) and NaV1.4 (IC50 = 4 μM). GpTx-1 sharesconsiderable sequence homology with HWTX-IV but wasslightly more potent against NaV1.7. Importantly, GpTx-1 hasexcellent inherent selectivity against NaV1.5 (∼1000-fold) withmoderate (20-fold) selectivity toward NaV1.4. KIIIA was mostpotent at inhibiting NaV1.4 (IC50 = 0.02 μM) and had muchweaker activity against NaV1.7 (IC50 = 0.46 μM). Overall, ourresults were in good agreement with those reported in the

literature.15,16,27 We also tested the commercially availablesynthetic centipede toxin peptide μ-SLPTX-Ssm6a (5, PeptidesInternational, KY, USA) and found it to be inactive againstNaV1.7 at concentrations up to 1 μM, in contrast to the reportfor the isolated natural peptide.18 We also chemicallysynthesized the reported sequence, and it was inactive up to1 μM. Given its native potency and selectivity profile, GpTx-1was determined to be a strong starting point for thedevelopment of NaV1.7 inhibitory peptides with selectivityagainst NaV1.5 and NaV1.4. The results obtained previously bythe manual patch clamp method were in good agreement withthose from the PX format, and further analogues were tested onthe latter platform due to its higher throughput.

Positional Alanine Scan of GpTx-1. To evaluate thestructure−activity relationships of GpTx-1, a series of alaninesubstitution analogues (6−34, Table 2) was prepared at eachamino acid position within the sequence, excluding thecysteines.28 These “alanine scan” mutants were tested foractivity against NaV1.7, NaV1.5, and NaV1.4 using the IWQassays. This Ala analoguing of GpTx-1 identified three residuesnear the C-terminus, namely Trp29 (29), Lys31 (30), and Phe34

(33), as being the most critical for potency against NaV1.7(Figure 9). Two other residues near or within this stretch ofamino acids at the C-terminus, His27 (27) and Tyr32 (31), werealso important for activity. Substitution of alanine into aseparate segment of amino acids near the N-terminus, at Phe5

(10) and at Arg7 (12), had a moderate impact on activity.Incorporation of alanine at the N-terminus (6 and 7) or withinthe sequence from Ile10−Lys15 (14−18) or Arg18−Pro19 (19−

Figure 3. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of fraction 31 from thefractionation of Grammostola porteri venom. The inset shows the low and mid mass ranges. The peak with an m/z ratio of 4074.9 was eventuallyidentified as GpTx-1 (average m/z ratio of 4073.9 Da), but the fraction is a mixture of at least four distinct peptides.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2302

Page 5: jm501765v (2)

20) had no significant effects on NaV1.7 inhibition. All of thecompounds retained excellent selectivity against NaV1.5 (IC50 >5 μM). Interestingly, the Ala substitution at position 5 to make[Ala5]GpTx-1 (10) was found to improve the NaV1.4selectivity of the peptide to 70-fold, compared to 30-fold forparent. The high-throughput IWQ platform was useful forrapidly testing the large set of Ala scan analogues, but thepopulation patch clamp method resulted in an approximately10-fold lower NaV1.7 potency (with a concomitant drop inselectivity) compared to the whole-cell patch clamp platforms.Compound 10 was tested in the PX assay format, revealing thatit retained activity against NaV1.7 with an IC50 value of 0.027 ±0.009 μM and was 300-fold selective against NaV1.4 (IC50 = 8.5± 3.3 μM), a significant improvement over native GpTx-1. Thismore selective GpTx-1 analogue was further analyzed bymanual electrophysiology, and the IC50 value of NaV1.7inhibition was 0.013 μM (Figure 10), confirming that[Ala5]GpTx-1 maintains potent inhibitory activity againstNaV1.7. Likewise, [Ala5]GpTx-1 was a potent and reversibleinhibitor of TTX-S current in mouse DRG neurons with anIC50 value of 0.023 μM (Figure 11). [Ala5]GpTx-1 was 99%

intact after 24 h incubation in human and mouse plasmas(Figure 12). These results suggest the potential of [Ala5]GpTx-1 as a tool for probing NaV1.7 inhibition in vivo.

Structure−Activity Relationship of GpTx-1. The linearpeptide sequence of GpTx-1 contains two stretches ofhydrophobic amino acids, one near the N-terminus(Phe5−Met6) and one near the C-terminus (Trp29−Phe34).Although relatively distant in the primary sequence, based onNMR structural analysis, these hydrophobic residues all comeinto close spatial proximity in the folded peptide. Held togetherby the three disulfide bonds, the overall conformation is furtherstabilized through the formation of a β-sheet by residues Val22

through Lys31. The C-terminal portion (His27-Phe34) iscomposed primarily of hydrophobic amino acids, while Phe5

and Met6 are located adjacent to that β-strand and form theremainder of a hydrophobic face. These same residues that areclustered on one face of GpTx-1, namely Phe5, Met6, His27,Trp29, Lys31, Tyr32, and Phe34, were also indicated as beingimportant for functional activity through alanine substitution.Because changes that alter the nature of this face reducepotency against NaV1.7, this hydrophobic region may be theportion of the molecule that interacts with the VGSCs at thebinding interface (see Figure 13A,B). Phe5 is situated at theperiphery of the putative binding face of GpTx-1 and mayinteract with a corresponding region on the channels that hassome variation between the different NaV isoforms, asreplacement with alanine has only a small impact on potency

Figure 4. (A) RP-HPLC subfractionation of the material in fraction 31 from the initial fractionation of Grammostola porteri venom. The tick marksalong the x-axis represent time slices of the subfractionation. (B) Activity of the isolated subfractions in the NaV1.7 IWQ assay. The major peak inthe RP-HPLC chromatogram was the most active in the NaV1.7 assay and was deconvoluted to identify GpTx-1.

Figure 5. Deconvoluted peptide sequence of GpTx-1 (1).

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2303

Page 6: jm501765v (2)

against NaV1.7 but greatly reduces activity against NaV1.4. Theincreased selectivity against NaV1.4 combined with the inherentselectivity against NaV1.5 make [Ala5]GpTx-1 (10) animportant tool for the elucidation of NaV biology and a startingpoint for the potential development of more selective GpTx-1peptide analogues.The NMR solution structure shows that GpTx-1 is

amphipathic in nature with a hydrophobic face on one sideof the molecule and a hydrophilic (mostly cationic) face on theopposite side (Figures 13C,D). The hydrophilic face of GpTx-1is comprised of residues Ile10−Lys15 and Arg18−Pro19, whosesubstitution has a negligible effect upon functional activity andmay be exposed to solvent during the binding interaction. Thisaspect could be exploited through peptide engineering to tunethe physical properties of the molecule and will be reported indue course.Position 5 Analogues of GpTx-1. The increase in

selectivity against NaV1.4 and retention of NaV1.7 potencyachieved with [Ala5]GpTx-1 (10) encouraged additionalinvestigation of amino acid residue 5 within the GpTx-1sequence. A set of peptide analogues (35−48) was prepared byvarying the size and shape of aliphatic and aromatic residues atthis position and tested against a small panel of NaV channels inthe PX format (Table 3). In general, it was observed thatsmaller aliphatic residues resulted in increases in selectivityagainst NaV1.4, while larger amino acids, especially aromaticresidues, caused a decrease in NaV1.7 specificity. Substitution ofglycine (35), methionine (39), or isoleucine (40) for the nativephenylalanine resulted in analogues that had equivalent orsuperior potency against NaV1.7 relative to native GpTx-1 with>200-fold selectivity against NaV1.4. Incorporation of 4-iodo-phenylalanine (4-I-Phe, 47) or biphenylalanine (Bip, 48) atposition 5 in GpTx-1 reduced potency against NaV1.7. TheSAR indicates that the residue in this position of GpTx-1 mayinteract with a corresponding site within the target that hassome variability among the different NaV isoforms.Position 6 Analogues of GpTx-1. In a parallel

optimization effort, we sought to replace the native methionine

in position 6 of GpTx-1 with a nonoxidizable residue. Thetendency of Met6 to oxidize to the corresponding methioninesulfoxide during peptide cleavage and folding was observed byLC-MS, which reduced yield and raised concerns over stability.Although the oxidized side product could be removed bypurification, an attempt was made to remove this liabilityaltogether through the preparation and screening of a smallseries of GpTx-1 position 6 analogues (49−55, Table 4).Incorporation of norleucine, 49, the most straightforwardstructural replacement for methionine, and phenylalanine, 52,resulted in a slight loss in selectivity against NaV1.4 and NaV1.5relative to GpTx-1. Incorporation of cyclohexylalanine (Cha,51) retained a NaV selectivity profile similar to GpTx-1, exceptwith increased potency against NaV1.7.The cooperative effects of substitution at positions 5 and 6

were explored through the synthesis and testing of five GpTx-1combination analogues (56−60, Table 4). The incorporation ofalanine at position 5, together with a nonoxidizable, hydro-phobic residue at position 6 such as norleucine (56) or leucine(57), produced analogues with NaV potency and selectivitysimilar to [Ala5]GpTx-1 but without the potential oxidativeliability.

