Engineering Potent and Selective Analogues of GpTx-1, a Tarantula

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Engineering Potent and Selective Analogs of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel Justin Keith Murray, Joseph Ligutti, Dong Liu, Anruo Zou, Leszek Poppe, Hongyan Li, Kristin L. Andrews, Bryan D Moyer, Stefan I McDonough, Philippe Favreau, Reto Stöcklin, and Les P Miranda J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 7, 2015

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Journal of Medicinal Chemistry

Engineering Potent and Selective Analogs of GpTx1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel Justin K. Murray,1 Joseph Ligutti,2 Dong Liu,2 Anruo Zou,2 Leszek Poppe,1 Hongyan Li,3 Kristin L. Andrews,4 Bryan D. Moyer,2 Stefan I. McDonough,5 Philippe Favreau,6 Reto Stöcklin,6 and Les P. Miranda1,* Departments of 1Therapeutic Discovery, 2Neuroscience, and 3Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA, 4

Therapeutic Discovery and 5Neuroscience, Amgen Inc., 360 Binney Street, Cambridge, MA

02142, USA, and 6Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland Voltage-gated sodium channel, NaV1.7, peptide antagonist, peptide toxin, venom

ACS Paragon Plus Environment

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Abstract NaV1.7 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 identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20x and 1000x over NaV1.4 and NaV1.5, respectively).

NMR structural analysis of the

chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp29, Lys31, and Phe34 near the Cterminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred three hundred-fold selectivity 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 >1000x selectivity against NaV1.4 and NaV1.5.


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Introduction Voltage-gated sodium channels (VGSCs or NaVs) 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 (I-IV), each domain containing six transmembrane helices (S1-S6).

The S5-S6 domains govern the main aspects of ion

permeation, and domains including most prominently fixed 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 (NaV1.1-NaV1.9). These subtypes show tissue specific localization and functional differences with NaV1.1, NaV1.2, and NaV1.3 found principally in the central nervous system, NaV1.6 located both centrally and peripherally, and NaV1.7, NaV1.8, and NaV1.9 expressed primarily in the peripheral nervous system.3 NaV1.4 is present in skeletal muscle, and NaV1.5 is found predominantly in cardiac muscle.4 Three VGSCs (NaV1.5, NaV 1.8, and NaV 1.9) are resistant to blockade by the sodium channel blocker tetrodotoxin (TTX),5 demonstrating subtype specificity within this 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 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, inflammatory, and chemical pain,8 and cell-specific deletion reduces responsiveness to several forms of pain.9 Based on such evidence, decreasing NaV1.7 channel activity in peripheral sensory neurons has been proposed as an effective pain treatment.10


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A role for NaV1.7 in itch also is suggested by clinical genetics.11 Broad NaV antagonists, such as TTX, lidocaine, bupivacaine, phenytoin, lamotrigine, and carbamazepine, have been shown to be useful for attenuating pain in humans and animal models but have a variety of side effects due to a lack of isoform specificity.12 A primary challenge in the development of a NaV1.7 antagonist as a therapeutic is attaining sufficient selectivity against NaV1.5, expressed in cardiac tissue, and NaV1.4, in skeletal muscle, so as not to impair normal cardiac and skeletal muscle function.13 Spider venoms contain many peptide toxins that target voltage-gated ion channels, including KV, CaV, and NaV channels and have been useful tools to study channel structure and function.14 Two well-characterized examples of NaV1.7 inhibitory peptides that display different NaV selectivity profiles and promiscuities toward other voltage-gated ion channel families are Huwentoxin-IV (HWTX-IV) from the venom of the 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 to the inhibitory cystine knot (ICK) peptide structural motif17 and inhibit channel activation by binding to the voltage sensor and locking 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 toxin peptide µ-SLPTX-Ssm6a,19 have been prepared and characterized 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 sodium channels,20 from the venom of the tarantula spider Grammostola porteri.21 GpTx-1 was first reported as a CaV channel blocker after isolation from the venom of the closely related Chilean tarantula Grammostola rosea and named GTx1-15 (UniproKB: Accession number, P0DJA9).22 It was later identified in the venom of Paraphysa scrofa (Phrixotrichus auratus).23 Based on its potency and desirable NaV subtype selectivity profile, we selected


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GpTx-1 as a lead in our effort to develop therapeutically useful NaV1.7 peptides. We describe a significant peptide medicinal chemistry effort to investigate the GpTx-1 structure-activity relationships and engineer analogs with improved levels of NaV1.7 potency and selectivity against the important off-target NaV isoforms NaV1.4 and NaV1.5. Results and Discussion High-Throughput Screening of Venom Fractions. To identify a novel peptide inhibitor with NaV potency, 84 venom fractions from the tarantula Grammostola porteri (Atheris Laboratories, Switzerland, Melusine Ref. MLU-020007) were screened for activity against NaV1.7 (Figure 1). A 384-well IonWorks Quattro (IWQ) platform, which evaluates receptor inhibition with a population patch clamp, was utilized for its high-throughput screening capability.

Several

venom fractions with significant (>80% inhibition of peak current) NaV1.7 inhibitory activity were identified, the first of which was fraction 31. A second aliquot of this fraction was tested in the NaV1.7 and NaV1.5 IWQ assays to confirm the activity of the hit and evaluate selectivity. All samples were tested for potency on sodium channels with electrophysiology to give a direct measure of receptor inhibition. The validated hit fraction was then analyzed by high-resolution electro-spray ionization (ESI-Q-TOF) and matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) mass spectrometry (MS), which indicated that the fraction was a mixture of at least four distinct peptide species (Figures 2 and 3, respectively). The active fraction was then separated by reversed phase (RP) HPLC, and the corresponding sub-fractions were screened for activity in the NaV1.7 and NaV1.5 IWQ assays. Sub-fraction 11 was the major peak in the RP-HPLC chromatogram and showed >90% inhibition of NaV1.7 activity (Figure 4). Deconvolution of sub-fraction 11 by Edman degradation and MS/MS sequencing revealed the primary peptide sequence of GpTx-1 (1, Figure 5). GpTx-1 is a 34 residue, C-terminally


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amidated polypeptide containing six cysteine residues engaged in 3 disulfide bonds and is a putative member of NaSpTx Family 1.24 To confirm its identity and activity, synthetic GpTx-1 was chemically synthesized using Fmoc solid-phase peptide synthesis (SPPS) to generate the linear peptide sequence, which was then oxidatively folded, purified by RP-HPLC to produce the final product (Figure 6), and tested.25 A co-elution of the synthetic and native products (1:1) was observed, confirming the authenticity of the synthetic versus native product (see Supporting Information). Results of Electrophysiology Studies. Chemically synthesized GpTx-1 (1) was characterized in a manual electrophysiology whole-cell patch clamp assay using human clones of several NaV subtypes (Figure 7). To test for inhibition or stabilization of as many channel gating states as possible, dose response curves were measured with voltage clamped to holding potentials that imposed steady 20% fractional inactivation.

The IC50 values of NaV1.8, NaV1.7, NaV1.5,

NaV1.4, and NaV1.3 inhibition for GpTx-1 were 12.2±2.2 µM, 0.0044±0.0020 µM, 4.20±0.09 µM, 0.301±0.041 µM, and 0.0203±0.0069 µM, respectively, confirming that GpTx-1 is a potent peptide inhibitor of NaV1.7 with moderate selectivity against NaV1.4 and excellent selectivity against the TTX-resistant (TTX-R) channels NaV1.5 and NaV1.8.

Manual patch clamp

electrophysiology was also performed with GpTx-1 on sensory neurons isolated from mouse dorsal root ganglia (DRG) to evaluate physiologic relevance. The TTX-S current in these neurons includes a component attributable to NaV1.7.8 The IC50 for inhibition of this current in DRG by GpTx-1 was 0.0063 µM.26 Taken together, these electrophysiology results confirm GpTx-1 as a potent NaV1.7 inhibitory peptide with a promising NaV subtype selectivity profile, making it a suitable starting point for further structure-activity relationship (SAR) investigation.


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NMR Structural Analysis of GpTx-1. To investigate the disulfide architecture of the folded peptide, the NMR solution structure of synthetic GpTx-1 was examined. The primarily β-type structure of the peptide (backbone RMSD of 0.1 Å) is stabilized by three disulfide bonds and 11 hydrogen bonds between backbone residues (see Figure 8 for the ensemble of the 10 lowest energy conformations). The secondary structural motifs are a Type II β-turn between Gly4 and Arg7, followed by a β-strand between Arg7 and Ile10, then a Type I β-turn between Ile10 and Asn13, with an α-turn between Cys17 and Leu21, and finally a β-hairpin between Val22 and Lys31. The NMR analysis was consistent with the assumed disulfide connectivity of the six cysteine residues as Cys2—Cys17, Cys9—Cys23, and Cys16—Cys30 or a C1—C4, C2—C5, C3—C6 pattern,27 revealing that GpTx-1 contains an inhibitory cystine knot (ICK) motif. Comparison to Other Nav1.7 Inhibitory Peptides. The peptide sequence, NaV1.7 potency, and NaV subtype selectivity of synthetic GpTx-1 (1) was compared to previously reported NaV1.7 inhibitory peptides. Two voltage gating modifier peptides from spider venom, HWTXIV (2) and ProTxII (3) and the pore-blocking µ-conotoxin KIIIA18 (4) were chemically synthesized 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 clamp automated 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 the other NaV isoforms. HWTX-IV had moderate potency against NaV1.7 (IC50 = 0.033 µM) with good selectivity against NaV1.5 (IC50 = 25 µM) and NaV1.4 (IC50 = 4 µM).

GpTx-1 shares

considerable sequence homology with HWTX-IV but was slightly more potent against NaV1.7. Importantly, GpTx-1 has excellent inherent selectivity against NaV1.5 (~1000-fold) with moderate (20-fold) selectivity toward NaV1.4. KIIIA was most potent at inhibiting NaV1.4 (IC50


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= 0.02 µM) and had much weaker activity against NaV1.7 (IC50 = 0.46 µM). Overall, our results were in good agreement with those reported in the literature.15,16,18

We also tested the

commercially available synthetic centipede toxin peptide µ-SLPTX-Ssm6a (5, Peptides International, KY, USA) and found it to be inactive against NaV1.7 at concentrations up to 1 µM, in contrast to the report for the isolated natural peptide.19 We also chemically synthesized the reported sequence, and it was inactive up to 1 µM. Given its native potency and selectivity profile, GpTx-1 was determined to be a strong starting point for the development of NaV1.7 inhibitory peptides with selectivity against NaV1.5 and NaV1.4. The results obtained previously by the manual patch clamp method were in good agreement with those from the PX format, and further analogs were tested on the latter platform due to its higher throughput.


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Table 1. Sequence and Activity of Synthetic NaV1.7 Inhibitory Peptides hNav1.7 PX IC50 (µM)

hNav1.5 PX IC50 (µM)


GpTx-1

0.010

>10

0.20

2

HWTX-IV

0.033

25

3.9

3

ProTxII

0.003

0.09

0.03

4

KIIIA

0.46

>25

0.02

5

µ-SLPTXSsm6a

>1.0

N.D.

N.D.

Compd

Peptide

1

Sequence

* - Denotes C-terminal amide. Positional Alanine Scan of GpTx-1. To evaluate the structure-activity relationships of GpTx1, a series of alanine substitution analogs (6-34, Table 2), was prepared at each amino acid position within the sequence, excluding the cysteines.28 These “alanine scan” mutants were tested for activity against NaV1.7, NaV1.5, and NaV1.4 using the IWQ assays.

This Ala

analoging of GpTx-1 identified three residues near 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 of amino acids at the C-terminus, His27 (27) and Tyr32 (31), were also important for activity. Substitution of alanine into a separate 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 within the sequence from Ile10-Lys15 (14-


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18) or Arg18-Pro19 (19-20) had no significant effects on NaV1.7 inhibition. All of the compounds 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 NaV1.4 selectivity of the peptide to 70-fold, compared to 30-fold for parent. The high-throughput IWQ platform was useful for rapidly testing the large set of Ala scan analogs, but the population patch clamp method resulted in an approximately 10-fold lower NaV1.7 potency (with a concomitant drop in selectivity) compared to the manual patch clamp platforms. Compound 10 was tested in the PX assay format, revealing that it 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. This more selective GpTx-1 analog was further analyzed by manual electrophysiology, and the IC50 value of NaV1.7 inhibition was 0.013 µM (Figure 10), confirming that [Ala5]GpTx-1 maintains potent inhibitory activity against NaV1.7. Likewise [Ala5]GpTx-1 was a potent and reversible inhibitor of TTX-S current in mouse DRG neurons with an IC50 value of 0.023 µM (Figure 11). [Ala5]GpTx-1 was 99% intact after 24 hour 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.


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Table 2. NaV Inhibitory Activity of GpTx-1 Analogs from Positional Scanning with Alanine*