Positions 26 and 28 and Combination Analogues ofGpTx-1. Additional positions around the periphery of theputative binding face on GpTx-1 were explored for potentialincreases in NaV potency and/or selectivity with substitutionanalogues 61−66 (Table 5). While several residues had beendirectly identified during the alanine scan as being critical to theinteraction of the peptide with the NaV channels and thenfound to be clustered on one face of the GpTx-1 structure, itwas position 5, located at the edge of that hydrophobic face,which had been found to exert the greatest impact on relativesubtype activity. Guided by the NMR structure, a number ofother GpTx-1 residues at the other edges of the “binding face”were selected for substitution with a variety of residues insearch of additional potential interactions (Figure 14A).Replacement of Thr26 in GpTx-1 with arginine (62) andhistidine (64) had little effect, but [Leu26]GpTx-1 (61)

Figure 6. HPLC chromatograms of crude linear GpTx-1 (top), crude folded GpTx-1 (middle), and purified folded GpTx-1 (bottom).

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2304

Page 7: jm501765v (2)

afforded a slight increase in NaV1.7 potency and NaV1.4selectivity relative to GpTx-1. The native lysine residue ofGpTx-1 at position 28 was independently substituted witharginine (65) and gave a small boost in selectivity and potencycompared to 1. Incorporation of glutamic acid at either position26 (63) or 28 (66) produced NaV activity profiles similar to[Ala5]GpTx-1.The SAR study revealed a number of GpTx-1 analogues (10,

35, 39, 40, 56, 57, 63, and 66) with reasonable potency againstNaV1.7 (IC50 values 0.01−0.03 μM), excellent selectivityagainst NaV1.5 (>500-fold), and improved selectivity againstNaV1.4 (∼200-fold). Combining the SAR of GpTx-1 at

Figure 7.Manual patch clamp electrophysiology of GpTx-1: (A) Timecourse of increasing concentrations of GpTx-1 against partiallyinactivated NaV1.7 channels, recorded from a single-cell with manualpatch clamp electrophysiology. Peak inward NaV1.7 currents weremeasured at −10 mV every 10 s in the presence of increasingconcentrations of GpTx-1; cells were held at a voltage where channelswere fully noninactivated (squares) and then switched to voltageyielding approximately 20% inactivation (circles). Testing with GpTx-1 showed inhibition of the NaV1.7 current, which was reversible uponwashout. (B) Currents in response to increasing concentrations ofGpTx-1, from the timecourse displayed. “Control” trace shows NaV1.7current before GpTx-1, and other traces show NaV1.7 current afterGpTx-1 addition at indicated concentrations. (C) Dose−responsecurves of GpTx-1 against NaV1.8, NaV1.7, NaV1.5, NaV1.4, and NaV1.3channels measured with the same protocol. Currents were normalizedwith 100 representing NaV current with no peptide addition and 0representing NaV current following complete block.

Figure 8. NMR solution structure of GpTx-1: (A) overlay of the 10lowest energy conformations of the peptide backbone, (B) overlay ofthe heavy atoms from the 10 lowest energy conformations of thepeptide, and (C) ribbon representation of the peptide backbone in thelowest energy conformation with secondary structure and numberedcysteine residues.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2305

Page 8: jm501765v (2)

positions 5, 6, 26, and 28 in a final set of analogues (67−71,Table 5) produced additive improvements in potency andselectivity. [Ala5,Phe6,Leu26,Arg28]GpTx-1 (71, Figure 14B)was found to be exceptionally potent and selective, with a

NaV1.7 IC50 of 0.0016 μM, >1000-fold selectivity againstNaV1.4, and >6000-fold selectivity against NaV1.5. In spite ofthe large number of substitutions, compound 71 behavedsimilarly to wild-type GpTx-1 during folding and has beenamenable to scale-up for future studies, which will be reportedin due course. GpTx-1 analogue 71 is to our knowledge16,29 theonly confirmed peptide sequence with single-digit nanomolarNaV1.7 inhibitory activity with >1000-fold selectivity against thetwo important VGSC subtypes NaV1.4 and NaV1.5. Theseadvances in NaV1.7 inhibitory peptide SAR will facilitate futureinterrogation of NaV biology.

■ CONCLUSION

The voltage gated ion channel NaV1.7 remains an importantand challenging target for the discovery and development ofpain therapeutics. We identified GpTx-1 as a peptide antagonistof NaV1.7 via the high-throughput screening of fractionatedvenom from the tarantula Grammostola porteri. Our manualelectrophysiological characterization of the native peptide toxinrevealed its potent inhibition of expressed human NaV1.7 andinherent selectivity against NaV1.5. We then optimized GpTx-1selectivity against NaV1.4, which governs excitability of skeletalmuscle, through an extensive SAR campaign. An NMRstructure confirmed the disulfide architecture and aided ourinterpretation of the screening data from an initial set of alaninescanning analogues. We identifed a putative binding face for theGpTx-1 peptide to the NaV1.7 channel but more importantlyfound that substitution of alanine for Phe5 (10) increasedselectivity against NaV1.4 without compromising NaV1.7activity. The location of this amino acid at the periphery ofthe binding face led us to explore other similar positions in thepeptide structure. After replacement of the native Met6 to avoidoxidation and combination with substitutions at positions 26and 28, we have identified a GpTx-1 analogue (71) that isnearly 10-fold more potent than wild-type, with >1000-foldselectivity against NaV1.5 and NaV1.4, two prominent VGSCsubtypes with the possible liabilities of side effects on the heartand skeletal muscle. The small but significant and additive gainsin selectivity through appropriate amino acid selection at theperipheral binding residues along with the tuning of thehydrophobic nature of the residue at position 6 demonstrate

Table 1. Sequence and Activity of Synthetic NaV1.7 Inhibitory Peptidesa

a*Denotes C-terminal amide.

Table 2. NaV Inhibitory Activity of GpTx-1 Analogues fromPositional Scanning with Alaninea

compd substitutionhNav1.7 IWQIC50 (μM)

hNav1.5 IWQIC50 (μM)

hNav1.4 IWQIC50 (μM)

1 wild type 0.09 ± 0.01 >5 2.7 ± 1.26 N-Term. Ala- 0.37 ± 0.02 >5 >4.87 Asp1Ala 0.10 ± 0.01 >5 1.8 ± 0.18 Leu3Ala 0.43 ± 0.13 >5 >3.09 Gly4Ala 0.27 ± 0.13 >5 3.2 ± 0.410 Phe5Ala 0.63 ± 0.12 27 ± 10 45 ± 311 Met6Ala 0.43 ± 0.05 >5 3.8 ± 0.512 Arg7Ala 0.94 ± 0.09 >5 >4.613 Lys8Ala 0.50 ± 0.01 >5 >3.914 Ile10Ala 0.21 ± 0 >5 3.0 ± 0.115 Pro11Ala 0.17 ± 0.07 >5 3.0 ± 0.516 Asp12Ala 0.12 ± 0.05 >5 0.9 ± 0.117 Asn13Ala 0.19 ± 0.04 >5 2.7 ± 0.218 Lys15Ala 0.23 ± 0.05 >5 4.7 ± 0.319 Arg18Ala 0.13 ± 0.02 >20 3.0 ± 0.620 Pro19Ala 0.09 ± 0.01 >20 1.5 ± 0.321 Asn20Ala 0.69 ± 0 >5 >522 Leu21Ala 0.19 ± 0.02 >20 3.0 ± 0.723 Val22Ala 0.38 ± 0.14 >5 3.7 ± 0.324 Ser24Ala 0.47 ± 0.15 >4.7 1.9 ± 0.625 Arg25Ala 0.33 ± 0.16 >5 >526 Thr26Ala 0.26 ± 0.05 >5 3.2 ± 0.127 His27Ala 0.97 ± 0.18 >5 4.1 ± 0.428 Lys28Ala 0.47 ± 0.05 >5 >4.929 Trp29Ala >5 >5 >4.530 Lys31Ala >5 >5 >531 Tyr32Ala 0.80 ± 0.24 >5 >532 Val33Ala 0.17 ± 0.02 >5 4.5 ± 0.533 Phe34Ala 1.20 ± 0.14 >5 >534 C-Term. -Ala 0.41 ± 0.03 >5 >5

aAll analogues were C-terminal peptide amides. Samples tested onIWQ platform (av ± SD, n ≥ 2).