Compound

Substitution

hNav1.7 IWQ IC50 (µM) 0.09±0.01

hNav1.5 IWQ IC50 (µM) >5

hNav1.4 IWQ IC50 (µM) 2.7±1.2

1

Wild Type

6

N-Term. Ala-

0.37±0.02

>5

>4.8

7

Asp1Ala

0.10±0.01

>5

1.8±0.1

8

Leu3Ala

0.43±0.13

>5

>3.0

9

Gly4Ala

0.27±0.13

>5

3.2±0.4

10

Phe5Ala

0.63±0.12

27±10

45±3

11

Met6Ala

0.43±0.05

>5

3.8±0.5

12

Arg7Ala

0.94±0.09

>5

>4.6

13

Lys8Ala

0.50±0.01

>5

>3.9

14

Ile10Ala

0.21±0

>5

3.0±0.1

15

Pro11Ala

0.17±0.07

>5

3.0±0.5

16

Asp12Ala

0.12±0.05

>5

0.9±0.1

17

Asn13Ala

0.19±0.04

>5

2.7±0.2

18

Lys15Ala

0.23±0.05

>5

4.7±0.3

19

Arg18Ala

0.13±0.02

>20

3.0±0.6

20

Pro19Ala

0.09±0.01

>20

1.5±0.3

21

Asn20Ala

0.69±0

>5

>5

22

Leu21Ala

0.19±0.02

>20

3.0±0.7

23

Val22Ala

0.38±0.14

>5

3.7±0.3

24

Ser24Ala

0.47±0.15

>4.7

1.9±0.6

25

Arg25Ala

0.33±0.16

>5

>5

26

Thr26Ala

0.26±0.05

>5

3.2±0.1

27

His27Ala

0.97±0.18

>5

4.1±0.4

28

Lys28Ala

0.47±0.05

>5

>4.9

29

Trp29Ala

>5

>5

>4.5

30

Lys31Ala

>5

>5

>5

31

Tyr32Ala

0.80±0.24

>5

>5

32

Val33Ala

0.17±0.02

>5

4.5±0.5

33

Phe34Ala

1.20±0.14

>5

>5

34

C-Term. -Ala

0.41±0.03

>5

>5

* - All analogs were C-terminal peptide amides. Samples tested on IWQ platform (Avg. ± SD, n ≥ 2). Structure-Activity Relationship of GpTx-1. The linear peptide sequence of GpTx-1 contains two stretches of hydrophobic amino acids, one near the N-terminus (Phe5-Met6) and one near the C-terminus (Trp29-Phe34). Though relatively distant in the primary sequence, based on NMR structural analysis, these hydrophobic residues all come into close spatial proximity in the folded


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peptide. Held together by the three disulfide bonds, the overall conformation is further stabilized through the formation of a β-sheet by residues Val22 through Lys31. The C-terminal portion (His27-Phe34) is composed primarily of hydrophobic amino acids, while Phe5 and Met6 are located adjacent to that β-strand and form the remainder of a hydrophobic face. These same residues that are clustered on one face of GpTx-1, namely Phe5, Met6, His27, Trp29, Lys31, Tyr32, and Phe34, were also indicated as being important for functional activity through alanine substitution. Because changes that alter the nature of this face reduce potency against NaV1.7, this hydrophobic region may be the portion of the molecule that interacts with the VGSCs at the binding interface. (See Figures 13A and B.) Phe5 is situated at the periphery of the putative binding face of GpTx-1, and may interact with a corresponding region on the channels that has some variation between the different NaV isoforms, as replacement with alanine has only a small impact on potency against NaV1.7 but greatly reduces activity against NaV1.4. The increased selectivity against NaV1.4 combined with the inherent selectivity against NaV1.5 make [Ala5]GpTx-1 (10) an important tool for the elucidation of NaV biology and a starting point for the potential development of more selective GpTx-1 peptide analogs. The NMR solution structure shows that GpTx-1 is amphipathic in nature with a hydrophobic face on one side of the molecule and a hydrophilic (mostly cationic) face on the opposite side (Figures 13C and D). The hydrophilic face of GpTx-1 is comprised of residues Ile10-Lys15 and Arg18-Pro19 whose substitution has a negligible effect upon functional activity and may be exposed to solvent during the binding interaction. This aspect could be exploited through peptide engineering to tune the physical properties of the molecule and will be reported in due course.


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Position 5 Analogs of GpTx-1. The increase in selectivity against NaV1.4 and retention of NaV1.7 potency achieved with [Ala5]GpTx-1 (10) encouraged additional investigation of amino acid residue 5 within the GpTx-1 sequence. A set of peptide analogs (35-48) was prepared varying the size and shape of aliphatic and aromatic residues at this position and tested against a small panel of NaV channels in the PX format (Table 3). In general, it was observed that smaller aliphatic residues resulted in increases in selectivity against NaV1.4, while larger amino acids, especially aromatic residues, caused a decrease in NaV1.7 specificity. Substitution of glycine (35), methionine (39), or isoleucine (40) for the native phenylalanine resulted in analogs that had equivalent or superior 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) at position 5 in GpTx-1 reduced potency against NaV1.7. The SAR indicates that the residue in this position of GpTx-1 may interact with a corresponding site within the target that has some variability among the different NaV isoforms.


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Table 3. NaV Inhibitory Activity of Position 5 Analogs of GpTx-1*

Compound

Phe5 Substitution

10 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Ala Gly Abu Nva Val Met Ile Leu NMe-Leu Tle Cha Cpg Chg 4-I-Phe Bip

hNav1.7 PX IC50 (µM) 0.027 0.009 0.079 0.039 0.019 0.002 0.009 0.005 0.025 0.024 0.004 0.005 0.006 0.236 0.086

hNav1.5 PX IC50 (µM) >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 3.5 >10 >10 >10 2.1

hNav1.4 PX IC50 (µM) 8.5 11 >10 4.6 4.1 0.9 2.0 0.9 7.7 4.7 0.1 1.7 1.5 1.1 0.1

* - Abu, L-2-aminobutyric acid; Bip, L-4-biphenylalanine; Cha, L-cyclohexylalanine; Chg, Lcyclohexylglycine; Cpg, L-cyclopentylglycine; NMe-Leu, L-N-methylleucine; Nva, L-norvaline; 4-I-Phe, L-4-iodo-phenylalanine; Tle, L-tert-butylglycine Position 6 Analogs of GpTx-1. In a parallel optimization effort, we sought to replace the native methionine in position 6 of GpTx-1 with a non-oxidizable residue. The tendency of Met6 to oxidize to the corresponding methionine sulfoxide during peptide cleavage and folding was observed by LC-MS, which reduced yield and raised concerns over stability. Although the oxidized side product could be removed by purification, an attempt was made to remove this liability altogether through the preparation and screening of a small series of GpTx-1 position 6 analogs (49-55, Table 4). Incorporation of norleucine, 49, the most straightforward structural replacement for methionine, and phenylalanine, 52, resulted in a slight loss in selectivity against NaV1.4 and NaV1.5 relative to GpTx-1. Incorporation of cyclohexylalanine (Cha, 51) retained a NaV selectivity profile similar to GpTx-1, except with 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-1 combination analogs (56-60, Table 4). The incorporation of alanine


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at position 5 together with a non-oxidizable, hydrophobic residue at position 6 such as norleucine (56) or leucine (57) produced analogs with NaV potency and selectivity similar to [Ala5]GpTx-1 but without the potential oxidative liability. Table 4. NaV Inhibitory Activity of Position 6 Analogs of GpTx-1*

Substitution Met6




Nle Leu Cha Phe Tyr Trp 1-Nal Nle Leu Phe Trp 1-Nal

0.010 0.027 0.008 0.024 0.004 0.019 0.063 0.023 0.004 0.010 0.028 0.013 0.059 0.003

>10 >10 1.1 >10 2.8 3.0 >10 3.4 0.4 >10 >10 >10 >10 3.6

0.20 8.5 0.07 0.41 0.09 0.14 3.4 0.24 0.11 2.6 9.9 0.74 6.1 0.50

Compound Phe5 1 10 49 50 51 52 53 54 55 56 57 58 59 60

Ala

Ala Ala Ala Ala Ala

* - Cha, L-cyclohexylalanine; 1-Nal, L-1-naphthylalanine; Nle, L-norleucine Positions 26 and 28 and Combination Analogs of GpTx-1. Additional positions around the periphery of the putative binding face on GpTx-1 were explored for potential increases in NaV potency and/or selectivity with substitution analogs 61-66 (Table 5). While several residues had been directly identified during the alanine scan as being critical to the interaction of the peptide with the NaV channels and then found to be clustered on one face of the GpTx-1 structure, it was position 5, located at the edge of that hydrophobic face, which had been found to exert the greatest impact on relative subtype activity. Guided by the NMR structure, a number of other GpTx-1 residues at the other edges of the “binding face” were selected for substitution with a variety of residues in search of additional potential interactions (Figure 14A). Replacement of