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2306

Page 9: jm501765v (2)

the power of structure-guided design together with systematicanaloguing to improve upon a natural scaffold. GpTx-1 andrelated analogues will be useful tools with which to probesodium channel biology and could potentially serve as the basisfor the development of a peptide therapeutic.

■ EXPERIMENTAL SECTIONMaterials. Nα-Fmoc protected amino acids were purchased from

Novabiochem (San Diego, CA), Bachem (Torrance, CA), or GLBiochem (Shanghai, China). Rink Amide MBHA resin was purchased

from Peptides International (Louisville, KY). SP Sepharose HighPerformance resin was purchased from GE Healthcare Life Sciences.The following compounds were purchased: N,N-diisopropylethyl-amine (DIEA), trifluoroacetic acid (TFA), acetic acid, piperidine, 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane, oxidized gluta-thione, and reduced glutathione (Sigma-Aldrich, Milwaukee, WI);dichloromethane (DCM, Mallinckrodt Baker, Inc.); N,N-dimethylfor-amide (DMF, Fisher Sc ient ific) ; 1 -cyano-2-e thoxy -2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexa-fluorophosphate (COMU, Matrix Innovation, Montreal, Canada);HPLC-quality water and acetonitrile (Burdick and Jackson); and 1.0M Tris-HCl pH 7.5 (Teknova). Stable cell lines expressing human (h)

Figure 9. Positional scanning with alanine; each bar represents the IWQ NaV1.7 IC50 of the analogue with alanine substituted at the indicatedposition of GpTx-1 (SD, n ≥ 2). Peak concentration tested was 5 μM.

Figure 10. Dose−response curve of [Ala5]GpTx-1 (10) againsthuman NaV1.7 channels by manual whole-cell patch clamp electro-physiology (n = 4). Peak inward NaV1.7 currents were measured at−10 mV in the presence of increasing concentrations of [Ala5]GpTx-1; cells were held at a potential yielding approximately 20%inactivation. Currents were plotted as percent of control.

Figure 11. Dose−response curves of GpTx-1 and [Ala5]GpTx-1against TTX-S NaV channels recorded from mouse sensory neurons (n= 2). Peak inward currents were measured at −10 mV in the presenceof increasing concentrations of peptide and plotted as percent ofcontrol; cells were held at −140 mV.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2307

Page 10: jm501765v (2)

voltage-gated sodium (NaV) channels (CHO-hNaV1.3, HEK293-hNaV1.4, HEK293-hNaV1.5, HEK293-hNaV1.7, and CHO-hNav1.8)were used for experiments.Isolation and Purification of GpTx-1 from Venom. Venom

from the tarantula Gammostola porteri was extracted via electricalstimulation of an anesthetized spider. Venom samples were collected,lyophilized, and dissolved in 0.1% trifluoroacetic acid (TFA) in waterto approximately 1 mg venom/mL. The crude venom solutions weredesalted by solid-phase extraction (SPE) with Sep-Pak C18 cartridges(Waters, Milford, MA, USA) equilibrated in 0.1% TFA, eluted with80% aqueous acetonitrile, freeze-dried, and stored at −30 °C.The crude venom was fractionated by reversed phase (RP) HPLC,

collecting 84 samples in time slices. The venom extract was dissolvedin 0.1% TFA to approximately 1 mg venom/mL, separated by C18 RPHPLC chromatography, and collected into approximately 1 min widefractions. HPLC method: solvent A (0.1% TFA in water) and solventB (90% acetonitrile/10% water containing 0.1% TFA) at 1 mL/minwith a 1% /min gradient 0−100% solvent B. The fractions weretransferred into a 384-well plate format, dried in vacuo, and stored at−30 °C.N-Terminal sequencing of peptides was performed by Edman

degradation.30 Phenylthiohydantoin (PTH) amino acid derivativeswere analyzed with an Applied Biosystems automatic 473A sequencer.De novo peptide sequencing was accomplished by tandem massspectrometry.31

Peptide Synthesis. GpTx-1 peptides were assembled using Nα-Fmoc solid-phase peptide synthesis (SPPS) methodologies withappropriate orthogonal protection and resin linker strategies. Thefollowing side chain protection strategies were employed for standardamino acid residues: Asn(Trt), Asp(OtBu), Arg(Pbf), Cys(Trt),Gln(Trt), Glu(OtBu), His(Trt), Lys(Nϵ-Boc), Ser(OtBu), Thr(OtBu),Trp(Boc), and Tyr(OtBu). The peptides were synthesized on a 0.012mmol scale using Rink Amide MBHA resin (100−200 mesh, 1% DVB,RFR-1063-PI, 0.52 mequiv/g initial loading, 408291, PeptidesInternational, Louisville, KY). Dry resin (17 mg per well) was addedto a Phenomenex deep well protein precipitation plate (CEO-7565,38710−1) using a resin loader (Radley). Amino acids were added tothe growing peptide chain by stepwise addition using standard solidphase methods on an automated peptide synthesizer (IntavisMultipep). Amino acids (5 mol equiv, 120 μL, 0.5 M in DMF)were preact ivated (1 min) with (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexa-

fluorophosphate (COMU, 5 mol equiv, 170 μL, 0.35 M in DMF)and N,N-diisopropylethylamine (DIEA, 7.5 mol equiv, 70 μL, 1.25 Min dichloromethane (DCM)). Preactivated amino acids were trans-ferred to the appropriate well. Resins were incubated for 30 min anddrained, and the cycle was repeated. Following the second amino acidincubation, the plates were drained and washed with DMF eight times(3 mL per column of 8 wells). The Fmoc groups were then removedby two sequential incubations in 500 μL of a 20% piperidine in DMFsolution. The first incubation was 5 min. The resin was drained, andthe second incubation was for 20 min. The resin was drained andwashed with DMF 10 times (3 mL per column of eight wells). Afterremoval of the final Fmoc protecting group, the resin was washed withDCM 5 times (3 mL per column of eight wells) and allowed to air-dry.

Side Chain Deprotection and Cleavage from Resin. To thebottom of the filter plate was affixed a drain port sealing mat(ArcticWhite, AWSM-1003DP). To the resin in each well was addedtriisopropylsilane (100 μL), DODT (100 μL), and water (100 μL)using a multichannel pipet. To the resin in each well was added TFA(1 mL) using a Dispensette Organic dispenser. To the resin was addeda triangular micro stir bar, and the mixture was stirred for 3 h. Thesealing mat was removed, and the cleavage solution was eluted into asolid bottom 96-well deep well plate. The resin in each well waswashed with an additional 1 mL of TFA. The solutions wereconcentrated using rotary evaporation (Genevac). To each well in anew 96-well filter plate with a bottom sealing mat attached was added1 mL of cold diethyl ether using a Dispensette Organic dispenser. Tothe ether was added dropwise the concentrated peptide solutions usinga multichannel pipet with wide bore tips. A white precipitate formed.The mixture was agitated with the pipet to ensure complete mixingand precipitation. The white solid was filtered, washed with another 1mL of cold ether, filtered, and dried under vacuum.