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Thr26 in GpTx-1 with arginine (62) and histidine (64) had little effect, but [Leu26]GpTx-1 (61) afforded a slight increase in NaV1.7 potency and NaV1.4 selectivity relative to GpTx-1. The native lysine residue of GpTx-1 at position 28 was independently substituted with arginine (65) and gave a small boost in selectivity and potency compared to 1. Incorporation of glutamic acid at either position 26 (63) or 28 (66) produced NaV activity profiles similar to [Ala5]GpTx-1. The SAR study revealed a number of GpTx-1 analogs (10, 35, 39, 40, 56, 57, 63, and 66) with reasonable potency against NaV1.7 (IC50 values 0.01-0.03 µM), excellent selectivity against NaV1.5 (>500-fold), and improved selectivity against NaV1.4 (~200-fold). Combining the SAR of GpTx-1 at positions 5, 6, 26, and 28 in a final set of analogs (67-71, Table 5) produced additive improvements in potency and selectivity. [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 against Nav1.4, and >6000-fold selectivity against Nav1.5. In spite of the large number of substitutions, compound 71 behaved similar to wild type GpTx-1 during folding and has been amenable to scale-up for future studies, which will be reported in due course. GpTx-1 analog 71 is to our knowledge16,29 the only confirmed peptide sequence with single digit nanomolar NaV1.7 inhibitory activity with >1000-fold selectivity against the two important VGSC subtypes NaV1.4 and NaV1.5. These advances in NaV1.7 inhibitory peptide SAR will facilitate future interrogation of NaV biology.


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Table 5. NaV Inhibitory Activity of Position 5 Analogs of GpTx-1

Substitution Compound Phe5 1 10 61 62 63 64 65 66 67 68 69 70 71

Met6

Thr26

Lys28

Ala Leu Arg Glu His Arg Glu Ala Ala Ala Ala Ala

Phe Phe Leu Phe

Leu Leu

Leu

Arg Arg Arg




0.010 0.027 0.004 0.005 0.034 0.004 0.008 0.029 0.054 0.004 0.011 0.008 0.0016

>10 >10 4.6 0.5 >10 4.5 8.8 >10 >10 >10 >10 >10 >10

0.20 8.5 0.53 0.11 4.9 0.24 0.80 14 >10 1.2 3.3 6.6 1.9

Conclusion The voltage gated ion channel NaV1.7 remains an important and challenging target for the discovery and development of pain therapeutics. We identified GpTx-1 as a peptide antagonist of NaV1.7 via the high-throughput screening of fractionated venom from the tarantula Grammostola porteri. Our manual electrophysiological characterization of the native peptide toxin revealed its potent inhibition of expressed human NaV1.7 and inherent selectivity against NaV1.5. We then optimized GpTx-1 selectivity against NaV1.4, which governs excitability of skeletal muscle, through an extensive SAR campaign.

An NMR structure confirmed the

disulfide architecture and aided our interpretation of the screening data from an initial set of alanine scanning analogs. We identifed a putative binding face for the GpTx-1 peptide to the NaV1.7 channel, but more importantly found that substitution of alanine for Phe5 (10) increased selectivity against NaV1.4 without compromising NaV1.7 activity. The location of this amino acid at the periphery of the binding face led us to explore other similar positions in the peptide


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structure.

Page 18 of 56

After replacement of the native Met6 to avoid oxidation and combination with

substitutions at positions 26 and 28, we have identified a GpTx-1 analog (71) that is nearly 10fold more potent than wild-type with >1000-fold selectivity against NaV1.5 and NaV1.4, two prominent VGSC subtypes with the possible liabilities of side effects on the heart and skeletal muscle. The small but significant and additive gains in selectivity through appropriate amino acid selection at the peripheral binding residues along with the tuning of the hydrophobic nature of the residue at position 6 demonstrate the power of structure-guided design together with systematic analoging to improve upon a natural scaffold. GpTx-1 and related analogs will be useful tools with which to probe sodium channel biology and could potentially serve as the basis for the development of a peptide therapeutic. Experimental Section Materials. Nα-Fmoc protected amino acids were purchased from Novabiochem (San Diego, CA), Bachem (Torrance, CA), or GL Biochem (Shanghai, China). Rink Amide MBHA resin was purchased from Peptides International (Louisville, KY). SP Sepharose High Performance resin was purchased from GE Healthcare Life Sciences.

The following compounds were

purchased: N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), acetic acid, piperidine, 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane, oxidized glutathione, and reduced glutathione (Sigma-Aldrich, Milwaukee, WI); dichloromethane (DCM, Mallinckrodt Baker,

Inc.);

N,N-dimethylforamide

(DMF,

Fisher

Scientific);

1-Cyano-2-ethoxy-2-

oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU®, Matrix Innovation, Montreal, Canada); HPLC-quality water and acetonitrile (Burdick and Jackson); 1.0 M Tris-HCl pH 7.5 (Teknova). Stable cell lines expressing human (h) voltage-


18

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gated sodium (NaV) channels (CHO-hNaV1.3, HEK293-hNaV1.4, HEK293-hNaV1.5, HEK293hNaV1.7, 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 electrical stimulation of an anesthetized spider. Venom samples were collected, lyophilized, and dissolved in 0.1% trifluoroacetic acid (TFA) in water to approximately 1 mg venom/mL. The crude venom solutions were desalted by solid-phase extraction (SPE) with Sep-Pak C18 cartridges (Waters, Milford, MA, USA) equilibrated in 0.1% TFA, and eluted with 80% aqueous acetonitrile and then freeze-dried and stored at -30oC. The crude venom was fractionated by reversed phase (RP) HPLC, collecting 84 samples in time slices. The venom extract was dissolved in 0.1% TFA to approximately 1 mg venom/mL, and then separated by C18 RP HPLC chromatography and collected into approximately 1 minute wide fractions.