Parallel Peptide Oxidative Folding. The oxidative folding of the96 peptide array was performed in parallel and at high dilution usingan array of 50 mL centrifuge tubes in the following manner. A sealingmat was affixed to the bottom of the 96-well filter plate containing thecrude, precipitated peptides. To the sample in each well was added 0.9mL of 50:50 water/acetonitrile with a multichannel pipet and a microstir bar. The mixture was stirred for 1 h to dissolve the solid. Thesealing mat was removed, the mixtures were filtered using a vacuummanifold, and the eluent was collected in a solid bottom 96-well deepwell plate. To the residual crude peptide in each well was added asecond 0.9 mL aliquot of 50:50 water/acetonitrile with a multichannel

Figure 12. Peptide stability of GpTx-1 (1) and [Ala5]GpTx-1 (10) in mouse, rat, and human plasmas at 37 °C. Intact peptide measured by LC-MSpeak area; each time point was an average of n = 4 samples.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2308

Page 11: jm501765v (2)

pipet. The mixture was again stirred and filtered, combining the eluentin the same solid bottom 96-well deep well plate. The peptidesolutions were set aside. In a separate 4 L bottle was prepared 4.0 L offolding buffer by combining 3.3 L of water, 300 mL of acetonitrile, 2.0g of oxidized glutathione, 1.0 g of reduced glutathione, and 400 mL of1 M Tris-HCl pH 7.5 and stirring until the solids completely dissolved.Then 96 individual 50 mL centrifuge tubes were positioned in a large 8× 12 matrix using HPLC fraction collection racks. To each tube in thearray was added 40 mL of peptide folding buffer using a largeDispensette liquid dispenser. To the folding buffer in each 50 mLcentrifuge tube was added the 1.8 mL of dissolved peptide from thecorresponding position in the 96-well deep well plate (well A1 → tubeA1, well B1 → tube B1, etc.) using a Tecan automated liquid handler.The pH of the folding solutions was measured to be about 7.7. Thearray of folding reactions was allowed to stand overnight. To each tubein the array was added 1 mL of glacial acetic acid to lower the pH to4.0 and quench the reaction. Ion exchange resin was used to capturethe folded peptide from the dilute solution and concentrate for

subsequent RP-HPLC purification. To each well in a new 96-well filterplate was added 1 mL of SP Sepharose High Performance resin (GEBiosciences) as a slurry with a multichannel pipet. Using a Tecanautomated liquid handler equipped with a vacuum manifold, the ion-exchange resin in each well was conditioned with folding buffer (3 ×0.9 mL with vacuum filtration after each addition), loaded with thepeptide folding solution (50 × 0.9 mL, tube A1 → well A1, tube B1 →well B1, etc.), and washed (4 × 0.9 mL, 20 mM NaOAc, pH = 4.0).The folded peptides were eluted from the resin in each well manuallywith 2 × 1 mL (1 M NaCl, 20 mM NaOAc, pH = 4.0) on a vacuummanifold, and the eluent was collected into a solid bottom 96-welldeep well plate.

Reversed Phase HPLC Purification and Analysis and MassSpectrometry. After concentration by ion exchange, the foldedpeptide (2 mL) was purified by mass-triggered semiprep HPLC(Agilent 1100/LEAP, Phenomenex Jupiter 5μ C18 300 Å, 100 mm ×10 mm 5 μm column) with a gradient of 15−35% B over 45 min, witha 5 min flush and 5 min equilibration at 8 mL/min. The collected

Figure 13. (A−C) GpTx-1 oriented with the hydrophobic and putative binding face formed by the C-terminal β-strand and residues Phe5 and Met6

oriented toward the reader. Connolly surface as calculated by the program MOE35 and colored by lipophilicity.36 (A) Partially transparent surfacerendering of the molecule. (B) Ribbon representation of the peptide backbone with the side chains of key residues depicted. (C) Hydrophobic andputative binding face of GpTx-1 with an opaque surface (green = hydrophobic and magenta = hydrophilic). (D) Molecule has been rotatedclockwise by 90° around the z-axis to show the topological contrast between the flat hydrophobic face and the hydrophilic (solvent-exposed) face ofthe peptide.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2309

Page 12: jm501765v (2)

fractions were pooled, concentrated, and reformatted into vials on aTecan automated liquid handler. Final QC (Phenomenex Jupiter 20mm × 2 mm, 100 Å, 5 μm column eluted with a 10 to 60% B over 10min gradient (A, water; B, acetonitrile, 0.1% TFA in each) at a 0.750mL/min flow rate monitoring absorbance at 220 nm) was performed.Peptide quantification was performed by chemiluminescent nitrogendetection (CLND) via correlation to a caffeine standard curve using anAntek 8060 HPLC-CLND detector and an Agilent Zorbax 3.5 μm300SB-C3 2.1 mm × 50 mm column eluted with a 1−100% B over 1.5min gradient (A, water; B, 2-propanol, 0.1% formic acid in each) at a0.25 mL/min flow rate. Peptides with >95% purity and correct m/zratio were screened (see Supporting Information for LC-MScharacterization of synthetic GpTx-1 and analogues).Ion-Works Quattro Population Patch Clamp Electrophysiol-

ogy. Adherent cells were isolated from tissue culture flasks using0.25% trypsin−EDTA treatment for 10 min and were resuspended inexternal solution consisting of 140 mM NaCl, 5.0 mM KCl, 10 mMHEPES, 2 mM CaCl2, 1 mM MgCl2, and 10 mM glucose, pH 7.4.

Internal solution consisted of 70 mM KCl, 70 mM KF, 10 mMHEPES, and 5 mM EDTA, pH 7.3. Cells were voltage clamped, usingthe perforated patch clamp configuration at room temperature (∼22°C), to −110 mV and depolarized to −10 mV before and 5 min aftertest compound addition. Compound dilutions contained 0.1% bovineserum albumin to minimize nonspecific binding. Peak inward currentswere measured from different wells for each compound concentration,and IC50 values were calculated with Excel software. All compoundswere tested in duplicate (n = 2). The IWQ platform was employed forthe screening of large sets of samples and resulted in a general ∼10-fold shift in NaV1.7 potency for GpTx-1 peptides perhaps due tointeraction of peptides with the thousands of “extra” cells in each IWQwell inherent to the population patch clamp technique.

PatchXpress 7000A Electrophysiology. Adherent cells wereisolated from tissue culture flasks using 1:10 diluted 0.25% trypsin−EDTA treatment for 2−3 min and then were incubated in completeculture medium containing 10% fetal bovine serum for at least 15 minprior to resuspension in external solution consisting of 70 mM NaCl,140 mM D-mannitol, 10 mM HEPES, 2 mM CaCl2, and 1 mM MgCl2,pH 7.4, with NaOH. Internal solution consisted of 62.5 mM CsCl, 75mM CsF, 10 mM HEPES, 5 mM EGTA, and 2.5 mM MgCl2, pH 7.25,with CsOH. Cells were voltage clamped using the whole-cell patchclamp configuration at room temperature (∼22 °C) at a holdingpotential of −125 mV with test potentials to −10 mV (hNaV1.2,hNaV1.3, hNaV1.4, and hNaV1.7), −20 mV (hNaV1.5), or 0 mV(hNav1.8). To record from partially inactivated channels, currentswere recorded with a holding voltage that yielded ∼20% channelinactivation, calculated automatically for each individual cell. Testcompounds were added, and NaV currents were monitored at 0.1 Hz atthe appropriate test potential. All compound dilutions contained 0.1%bovine serum albumin to minimize nonspecific binding. Cells wereused for additional compound testing if currents recovered to >80% ofstarting values following compound washout. At least four differentconcentrations of test compound at half log units were appliedindividually, with washout, recovery of current, and resetting ofholding voltage between each individual concentration. Percentinhibition as a function of compound concentration was pooledfrom at least n = 10 different cells, with two to three data points perconcentration, and fitting the resulting data set with a Hill (4-parameter logistic) fit in DataXpress 2.0 software to produce a singleIC50 curve.

32

Whole-Cell Patch Clamp Electrophysiology. Cells were voltageclamped using the whole-cell patch clamp configuration at roomtemperature (∼22 °C). Pipette resistances were between 1.5 and 2.0MΩ. Whole-cell capacitance and series resistance were uncompen-sated. Currents were digitized at 50 kHz and filtered (4-pole Bessel) at10 kHz using pClamp10.2. Cells were lifted off the culture dish and

Table 3. NaV Inhibitory Activity of Position 5 Analogues ofGpTx-1a

compdPhe5

substitutionhNav1.7 PXIC50 (μM)

hNav1.5 PXIC50 (μM)

hNav1.4 PXIC50 (μM)

10 Ala 0.027 >10 8.535 Gly 0.009 >10 1136 Abu 0.079 >10 >1037 Nva 0.039 >10 4.638 Val 0.019 >10 4.139 Met 0.002 >10 0.940 Ile 0.009 >10 2.041 Leu 0.005 >10 0.942 NMe-Leu 0.025 >10 7.743 Tle 0.024 >10 4.744 Cha 0.004 3.5 0.145 Cpg 0.005 >10 1.746 Chg 0.006 >10 1.547 4-I-Phe 0.236 >10 1.148 Bip 0.086 2.1 0.1

aAbu, L-2-aminobutyric acid; Bip, L-4-biphenylalanine; Cha, L-cyclohexylalanine; Chg, L-cyclohexylglycine; Cpg, L-cyclopentylglycine;NMe-Leu, L-N-methylleucine; Nva, L-norvaline; 4-I-Phe, L-4-iodo-phenylalanine; Tle, L-tert-butylglycine.

Table 4. NaV Inhibitory Activity of Position 6 Analogues ofGpTx-1a

substitution

compd Phe5 Met6hNav1.7 PXIC50 (μM)

hNav1.5 PXIC50 (μM)

hNav1.4 PXIC50 (μM)

1 0.010 >10 0.2010 Ala 0.027 >10 8.549 Nle 0.008 1.1 0.0750 Leu 0.024 >10 0.4151 Cha 0.004 2.8 0.0952 Phe 0.019 3.0 0.1453 Tyr 0.063 >10 3.454 Trp 0.023 3.4 0.2455 1-Nal 0.004 0.4 0.1156 Ala Nle 0.010 >10 2.657 Ala Leu 0.028 >10 9.958 Ala Phe 0.013 >10 0.7459 Ala Trp 0.059 >10 6.160 Ala 1-Nal 0.003 3.6 0.50

aCha, L-cyclohexylalanine; 1-Nal, L-1-naphthylalanine; Nle, L-norleu-cine.

Table 5. NaV Inhibitory Activity of Position 5 Analogues ofGpTx-1

substitution

compd Phe5 Met6 Thr26 Lys28

hNav1.7PX IC50(μM)

hNav1.5PX IC50(μM)

hNav1.4PX IC50(μM)

1 0.010 >10 0.2010 Ala 0.027 >10 8.561 Leu 0.004 4.6 0.5362 Arg 0.005 0.5 0.1163 Glu 0.034 >10 4.964 His 0.004 4.5 0.2465 Arg 0.008 8.8 0.8066 Glu 0.029 >10 1467 Ala Leu 0.054 >10 >1068 Ala Phe Leu 0.004 >10 1.269 Ala Phe Arg 0.011 >10 3.370 Ala Leu Arg 0.008 >10 6.671 Ala Phe Leu Arg 0.0016 >10 1.9

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2310

Page 13: jm501765v (2)

positioned directly in front of a micropipette connected to a solutionexchange manifold for compound perfusion. To record fromnoninactivated channels, cells were held at −140 mV and depolarizedto −10 mV (0 mV for hNaV1.8). To record from partially inactivatedchannels, cells were held at −140 mV until currents stabilized and thenswitched to a voltage that yielded ∼20% channel inactivation. Then 10ms pulses were delivered every 10 s and peak inward currents wererecorded before and after compound addition. Compound dilutionscontained 0.1% bovine serum albumin to minimize nonspecificbinding. For hNaV1.8 channel recordings, tetrodotoxin (TTX, 0.5uM) was added to inhibit endogenous TTX-sensitive voltage-gatedsodium channels and record only NaV1.8-mediated TTX-resistantcurrents. External solution consisted of: 140 mM NaCl, 5.0 mM KCl,2.0 mM CaCl2, 1.0 mM MgCl2, 10 mM HEPES, and 11 mM glucose,pH 7.4, by NaOH. Internal solution consisted of: 62.5 mM CsCl, 75mM CsF, 2.5 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.25,by CsOH. Escalating compound concentrations were analyzed on thesame cell, and IC50 values were calculated with Clampfit 10.2 softwareand by fitting the resulting data set with a Hill (4-parameter logistic) fitin Origin Pro 8 software.DRG Neuron Isolation and Manual Patch Clamp Electro-

physiology. Adult male and female C57BL/6 mice (HarlanLaboratories, Indianapolis, IN) were euthanized with sodiumpentobarbital (Nembutal, 80 mg/kg, ip, Western Med Supply, Arcadia,CA) followed by decapitation. DRG from cervical, thoracic, andlumbar regions were removed, placed in Ca2+ and Mg2+-free Hanks’Balanced Salt Solution (Invitrogen, Carlsbad, CA), and trimmed ofattached fibers under a dissecting microscope. DRG were sequentiallydigested at 37 °C with papain (20 U/mL, Worthington BiochemicalCorporation, Lakewood, NJ), L-cysteine (25 μM) in Ca2+ and Mg2+-free Hanks’ (pH 7.4) for 20−30 min, and then with collagenase type 2(0.9% w/v, Worthington Biochemical Corporation) for 20−30 min.Digestions were quenched with a 1:1 mixture of DMEM and Ham’s F-12 Nutrient Mixture (Invitrogen) supplemented with 10% calf serum(Invitrogen), and cells were triturated with a fire-polished Pasteurpipet prior to plating on poly-D-lysine-coated glass coverslips (Cole-Parmer, Vernon Hills, IL). Cells were maintained in a humidifiedincubator at 28 °C with 5% CO2 for 3−7 days in the presence of 1%NSF-1 (Lonza, Basel, Switzerland) to increase the expression oftetrodotoxin-sensitive sodium channel currents.DRG neurons were voltage clamped using the whole-cell patch

clamp configuration at room temperature (21−24 °C) using anAxopatch 200 B or MultiClamp 700 B amplifier and DIGIDATA1322A with pCLAMP software (Molecular Devices, Sunnyvale, CA).Pipettes, pulled from borosilicate glass capillaries (World PrecisionInstruments, Sarasota, FL), had resistances between 1.0 and 3.0 MΩ.Voltage errors were minimized using >80% series resistance

compensation. A P/4 protocol was used for leak subtraction. Currentswere digitized at 50 kHz and filtered (4-pole Bessel) at 10 kHz. Cellswere lifted off the culture dish and positioned directly in front of amicropipette connected to a solution exchange manifold forcompound perfusion. Cells were held at −140 mV or a voltageyielding approximately 20% inactivation and depolarized to −10 mVfor 40 ms every 10 s. Tetrodotoxin (TTX, Sigma) was used followingpeptide addition to block any residual TTX-sensitive sodium currents.Pipette solution contained 62.5 mM CsCl, 75 mM CsF, 2.5 mMMgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.25, by CsOH. Bathsolution contained 70 mM NaCl, mM 5.0 KCl, 2.0 mM CaCl2, 1.0mM MgCl2, 10 mM HEPES, 11 mM glucose, and 140 mM mannitol,pH 7.4, by NaOH. Data were analyzed with Clampfit and Origin Pro 8(OriginLab Corp, Northampton, MA).

NMR Structural Analysis of GpTx-1. The structure of GpTx-1was obtained by high resolution NMR spectroscopy in 95% water and5% D2O at pH ∼3 and T = 298 K. The data were collected on aBruker Avance III 800 MHz spectrometer using standard 2Dexperiments.33 The 2D diffusion edited NOESY experiment wasrecorded with the PGSTE element34 to eliminate water resonance andfacilitate detection of all the α protons (Supporting Information). Thestructure was calculated from 500 NOE constraints (216 long-range),45 dihedral angle constraints, 11 hydrogen-bond constraints, and threedisulfide-bond constraints using Cyana 2.1 software. The final RMSDfor the backbone atoms was 0.1 ± 0.05 and 0.74 ± 0.12 Å for all heavyatoms. The disulfide connectivity was confirmed by the PADLOC27

calculations, which gave a probability of one to the Cys2Cys17,Cys9Cys23, and Cys16Cys30 pattern and zero probability to thealternative Cys2Cys16, Cys9Cys23, and Cys17Cys30 disulfidepattern.