HPLC method: Solvent A (0.1% TFA in water) and solvent B (90%

acetonitrile/10% water containing 0.1% TFA) at 1 mL/min with a 1% /min gradient 0-100% solvent B. The fractions were transferred into a 384-well plate format, dried in vacuo, and stored at -30oC. N-terminal

sequencing

of

peptides

was

performed

by

Edman

degradation.30

Phenylthiohydantoin (PTH)-amino acid derivatives were analyzed with an Applied Biosystems automatic 473A sequencer. De novo peptide sequencing was accomplished by tandem mass spectrometry.31 Peptide Synthesis. GpTx-1 peptides were assembled using Nα-Fmoc solid-phase peptide synthesis (SPPS) methodologies with appropriate orthogonal protection and resin linker strategies. The following side chain protection strategies were employed for standard amino acid


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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 0.012 mmol scale using Rink Amide MBHA resin (100-200 mesh, 1% DVB, RFR-1063-PI, 0.52 meq/g initial loading, 408291, Peptides International, Louisville, KY). Dry resin (17 mg per well) was added to a Phenomenex deep well protein precipitation plate (CEO-7565, 38710-1) using a resin loader (Radley). Amino acids were added to the growing peptide chain by stepwise addition using standard solid phase methods on an automated peptide synthesizer (Intavis Multipep). Amino acids (5 molar equivalents, 120 µL, 0.5 M in DMF) were pre-activated (1 min) with (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU®, 5 molar equivalents, 170 µL, 0.35 M in dimethylformamide (DMF)) and N,N-diisopropylethylamine (DIEA, 7.5 molar equivalents, 70 µL, 1.25 M in dichloromethane (DCM)). Pre-activated amino acids were transferred to the appropriate well. Resins were incubated for 30 min, drained, and the cycle was repeated. Following the 2nd amino acid incubation, the plates were drained and washed with DMF 8 times (3 mL per column of 8 wells). The Fmoc groups were then removed by 2 sequential incubations in 500 µL of a 20% piperidine in DMF solution. The 1st incubation was 5 min, the resin drained, and the 2nd incubation was for 20 min. The resin was drained and washed with DMF 10 times (3 mL per column of 8 wells). After removal of the final Fmoc protecting group, the resin was washed with DCM 5 times (3 mL per column of 8 wells) and allowed to air dry. Side Chain Deprotection and Cleavage from Resin. To the bottom of the filter plate was affixed a drain port sealing mat (ArcticWhite, AWSM-1003DP). To the resin in each well was added triisopropylsilane (100 µL), DODT (100 µL), and water (100 µL) using a multichannel pipette. To the resin in each well was added TFA (1 mL) using a Dispensette Organic dispenser.


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To the resin was added a triangular micro stir bar, and the mixture was stirred for 3 h. The sealing mat was removed, and the cleavage solution was eluted into a solid bottom 96-well deep well plate. The resin in each well was washed with an additional 1 mL of TFA. The solutions were concentrated using rotary evaporation (Genevac). To each well in a new 96-well filter plate with a bottom sealing mat attached was added 1 mL of cold diethyl ether using a Dispensette Organic dispenser. To the ether was added dropwise the concentrated peptide solutions using a multichannel pipette with wide bore tips. A white precipitate formed. The mixture was agitated with the pipette to ensure complete mixing and precipitation. The white solid was filtered, washed with another 1 mL of cold ether, filtered, and dried under vacuum. Parallel Peptide Oxidative Folding. The oxidative folding of the 96 peptide array was performed in parallel and at high dilution using an array of 50 mL centrifuge tubes in the following manner. A sealing mat was affixed to the bottom of the 96-well filter plate containing the crude, precipitated peptides. To the sample in each well was added 0.9 mL of 50:50 water/acetonitrile with a multichannel pipette and a micro stir bar. The mixture was stirred for 1 h to dissolve the solid. The sealing mat was removed, the mixtures were filtered using a vacuum manifold, and the eluent was collected in a solid bottom 96-well deep well plate. To the residual crude peptide in each well was added a second 0.9 mL aliquot of 50:50 water/acetonitrile with a multichannel pipette. The mixture was again stirred and filtered, combining the eluent in the same solid bottom 96-well deep well plate. The peptide solutions were set aside. In a separate 4 L bottle was prepared 4.0 L of folding buffer by combining 3.3 L of water, 300 mL of acetonitrile, 2.0 g of oxidized glutathione, 1.0 g of reduced glutathione, and 400 mL of 1 M TrisHCl pH 7.5 and stirring until the solids completely dissolved. 96 individual 50 mL centrifuge tubes were positioned in a large 8 x 12 matrix using HPLC fraction collection racks. To each


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tube in the array was added 40 mL of peptide folding buffer using a large Dispensette liquid dispenser. To the folding buffer in each 50 mL centrifuge tube was added the 1.8 mL of dissolved peptide from the corresponding position in the 96-well deep well plate (well A1 tube A1, well B1 tube B1, etc.) using a Tecan automated liquid handler. The pH of the folding solutions was measured to be about 7.7. The array of folding reactions was allowed to stand overnight. To each tube in the array was added 1 mL of glacial acetic acid to lower the pH to 4.0 and quench the reaction. Ion exchange resin was used to capture the folded peptide from the dilute solution and concentrate for subsequent RP-HPLC purification. To each well in a new 96-well filter plate was added 1 mL of SP Sepharose High Performance resin (GE Biosciences) as a slurry with a multichannel pipette. Using a Tecan automated liquid handler equipped with a vacuum manifold, the ion exchange resin in each well was conditioned with folding buffer (3 x 0.9 mL with vacuum filtration after each addition), loaded with the peptide folding solution (50 x 0.9 mL, tube A1 well A1, tube B1 well B1, etc.), and washed (4 x 0.9 mL, 20 mM NaOAc, pH = 4.0). The folded peptides were eluted from the resin in each well manually with 2 x 1 mL (1 M NaCl, 20 mM NaOAc, pH = 4.0) on a vacuum manifold, and the eluent was collected into a solid bottom 96-well deep well plate. Reversed Phase HPLC Purification and Analysis and Mass Spectrometry.

After

concentration by ion exchange, the folded peptide (2 mL) was purified by mass-triggered semiprep HPLC (Agilent 1100/LEAP, Phenomenex Jupiter 5u C18 300 Å, 100 x 10 mm 5 micron column) with a gradient of 15-35% B over 45 min, with a 5 min flush and 5 min equilibration at 8 mL/min. The collected fractions were pooled and reformatted into vials on a Tecan automated liquid handler. Final QC (Phenomenex Jupiter 20 × 2 mm, 100 Å, 5 micron column eluted with a 10 to 60% B over 10 min gradient (A: water and B: acetonitrile, 0.1% TFA in each) at a 0.750


22

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mL/min flow rate monitoring absorbance at 220 nm) was performed. Peptide quantification was performed by chemiluminescent nitrogen detection (CLND) via correlation to a caffeine standard curve using an Antek 8060 HPLC-CLND detector and an Agilent Zorbax 3.5 µm 300SB-C3 2.1 x 50 mm column eluted with a 1-100% B over 1.5 min gradient (A: water and B: isopropanol, 0.1% formic acid in each) at a 0.25 mL/min flow rate. Peptides with > 95% purity and correct (m/z) ratio were screened. (See Supporting Information for LC-MS characterization of synthetic GpTx-1 and analogs). Ion-Works® Quattro Population Patch Clamp Electrophysiology. Adherent cells were isolated from tissue culture flasks using 0.25% trypsin-EDTA treatment for 10 minutes and were resuspended in external solution consisting of 140 mM NaCl, 5.0 mM KCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, pH 7.4. Internal solution consisted of 70 mM KCl, 70 mM KF, 10 HEPES, 5 mM EDTA, pH 7.3. Cells were voltage clamped, using the perforated patch clamp configuration at room temperature (~22°C), to -110 mV and depolarized to -10 mV before and 5 min after test compound addition. Compound dilutions contained 0.1% bovine serum albumin to minimize non-specific binding. Peak inward currents were measured from different wells for each compound concentration and IC50 values were calculated with Excel software. All compounds were tested in duplicate (n = 2). The IWQ platform was employed for the screening of large sets of samples and resulted in a general ~10-fold shift in NaV1.7 potency for GpTx-1 peptides perhaps due to interaction of peptides with the thousands of ‘extra’ cells in each IWQ well inherent to the population patch clamp technique. PatchXpress® 7000A Electrophysiology. Adherent cells were isolated from tissue culture flasks using 1:10 diluted 0.25% trypsin-EDTA treatment for 2-3 minutes and then were incubated in complete culture medium containing 10% fetal bovine serum for at least 15 minutes