Plasma Stability Studies. The stability of GpTx-1 (1) and[Ala5]GpTx-1 (10) was studied in human, rat, and mouse plasmas.Peptide stock solutions were made from GpTx-1 peptide and[Ala5]GpTx-1 peptide analogue reference standards in 50/50 (v/v)methanol/water and stored at −20 °C. Peptide stock solutions (1 mg/mL) were used to prepare 20 μg/mL peptide working solutions inHPLC grade water. The peptide working solutions were stored in arefrigerator at 2−8 °C prior to use. Stability samples were prepared byadding 225 μL of plasma into the vials containing 25 μL of 20 μg/mLpeptide working solution and incubating at 37 °C. The initialconcentration was 2 μg/mL for each peptide in human, rat, or mouseplasma. Aliquots of plasma (25 μL) at five time points (0, 2, 4, 8, and24 h) were transferred into the appropriate well of a 96-well plate,followed by the addition of 25 μL of internal standard solution (100ng/mL, peptide analogue made in 50/50 methanol/water) and 100 μLof 0.1% formic acid, and the samples were vortex mixed. An OasisHLB μElution 96-well solid phase extraction plate was used to extract

Figure 14. (A) Surface rendering of NMR structure of GpTx-1 (1) with key binding residues colored in green and residues impacting selectivitycolored magenta. (B) Surface rendering of a homology model of [Ala5,Phe6,Leu26,Arg28]GpTx-1 (71) with key binding residues colored in greenand substituted residues improving potency, stability, and/or selectivity in yellow. Figures generated using PyMOL.37

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2311

Page 14: jm501765v (2)

GpTx-1 or [Ala5]GpTx-1 from the pretreated plasma samples and theextracts were injected (10 μL) onto the LC-MS/MS system foranalysis. The LC-MS/MS consisted of an Acquity UPLC system(Waters, Milford, MA) coupled to a 5500 QTRAP mass spectrometer(AB Sciex, Toronto, Canada) with a Turbo IonSpray ionizationsource. The analytical column was an Acquity UPLC BEH C18 2.1 mm× 50 mm column. The mobile phases were 0.1% formic acid inacetonitrile/water (5/95, v/v, mobile phase A) and 0.1% formic acid inacetonitrile/water (95/5, v/v, mobile phase B). Data was collected andprocessed using AB Sciex Analyst software (version 1.5). The plasmastability of the tested peptides were derived from the peak area ratioscorresponding to peptides and internal standard obtained from theLC-MS/MS analysis; all data were normalized to the value at 0 h timepoint.

■ ASSOCIATED CONTENT*S Supporting InformationCharacterization of synthetic GpTx-1 and comparison to nativepeptide, NMR chemical shifts of GpTx-1, analytical character-ization of peptide analogues, and dose−response curves for keycompounds against human NaV1.7. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 1-805-447-9397. Fax: 1-805-480-3015. E-mail: [email protected].

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe gratefully thank Jennifer Aral, Jason Long, StephanieDiamond, Ryan Holder, and Jingwen Zhang for peptidesynthesis support, Xiaoyang Xia for molecular modelingsupport, and Kaustav Biswas and Elizabeth Doherty foreditorial assistance.

■ ABBREVIATIONS USEDAbu, L-2-aminobutyric acid; Bip, L-4-biphenylalanine; Boc, tert-butoxycarbonyl; Cha, L-cyclohexylalanine; Chg, L-cyclohexyl-glycine; Cpg, L-cyclopentylglycine; Fmoc, Nα-9-fluorenylme-thoxycarbonyl; 1-Nal, L-1-naphthylalanine; Nle, L-norleucine;NMe-Leu, L-N-methylleucine; Nva, L-norvaline; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; 4-I-Phe, L-4-iodo-phenylalanine; tBu, tert-butyl; Tle, L-tert-butylglycine; Trt, trityl

■ REFERENCES(1) Yu, F. H.; Yarov-Yarovoy, V.; Gutman, G. A.; Catterall, W. A.Overview of molecular relationships in the voltage-gated ion channelsuperfamily. Pharmacol. Rev. 2005, 57, 387−395.(2) (a) Noda, M.; Ikeda, T.; Suzuki, H.; Takeshima, H.; Takahashi,T.; Kuno, M.; Numa, S. Expression of functional sodium channelsfrom cloned cDNA. Nature 1986, 322, 826−828. (b) Noda, M.;Numa, S. Structure and Function of Sodium Channel. J. Recept. Res.1987, 7, 467−497.(3) (a) Goldin, A. L. Resurgence of sodium channel research. Annu.Rev. Physiol. 2001, 63, 871−894. (b) Fischer, T. Z.; Waxman, S. G.Familial pain syndromes from mutations of the NaV1.7 sodiumchannel. Ann. N. Y. Acad. Sci. 2010, 1184, 196−207.(4) French, R. J.; Terlau, H. Sodium channel toxins receptor targetingand therapeutic potential. Curr. Med. Chem. 2004, 11, 3053−3064.(5) Lee, C. H.; Ruben, P. C. Interaction between voltage-gatedsodium channels and the neurotoxin, tetrodotoxin. Channels 2008, 2,407−412.

(6) (a) Yang, Y.; Wang, Y.; Li, S.; Xu, Z.; Li, H.; Ma, L.; Fan, J.; Bu,D.; Liu, B.; Fan, Z.; Wu, G.; Jin, J.; Ding, B.; Zhu, X.; Shen, Y.Mutations in SCN9A, encoding a sodium channel alpha subunit, inpatients with primary erythermalgia. J. Med. Genet. 2004, 41, 171−174.(b) Harty, T. P.; Dib-Hajj, S. D.; Tyrrell, L.; Blackman, R.; Hisama, F.M.; Rose, J. B.; Waxman, S. G. NaV1.7 mutant A863P inerythromelalgia: effects of altered activation and steady-stateinactivation on excitability of nociceptive dorsal root ganglion neurons.J. Neurosci. 2006, 26, 12566−12575. (c) Estacion, M.; Dib-Hajj, S. D.;Benke, P. J.; te Morsche, R. H. M.; Eastman, E. M.; Macala, L. J.;Drenth, J. P. H.; Waxman, S. G. NaV1.7 gain-of-function mutations as acontinuum: A1632E displays physiological changes associated witherythromelalgia and paroxysmal extreme pain disorder mutations andproduces symptoms of both disorders. J. Neurosci. 2008, 28, 11079−11088.(7) (a) Cox, J. J.; Reimann, F.; Nicholas, A. K.; Thornton, G.;Roberts, E.; Springell, K.; Karbani, G.; Jafri, H.; Mannan, J.; Raashid,Y.; Al-Gazali, L.; Hamamy, H.; Valente, E. M.; Gorman, S.; Williams,R.; McHale, D. P.; Wood, J. N.; Gribble, F. M.; Woods, C. G. AnSCN9A channelopathy causes congenital inability to experience pain.Nature 2006, 444, 894−898. (b) Ahmad, S.; Dahllund, L.; Eriksson, A.B.; Hellgren, D.; Karlsson, U.; Lund, P.-E.; Meijer, I. A.; Meury, L.;Mills, T.; Moody, A.; Morinville, A.; Morten, J.; O’Donnell, D.;Raynoschek, C.; Salter, H.; Rouleau, G. A.; Krupp, J. J. A stop codonmutation in SCN9A causes lack of pain sensation. Hum. Mol. Genet.2007, 16, 2114−2121. (c) Goldberg, Y. P.; MacFarlane, J.;MacDonald, M. L.; Thompson, J.; Dube, M.-P.; Mattice, M.; Fraser,R.; Young, C.; Hossain, S.; Pape, T.; Payne, B.; Radomski, C.;Donaldson, G.; Ives, E.; Cox, J.; Younghusband, H. B.; Green, R.; Duff,A.; Boltshauser, E.; Grinspan, G. A.; Dimon, J. H.; Sibley, B. G.;Andria, G.; Toscano, E.; Kerdraon, J.; Bowsher, D.; Pimstone, S. N.;Samuels, M. E.; Sherrington, R.; Hayden, M. R. Loss-of-functionmutations in the NaV1.7 gene underlie congenital indifference to painin multiple human populations. Clin. Genet. 2007, 71, 311−319.(8) Gingras, J.; Smith, S.; Matson, D. J.; Johnson, D.; Nye, K.;Couture, L.; Feric, E.; Yin, R.; Moyer, B. D.; Peterson, M. L.; Rottman,J. B.; Beiler, R. J.; Malmberg, A. B.; McDonough, S. I. Global NaV1.7knockout mice recapitulate the phenotype of human congenitalindifference to pain. PLoS One 2014, 9 (9), e105895 DOI: 10.1371/journal.pone.0105895.(9) (a) Nassar, M. A.; Stirling, L. C.; Forlani, G.; Baker, M. D.;Matthews, E. A.; Dickenson, A. H.; Wood, J. N. Nociceptor-specificgene deletion reveals a major role for NaV1.7 (PN1) in acute andinflammatory pain. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12706−12711. (b) Minett, M. S.; Nassar, M. A.; Clark, A. K.; Passmore, G.;Dickenson, A. H.; Wang, F.; Malcangio, M.; Wood, J. N. DistinctNaV1.7-dependent pain sensations require different sets of sensory andsympathetic neurons. Nature Commun. 2012, 3, 1795/1−1795/9.(c) Minett, M. S.; Falk, S.; Santana-Varela, S.; Bogdanov, Y. D.; Nassar,M. A.; Heegaard, A.-M.; Wood, J. N. Pain without Nociceptors?NaV1.7-Independent Pain Mechanisms. Cell Rep. 2014, 6, 301−312.(10) (a) Thakker, D.; Burright, E. Suppression of SCN9A geneexpression and/or function for the treatment of pain. PCT. Int. Appl.WO 2009033027 A2 20090312, 2009. (b) Yeomans, D. C.; Levinson,S. R.; Peters, M. C.; Koszowski, A. G.; Tzabazis, A. Z.; Gilly, W. F.;Wilson, S. P. Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of NaV1.7 sodium channels in primary afferents.Hum. Gene Ther. 2005, 16, 271−277. (c) Fraser, R. A.; Sherrington, R.;MacDonald, M. L.; Samuels, M.; Newman, S.; Fu, J.-M.; Kamboj, R.Methods for identification of selective NaV1.7 sodium channel blockersfor treatment of pain. PCT. Int. Appl. WO 2007109324 A2 20070927,2007. (d) Hoyt, S. B.; London, C.; Gorin, D.; Wyvratt, M. J.; Fisher,M. H.; Abbadie, C.; Felix, J. P.; Garcia, M. L.; Li, X.; Lyons, K. A.;McGowan, E.; MacIntyre, D. E.; Martin, W. J.; Priest, B. T.; Ritter, A.;Smith, M. M.; Warren, V. A.; Williams, B. S.; Kaczorowskic, G. J.;Parsons, W. H. Discovery of a novel class of benzazepinone NaV1.7blockers: potential treatments for neuropathic pain. Bioorg. Med. Chem.Lett. 2007, 17, 4630−4634. (e) Hoyt, S. B.; London, C.; Ok, H.;Gonzalez, E.; Duffy, J. L.; Abbadie, C.; Dean, B.; Felix, J. P.; Garcia, M.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2312