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Page 24 of 56

prior 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, 75 mM 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 patch clamp configuration at room temperature (~22°C) at a holding potential 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, currents were recorded with a holding voltage that yielded ~20% channel inactivation, calculated automatically for each individual cell. Test compounds were added and NaV currents were monitored at 0.1 Hz at the appropriate test potential.

All

compound dilutions contained 0.1% bovine serum albumin to minimize non-specific binding. Cells were used for additional compound testing if currents recovered to >80% of starting values following compound washout. At least four different concentrations of test compound at half log units were applied individually, with washout, recovery of current, and resetting of holding voltage between each individual concentration. Percent inhibition as a function of compound concentration was pooled from at least n = 10 different cells, with two to three data points per concentration, and fitting the resulting dataset with a Hill (4-parameter logistic) fit in DataXpress 2.0 software to produce a single IC50 curve.32 Whole-Cell Patch Clamp Electrophysiology. Cells were voltage clamped using the wholecell patch clamp configuration at room temperature (~22°C). Pipette resistances were between 1.5 and 2.0 MΩ. Whole-cell capacitance and series resistance were uncompensated. Currents were digitized at 50 kHz and filtered (4-pole Bessel) at 10 kHz using pClamp10.2. Cells were lifted off the culture dish and positioned directly in front of a micropipette connected to a solution exchange manifold for compound perfusion. To record from non-inactivated channels,


24

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cells were held at -140 mV and depolarized to -10 mV (0 mV for hNaV1.8). To record from partially inactivated channels, cells were held at -140 mV until currents stabilized and then switched to a voltage that yielded ~20% channel inactivation. 10 ms pulses were delivered every 10 seconds and peak inward currents were recorded before and after compound addition. Compound dilutions contained 0.1% bovine serum albumin to minimize non-specific binding. For hNaV1.8 channel recordings, tetrodotoxin (TTX, 0.5 uM) was added to inhibit endogenous TTX-sensitive voltage-gated sodium channels and record only NaV1.8-mediated TTX-resistant currents. 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, 75 mM CsF, 2.5 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.25 by CsOH. Escalating compound concentrations were analyzed on the same cell and IC50 values were calculated with Clampfit 10.2 software and by fitting the resulting dataset with a Hill (4parameter logistic) fit in Origin Pro 8 software. DRG neuron isolation and Manual Patch Clamp Electrophysiology. Adult male and female C57BL/6 mice (Harlan Laboratories, Indianapolis, IN) were euthanized with sodium pentobarbital (Nembutal, 80 mg/kg, i.p., Western Med Supply, Arcadia, CA) followed by decapitation. DRG from cervical, thoracic and lumbar regions were removed, placed in Ca2+ and Mg2+-free Hanks’ Balanced Salt Solution (Invitrogen, Carlsbad, CA), and trimmed of attached fibers under a dissecting microscope. DRG were sequentially digested at 37°C with papain (20 U/mL, Worthington Biochemical Corporation, Lakewood, NJ) and 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


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Page 26 of 56

serum (Invitrogen), and cells were triturated with a fire-polished Pasteur pipette prior to plating on Poly-D-Lysine-coated glass coverslips (Cole-Parmer, Vernon Hills, IL).

Cells were

maintained in a humidified incubator at 28°C with 5% CO2 for 3-7 days in the presence of 1% NSF-1 (Lonza, Basel, Switzerland) to increase the expression of tetrodotoxin-sensitive sodium channel currents. DRG neurons were voltage clamped using the whole-cell patch clamp configuration at room temperature (21-24oC) using an Axopatch 200 B or MultiClamp 700 B amplifier and DIGIDATA 1322A with pCLAMP software (Molecular Devices, Sunnyvale, CA). Pipettes, pulled from borosilicate glass capillaries (World Precision Instruments, 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. Currents were digitized at 50 kHz and filtered (4-pole Bessel) at 10 kHz. Cells were lifted off the culture dish and positioned directly in front of a micropipette connected to a solution exchange manifold for compound perfusion. Cells were held at -140 mV or a voltage yielding approximately 20% inactivation and depolarized to -10 mV for 40 msec every 10 seconds. Tetrodotoxin (TTX, Sigma) was used following peptide addition to block any residual TTX-sensitive sodium currents. Pipette solution contained 62.5 mM CsCl, 75 mM CsF, 2.5 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.25 by CsOH. Bath solution contained 70 mM NaCl, mM 5.0 KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 10 mM HEPES, 11 mM glucose, and 140 mM mannitol, pH 7.4 by NaOH. Data were analyzed with Clampfit and Origin Pro8 (OriginLab Corp, Northampton, MA). NMR structural analysis of GpTx-1.

The structure of GpTx-1 was obtained by high

resolution NMR spectroscopy in 95% water and 5% D2O at pH ~3 and T = 298 K. The data


26

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were collected on a Bruker Avance III 800 MHz spectrometer using standard 2D experiments.33 The 2D diffusion edited NOESY experiment was recorded with the PGSTE element34 to eliminate water resonance and facilitate detection of all the alpha protons (Supporting Information). The structure was calculated from 500 NOE constraints (216 long-range), 45 dihedral angle constraints, 11 hydrogen-bond constraints, and 3 disulfide-bond constraints using Cyana 2.1 software. The final RMSD for the backbone atoms was 0.1 ± 0.05 Å and 0.74 ± 0.12 Å for all heavy atoms. The disulfide connectivity was confirmed by the PADLOC27 calculations, which gave probability of one to the Cys2—Cys17, Cys9—Cys23, and Cys16—Cys30 pattern and zero probability to the alternative Cys2—Cys16, Cys9—Cys23, and Cys17—Cys30 disulfide pattern. 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 analog reference standards in 50/50 (v/v) methanol/water and stored at 20oC. Peptide stock solutions (1 mg/mL) were used to prepare 20 µg/mL peptide working solutions in HPLC grade water. The peptide working solutions were stored in a refrigerator at 2 to 8°C prior to use. Stability samples were prepared by adding 225 µl plasma into the vials containing 25µl of 20 µg/mL peptide working solutions and were incubated at 37oC. The initial concentration was 2 µg/mL for each peptide in human, rat, or mouse plasma. Aliquots of plasma (25 µL) at five time points (0, 2, 4, 8 and 24 hours) were transferred into the appropriate well of a 96-well plate, followed by the addition of 25 µL of internal standard solution (100 ng/mL, peptide analog made in 50/50 methanol/water) and 100 µL of 0.1% formic acid, and the samples were vortex mixed. An Oasis HLB µElution 96-well solid phase extraction plate was used to extract GpTx-1 or [Ala5]GpTx-1 from the pretreated plasma samples and the extracts were injected (10 µL) onto the LC-MS/MS system for analysis. The LC-MS/MS consisted of an