Page 15: jm501765v (2)

L.; Jochnowitz, N.; Karanam, B. V.; Li, X.; Lyons, K. A.; McGowan, E.;MacIntyre, D. E.; Martin, W. J.; Priest, B. T.; Smith, M. M.; Tschirret-Guth, R.; Warren, V. A.; Williams, B. S.; Kaczorowskic, G. J.; Parsons,W. H. Benzazepinone NaV1.7 blockers: potential treatments forneuropathic pain. Bioorg. Med. Chem. Lett. 2007, 17, 6172−6177.(11) Devigili, G.; Eleopra, R.; Pierro, T.; Lombardi, R.; Rinaldo, S.;Lettieri, C.; Faber, C. G.; Merkies, I. S. J.; Waxman, S. G.; Lauria, G.Paroxysmal itch caused by gain-of-function NaV1.7 mutation. Pain2014, 155, 1702−1707.(12) (a) Mao, J.; Chen, L. L. Systemic lidocaine for neuropathic painrelief. Pain 2000, 87, 7−17. (b) Jensen, T. S. Anticonvulsants inneuropathic pain: rationale and clinical evidence. Eur. J. Pain. 2002, 6(Suppl A), 61−68. (c) Rozen, T. D. Antiepileptic drugs in themanagement of cluster headache and trigeminal neuralgia. Headache2001, 41 (Suppl1), S25−S32. (d) Backonja, M. M. Use ofanticonvulsants for treatment of neuropathic pain. Neurology 2002,59 (5 Suppl 2), S14−S17.(13) Bagal, S. K.; Chapman, M. L.; Marron, B. E.; Prime, R.; Storer,R. I.; Swain, N. A. Recent progress in sodium channel modulators forpain. Bioorg. Med. Chem. Lett. 2014, 24, 3690−3699.(14) (a) Bosmans, F.; Rash, L.; Zhu, S.; Diochot, S.; Lazdunski, M.;Escoubas, P.; Tytgat, J. Four novel tarantula toxins as selectivemodulators of voltage-gated sodium channel subtypes. Mol. Pharm.2005, 69, 419−429. (b) Oldrati, V.; Bianchi, E.; Stocklin, R. Spidervenom components as drug candidates. In Spider Ecophysiology;Nentwig, W., Ed.; Springer Verlag: Heidelberg, Germany, 2012; pp491−503. (c) Kuhn-Nentwig, L.; Stocklin, R.; Nentwig, W. Venomcomposition and strategies in spiders: is everything possible? Adv.Insect Physiol. 2011, 40, 1−86. (d) Kalia, J.; Milescu, M.; Salvatierra, J.;Wagner, J.; Klint, J. K.; King, G. F.; Olivera, B. M.; Bosmans, F. Fromfoe to friend: using animal toxins to investigate ion channel function. J.Mol. Biol. 2015, 427, 158−175.(15) (a) Peng, K.; Shu, Q.; Liu, Z.; Liang, S. Function and solutionstructure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from chinese bird spiderSelenocosmia huwena. J. Biol. Chem. 2002, 277, 47564−47571.(b) Xiao, Y.; Bingham, J.-P.; Zhu, W.; Moczydlowski, E.; Liang, S.;Cummins, T. R. Tarantula huwentoxin-IV inhibits neuronal sodiumchannels by binding to receptor site 4 and trapping the domain IIvoltage sensor in the closed configuration. J. Biol. Chem. 2008, 283,27300−27313.(16) (a) Middleton, R. E.; Warren, V. A.; Kraus, R. L.; Hwang, J. C.;Liu, C. J.; Dai, G.; Brochu, R. M.; Kohler, M. G.; Gao, Y.-D.; Garsky,V. M.; Bogusky, M. J.; Mehl, J. T.; Cohen, C. J.; Smith, M. M. Twotarantula peptides inhibit activation of multiple sodium channels.Biochemistry 2002, 41, 14734−14747. (b) Priest, B. T.; Blumenthal, K.M.; Smith, J. J.; Warren, V. A.; Smith, M. M. ProTx-I and ProTxII:gating modifiers of voltage-gated sodium channels. Toxicon 2007, 49,194−201. (c) 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. ProTxII, a selective inhibitor of NaV1.7sodium channels, blocks action potential propagation in nociceptors.Mol. Pharmacol. 2008, 74, 1476−1484. (d) Edgerton, G. B.;Blumenthal, K. M.; Hanck, D. A. Inhibition of the activation pathwayof the T-type calcium channel CaV3.1 by ProTxII. Toxicon 2010, 56,624−636. (e) Smith, J. J.; Cummins, T. R.; Alphy, S.; Blumenthal, K.M. Molecular interactions of the gating modifier toxin ProTxII withNaV1.5: implied existence of a novel toxin binding site coupled toactivation. J. Biol. Chem. 2007, 282, 12687−12697. (f) Park, J. H.;Carlin, K. P.; Wu, G.; Ilyin, V. I.; Kyle, D. J. Cysteine racemizationduring the Fmoc solid phase peptide synthesis of the NaV1.7-selectivepeptide−protoxin II. J. Pept. Sci. 2012, 18, 442−448.(17) (a) Norton, R. S.; Pallaghy, P. K. The cystine knot structure ofion channel toxins and related polypeptides. Toxicon 1998, 36, 1573−1583. (b) Pallaghy, P. K.; Nielsen, K. J.; Craik, D. J.; Norton, R. Acommon structural motif incorporating a cystine knot and a triple-stranded β-sheet in toxic and inhibitory polypeptides. Protein Sci. 1994,3, 1833−1836.

(18) (a) Bulaj, G.; West, P. J.; Garrett, J. E.; Watkins, M.; Zhang, M.-M.; Norton, R. S.; Smith, B. J.; Yoshikami, D.; Olivera, B. M. Novelconotoxins from Conus striatus and Conus kinoshitai selectively blockTTX-resistant sodium channels. Biochemistry 2005, 44, 7259−7265.(b) Zhang, M.-M.; Green, B. R.; Catlin, P.; Fiedler, B.; Azam, L.;Chadwick, A.; Terlau, H.; McArthur, J. R.; French, R. J.; Gulyas, J.;Rivier, J. E.; Smith, B. J.; Norton, R. S.; Olivera, B. M.; Yoshikami, D.;Bulaj, G. Structure/function characterization of μ-conotoxin KIIIA, ananalgesic, nearly irreversible blocker of mammalian neuronal sodiumchannels. J. Biol. Chem. 2007, 282, 30699−30706. (c) Khoo, K. K.;Gupta, K.; Green, B. R.; Zhang, M.-M.; Watkins, M.; Olivera, B. M.;Balaram, P.; Yoshikami, D.; Bulaj, G.; Norton, R. S. Distinct disulfideisomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodiumchannels. Biochemistry 2012, 51, 9826−9835.(19) Yang, S.; Xiao, Y.; Kang, D.; Liu, J.; Li, Y.; Undheim, E. A. B.;Klint, J. K.; Rong, M.; Lai, R.; King, G. F. Discovery of a selectiveNaV1.7 inhibitor from centipede venom with analgesic efficacyexceeding morphine in rodent pain models. Proc. Natl. Acad. Sci. U.S. A. 2013, 110, 17534−17539.(20) Cherki, R. S.; Kolb, E.; Langut, Y.; Tsveyer, L.; Bajayo, N.; Meir,A. Two tarantula venom peptides as potent and differential NaVchannel blockers. Toxicon 2013, 77, 58−67.(21) Murray, J. K.; Miranda, L. P.; McDonough, S. I. Potent andselective polypeptide inhibitors of NaV1.3 and NaV1.7 sodiumchannels. PCT Int. Appl. WO 2012125973 A2 20120920, 2012.(22) Ono, S.; Kimura, T.; Kubo, T. Characterization of voltage-dependent calcium channel blocking peptides from the venom of thetarantula Grammostola rosea. Toxicon 2011, 58, 265−276.(23) Meir, A.; Cherki, R. S.; Kolb, E.; Langut, Y.; Bajayo, N. Ionchannel-blocking peptides from spider venom and their use asanalgesics. U.S. Pat. Appl. Publ. US 20110065647 A1 20110317, 2011.(24) Klint, J. K.; Senff, S.; Rupasinghe, D. B.; Er, S. Y.; Herzif, V.;Nicholson, G. M.; King, G. F. Spider-venom peptides that targetvoltage-gated sodium channels: pharmacological tools and potentialtherapeutic leads. Toxicon 2012, 60, 478−491.(25) (a) Steiner, A. M.; Bulaj, G. Optimization of oxidative foldingmethods for cysteine-rich peptides: a study of conotoxins containingthree disulfide bridges. J. Pept. Sci. 2011, 17, 1−7. (b) Gongora-Benítez, M.; Tulla-Puche, J.; Paradis-Bas, M.; Werbitzky, O.; Giraud,M.; Albericio, F. Optimized Fmoc solid-phase synthesis of thecysteine-rich peptide linaclotide. Biopolymers Pept. Sci. 2011, 96, 69−80.(26) Repeating the experiment at a holding voltage that produced20% inactivation yielded similar results (IC50 = 0.0036 μM).(27) Poppe, L.; Hui, J. O.; Ligutti, J.; Murray, J. K.; Schnier, P. D.PADLOC: A powerful tool to assign disulfide bond connectivities inpeptides and proteins by NMR spectroscopy. Anal. Chem. 2012, 84,262−266.(28) The Asp14Ala mutant had a complex LC-MS profile of multiplepeaks with the same MW after folding of the purified linear peptideand did not yield a suitable sample for assay after purification.(29) (a) Revell, J. D.; Lund, P.-E.; Linley, J. E.; Metcalfe, J.;Burmeister, N.; Sridharan, S.; Jones, C.; Jermutus, L.; Bednarek, M. A.Potency optimization of Huwentoxin-IV on hNaV1.7: a neurotoxinTTX-S sodium-channel antagonist from the venom of the Chinesebird-eating spider Selenocosmia huwena. Peptides 2013, 44, 40−46.(b) Minassian, N. A.; Gibbs, A.; Shih, A. Y.; Liu, Y.; Neff, R. A.; Sutton,S. W.; Mirzadegan, T.; Connor, J.; Fellows, R.; Husovsky, M.; Nelson,S.; Hunter, M. J.; Flinspach, M.; Wickenden, A. D. Analysis of thestructural and molecular basis of voltage-sensitive sodium channelinhibition by the spider toxin huwentoxin-IV (μ-TRTX-Hh2a). J. Biol.Chem. 2013, 288, 22707−22720. (c) Park, J. H.; Carlin, K. P.; Wu, G.;Ilyin, V. I.; Musza, L. L.; Blake, P. R.; Kyle, D. J. Studies examining thereationship between the chemical structure of Protoxin II and itsactivity on voltage gated sodium channels. J. Med. Chem. 2014, 57,6623−6631.(30) (a) Edman, P. Method for determination of the amino acidsequence in peptides. Acta Chem. Scand. 1950, 4, 283−293. (b) Niall,

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2313

Page 16: jm501765v (2)

H. D. Automated Edman degradation: the protein sequenator.Methods Enzymol. 1973, 27, 942−1010.(31) (a) Dancík, V.; Addona, T. A.; Clauser, K. R.; Vath, J. E.;Pevzner, P. A. De novo peptide sequencing via tandem massspectrometry. J. Comp. Biol. 1999, 6, 327−342. (b) Favreau, P.;Menin, L.; Michalet, S.; Perret, F.; Cheneval, O.; Stocklin, M.; Bulet,A.; Stocklin, R. Mass spectrometry strategies for venom mapping andpeptide sequencing from crude venoms: case applications with singlearthropod specimen. Toxicon 2006, 47, 676−687. (c) Favreau, P.;Cheneval, O.; Menin, L.; Michalet, S.; Gaertner, H.; Principaud, F.;Thai, R.; Menez, A.; Bulet, P.; Stocklin, R. The venom of the snakegenus Atheris contains a new class of peptides with clusters of histidineand glycine residues. Rapid Commun. Mass Spectrom. 2007, 21, 406−412. (d) Violette, A.; Biass, D.; Dutertre, S.; Koua, D.; Piquemal, D.;Pierrat, F.; Stocklin, R.; Favreau, P. Large-scale discovery ofconopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomicapproach. J. Proteomics 2012, 75, 5215−5225.(32) Bregman, H.; Berry, L.; Buchanan, J. L.; Chen, A.; Du, B.; Feric,E.; Hierl, M.; Huang, L.; Immke, D.; Janosky, B.; Johnson, D.; Li, X.;Ligutti, J.; Liu, D.; Malmberg, A.; Matson, D.; McDermott, J.; Miu, P.;Nguyen, H. N.; Patel, V. F.; Waldon, D.; Wilenkin, B.; Zheng, X. M.;Zou, A.; McDonough, S. I.; DiMauro, E. F. Identification of a potent,state-dependent inhibitor of NaV1.7 with oral efficacy in the formalinmodel of persistent pain. J. Med. Chem. 2011, 54, 4427−4445.(33) Wuthrich, K. NMR of Proteins and Nucleic Acids; John Wiley &Sons: Mississauga, Ontario, 1986.(34) Cotts, R. M.; Hoch, M. J. R.; Sun, T.; Markert, J. T. Pulsed fieldgradient stimulated echo methods for improved NMR diffusionmeasurements in heterogeneous systems. J. Magn. Reson. 1989, 83,252−266.(35) Molecular Operating Environment (MOE), 2013.08; ChemicalComputing Group Inc.: 1010 Sherbooke Street West, Suite #910,Montreal, QC, Canada, H3A 2R7, 2014.(36) Wildman, S. A.; Crippen, G. M. Prediction of physiochemicalparameters by atomic contributions. J. Chem. Inf. Comput. Sci. 1999,39, 868−873.(37) The PyMOL Molecular Graphics System, version 1.7.2;Schrodinger, LLC: New York, 2014.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm501765vJ. Med. Chem. 2015, 58, 2299−2314

2314