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Page 28 of 56

Acquity UPLC system (Waters, Milford, MA) coupled to a 5500 QTRAP mass spectrometer (AB Sciex, Toronto, Canada) with a Turbo IonSpray® ionization source. The analytical column was an Acquity UPLC BEH C18 2.1 mm × 50 mm column. The mobile phases were 0.1% formic acid in acetonitrile/water (5/95, v/v, mobile phase A) and 0.1% formic acid in acetonitrile/water (95/5, v/v, mobile phase B). Data was collected and processed using AB Sciex Analyst® software (version 1.5). The plasma stability of the tested peptides were derived from the peak area ratios corresponding to peptides and internal standard obtained from the LC-MS/MS analysis, all data were normalized to the value at 0-hr time point. Supporting Information Available: Characterization of synthetic GpTx-1 and comparison to native peptide, NMR chemical shifts of GpTx-1, analytical characterization of peptide analogs, and dose-response curves for key compounds against human NaV1.7. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author * To whom correspondence should be addressed. Phone: 1-805-447-9397. Fax: 1-805-4803015. E-mail: [email protected]. Acknowledgement. We gratefully thank Jennifer Aral, Jason Long, Stephanie Diamond, Ryan Holder, and Jingwen Zhang for peptide synthesis support, Xiaoyang Xia for molecular modeling support, and Kaustav Biswas and Elizabeth Doherty for editorial assistance.

Abbreviations Used. Abu, L-2-aminobutyric acid; Bip, L-4-biphenylalanine; Boc, tert-butoxycarbonyl; Cha, Lcyclohexylalanine; Chg, L-cyclohexylglycine; Cpg, L-cyclopentylglycine; Fmoc, Nα-9-


28

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fluorenylmethoxycarbonyl; 1-Nal, L-1-naphthylalanine; Nle, L-norleucine; NMe-Leu, L-Nmethylleucine; Nva, L-norvaline; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; 4-IPhe, L-4-iodo-phenylalanine; tBu, tert-butyl; Tle, L-tert-butylglycine; Trt, trityl References and Notes.

1 Yu, F. H.; Yarov-Yarovoy, V.; Gutman, G. A.; Catterall, W. A. Overview of molecular relationships in the voltage-gated ion channel superfamily. 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 channels from cloned cDNA. Nature. 1986, 322, 826-828. (b) Noda, M.; Numa, S. Structure and Function of Sodium Channel. J. Recept. Res. 1987, 7, 467497. 3 (a) Goldin, A. L. Resurgence of sodium channel research. Annu. Rev. Physiol. 2001, 63, 871894. (b) Fischer, T. Z.; Waxman, S. G. Familial pain syndromes from mutations of the NaV1.7 sodium channel. Ann. N. Y. Acad. Sci. 2010, 1184, 196-207. 4 French, R. J.; Terlau, H. Sodium channel toxins receptor targeting and therapeutic potential. Curr. Med. Chem. 2004, 11, 3053-3064. 5 Lee, C. H.; Ruben, P. C. Interaction between voltage-gated sodium 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, in patients 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 in erythromelalgia: effects of altered activation and steady-state


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inactivation 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 a continuum: A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces 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. An SCN9A 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 codon mutation 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-function mutations in the NaV1.7 gene underlie congenital indifference to pain in 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.


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Global NaV1.7 knockout mice recapitulate the phenotype of human congenital indifference 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-specific gene deletion reveals a major role for NaV1.7 (PN1) in acute and inflammatory 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. Distinct NaV1.7-dependent pain sensations require different sets of sensory and sympathetic neurons. Nature Comm. 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 gene expression 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 blockers for 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 NaV1.7 blockers: potential treatments for neuropathic pain. Bioorg. Med. Chem. Lett. 2007, 17, 46304634. (e) Hoyt, S. B.; London, C.; Ok, H.; Gonzalez, E.; Duffy, J. L.; Abbadie, C.; Dean, B.;


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Felix, J. P.; Garcia, M. 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 for neuropathic 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. Pain. 2014, 155, 1702-1707. 12 (a) Mao, J.; Chen, L. L. Systemic lidocaine for neuropathic pain relief. Pain. 2000, 87, 7-17. (b) Jensen, T. S. Anticonvulsants in neuropathic pain: rationale and clinical evidence. Eur. J. Pain. 2002, 6 (Suppl A), 61-68. (c) Rozen, T. D. Antiepileptic drugs in the management of cluster headache and trigeminal neuralgia. Headache. 2001, 41 (Suppl 1), S25-32. (d) Backonja, M. M. Use of anticonvulsants for treatment of neuropathic pain. Neurology. 2002, 59 (5 Suppl 2), S14-17. 13 Bagal, S. K.; Chapman, M. L.; Marron, B. E.; Prime, R.; Storer, R. I.; Swain, N. A. Recent progress in sodium channel modulators for pain. 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 selective modulators of voltage-gated sodium channel subtypes. Mol. Pharm. 2005, 69, 419-429. (b) Oldrati, V.; Bianchi, E; Stöcklin, R. Spider venom components as drug candidates. In Spider Ecophysiology; Nentwig, W., Ed.; Springer Verlag: Heidelberg, Germany, 2012; pp 491-503. (c) Kuhn-Nentwig, L.; Stöcklin, R.; Nentwig, W. Venom composition 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.;


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Bosmans, F. From foe 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 solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from chinese bird spider Selenocosmia 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 sodium channels by binding to receptor site 4 and trapping the domain II voltage 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. Two tarantula 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 NaV1.7 sodium 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 pathway of 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 with NaV1.5: implied existence of a novel toxin binding site coupled to activation. J. Biol. Chem. 2007, 282, 12687-12697. (f) Park, J. H.; Carlin, K. P.; Wu, G.; Ilyin, V. I.; Kyle, D. J. Cysteine racemization during the Fmoc solid


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phase peptide synthesis of the NaV1.7-selective peptide - protoxin II. J. Pept. Sci. 2012, 18, 442448. 17 (a) Norton, R. S.; Pallaghy, P. K. The cystine knot structure of ion channel toxins and related polypeptides. Toxicon. 1998, 36, 1573-1583. (b) Pallaghy, P. K.; Nielsen, K. J.; Craik, D. J.; Norton, R. A common structural motif incorporating a cystine knot and a triple-stranded β-sheet in toxic and inhibitory polypeptides. Prot. 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. Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-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, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels. J. Biol. Chem. 2007, 282, 3069930706. (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 disulfide isomers of µ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels. 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 selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models. Proc. Nat. 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 NaV channel blockers. Toxicon. 2013, 77, 58-67.


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21 Murray, J. K.; Miranda, L. P.; McDonough, S. I. Potent and selective polypeptide inhibitors of NaV1.3 and NaV1.7 sodium channels. 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 the tarantula Grammostola rosea. Toxicon. 2011, 58, 265276. 23 Meir, A.; Cherki, R. S.; Kolb, E.; Langut, Y.; Bajayo, N. Ion channel-blocking peptides from spider venom and their use as analgesics. 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 target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. Toxicon. 2012, 60, 478-491. 25 (a) Steiner, A. M.; Bulaj, G. Optimization of oxidative folding methods for cysteine-rich peptides: a study of conotoxins containing three disulfide bridges. J. Pept. Sci. 2011, 17, 1-7. (b) Góngora-Benítez, M.; Tulla-Puche, J.; Paradis-Bas, M.; Werbitzky, O.; Giraud, M.; Albericio, F. Optimized Fmoc solid-phase synthesis of the cysteine-rich peptide linaclotide. Biopolymers Pept. Sci. 2011, 96, 69-80. 26 Repeating the experiment at a holding voltage that produced 20% 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 in peptides and proteins by NMR spectroscopy. Anal. Chem. 2012, 84, 262-266.


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28 The Asp14Ala mutant had a complex LC-MS profile of multiple peaks with the same MW after folding of the purified linear peptide and 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 neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-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 the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxinIV (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 the reationship between the chemical structure of Protoxin II and its activity on Voltage Gated Sodium Channels. J. Med. Chem. 2014, 57, 6623-6631. 30 (a) Edman, P. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 1950, 4, 283–293. (b) Niall, H.D. Automated Edman degradation: the protein sequenator. Meth. Enzymol. 1973, 27, 942–1010. 31 (a) Dančík, V.; Addona, T. A.; Clauser, K. R.; Vath, J. E.; Pevzner, P. A. De novo peptide sequencing via tandem mass spectrometry. J. Comp. Biol. 1999, 6, 327-342. (b) Favreau, P.; Menin, L.; Michalet, S.; Perret, F.; Cheneval, O.; Stöcklin, M.; Bulet, A.; Stöcklin, R. Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. Toxicon. 2006, 47, 676-687. (c) Favreau, P.; Cheneval, O.; Menin, L.; Michalet, S.; Gaertner, H.; Principaud, F.; Thai, R.; Ménez, A.; Bulet,


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P.; Stöcklin, R. The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues. Rapid Comm. Mass Spec. 2007, 21, 406–412. (d) Violette, A.; Biass, D.; Dutertre, S.; Koua, D.; Piquemal, D.; Pierrat, F.; Stöcklin, R.; Favreau, P. Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fishhunting cone snail using a combined proteomic and transcriptomic approach. 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 formalin model of persistent pain. J. Med. Chem. 2011, 54, 4427-4445. 33 Wüthrich, K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Canada, 1986. 34 Cotts, R. M.; Hoch M. J. R.; Sun, T.; Markert, J. T. Pulsed field gradient stimulated echo methods for improved NMR diffusion measurements in heterogeneous systems. J. Magn. Reson. 1989, 83, 252-266.


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Figure 1. A) Reversed phase (RP) HPLC fractionation of crude venom extracted from Grammostola porteri. The tick marks along the x-axis represent 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 ion channel assay, and was later identified as GpTx-1.


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Figure 2. High resolution electrospray ionization-mass spectrometry (ESI-MS) analysis of fraction 31 from the initial fractionation of Grammostola porteri 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. Additional peaks in the mass spectrum indicate that the venom fraction is a mixture of at least four distinct peptides.


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


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Figure 4. A) RP-HPLC sub-fractionation of the material in fraction 31 from the intial fraction of Grammostola porteri venom.

The tick marks along the x-axis represent time slices of the sub-

fractionation. B) Activity of the isolated sub-fractions in the NaV1.7 IWQ assay. The major peak in the RP-HPLC chromatogram was the most active in the NaV1.7 assay and was deconvoluted to identify GpTx-1.


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DCLGF MRKCI PDNDK CCRPN LVCSR THKWC KYVF-NH2 5

10

15

20

25

30

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

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


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 7. Manual patch clamp electrophysiology of GpTx-1: A) Time course of increasing concentrations of GpTx-1 against partially inactivated NaV1.7 channels, recorded from a singlecell with manual patch clamp electrophysiology. Peak inward NaV1.7 currents were measured at -10 mV every 10 seconds in the presence of increasing concentrations of GpTx-1; cells were held at a voltage where channels were fully non-inactivated (squares) and then switched to voltage yielding approximately 20% inactivation (circles).

Testing with GpTx-1 showed

inhibition of the NaV1.7 current, which was reversible upon washout. B) Currents in response to increasing concentrations of GpTx-1, from the timecourse displayed.

“Control” trace shows

NaV1.7 current before GpTx-1, and other traces show NaV1.7 current after GpTx-1 addition at


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indicated concentrations. C) Dose-response curves of GpTx-1 against NaV1.8, NaV1.7, NaV1.5, NaV1.4, and NaV1.3 channels measured with the same protocol. Currents were normalized with 100 representing NaV current with no peptide addition and 0 representing NaV current following complete block.


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Figure 8. NMR solution structure of GpTx-1: A) overlay of the 10 lowest energy conformations of the peptide backbone; B) overlay of the heavy atoms from the 10 lowest energy conformations of the peptide; and C) ribbon representation of the peptide backbone in the lowest energy conformation with secondary structure and numbered cysteine residues.


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Figure 9. Positional scanning with alanine; each bar represents the IWQ NaV1.7 IC50 of the analog with alanine substituted at the indicated position of GpTx-1 (SD, n ≥ 2).

Peak

concentration tested was 5 µM.


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100 IC50 = 13.2 nM 80 I/I0 x 100

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60 40 20 0 1

10

100

1000

Conc. [Ala5]GpTx-1 (nM)

Figure 10. Dose-response curve of [Ala5]GpTx-1 (10) against human NaV1.7 channels by manual whole-cell patch clamp electrophysiology (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.


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


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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-MS peak area; each time point was an average of n=4 samples.


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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 MOE (Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2014.) and colored by lipophilicity (Wildman, S.A.; Crippen, G.M. Prediction of


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physiochemical parameters by atomic contributions. J. Chem. Inf. Comput. Sci. 1999, 39, 868– 873.). A) Partially transparent surface rendering of the molecule. B) Ribbon representation of the peptide backbone with the side chains of key residues depicted. C) Hydrophobic and putative binding face of GpTx-1 with an opaque surface (green = hydrophobic and magenta = hydrophilic). D) molecule has been rotated clockwise by 90° around the z-axis to show the topological contrast between the flat hydrophobic face and the hydrophilic (solvent-exposed) face of the peptide.


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Figure 14. A) Surface rendering of NMR structure of GpTx-1 (1) with key binding residues colored in green and residues impacting selectivity colored magenta. B) Surface rendering of a homology model of [Ala5,Phe6,Leu26,Arg28]GpTx-1 (71) with key binding residues colored in green and substituted residues improving potency, stability, and/or selectivity in yellow. Figures generated using PyMOL (The PyMOL Molecular Graphics System, Version 1.7.2 Schrödinger, LLC.).


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TOC Graphic


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Engineering Potent and Selective Analogues of GpTx-1, a Tarantula

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