jm501765v (2)

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula
Venom Peptide Antagonist of the NaV1.7 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 Stöcklin,#
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: 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 (20×
and 1000× 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 C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-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 >1000× selectivity against
NaV1.4 and NaV1.5.
■ 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, NaV1.8, and NaV1.9)
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
On the basis of such evidence, decreasing NaV1.7 channel
activity in peripheral sensory neurons has been proposed as an
effective pain treatment.10
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
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, 2299−2314

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 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 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 no. P0DJA9).22
It was later identified
in the venom of Paraphysa scrofa (Phrixotrichus auratus).23
On
the basis of its potency and desirable NaV subtype selectivity
profile, we selected 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 analogues 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
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.
Journal of Medicinal Chemistry Article
DOI: 10.1021/jm501765v
J. Med. Chem. 2015, 58, 2299−2314
2300

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 electrospray ionization (ESI)
and matrix-assisted laser desorption ionization time-of-flight
(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 subfractions were screened for activity in the
NaV1.7 and NaV1.5 IWQ assays. Subfraction 11 was the major
peak in the RP-HPLC chromatogram and showed >90%
inhibition of NaV1.7 activity (Figure 4). Deconvolution of
subfraction 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 amidated polypeptide
containing six cysteine residues engaged in three 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 syn-
thesis (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 coelution 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,
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 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.
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
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.
DOI: 10.1021/jm501765v
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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, HWTX-IV (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 = 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.15,16,27
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.18
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 analogues were tested on
the latter platform due to its higher throughput.
Positional Alanine Scan of GpTx-1. To evaluate the
structure−activity relationships of GpTx-1, a series of alanine
substitution analogues (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 analoguing 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
(14−18) or Arg18
−Pro19
(19−
Figure 3. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of fraction 31 from 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.
DOI: 10.1021/jm501765v
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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 analogues, 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 whole-cell 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 analogue 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 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 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
).
Although relatively distant in the primary sequence, based on
NMR structural analysis, these hydrophobic residues all come
into close spatial proximity in the folded 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 Figure 13A,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
Figure 4. (A) RP-HPLC subfractionation of the material in fraction 31 from the initial fractionation of Grammostola porteri venom. The tick marks
along the x-axis represent time slices of the subfractionation. (B) Activity of the isolated subfractions 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.
Figure 5. Deconvoluted peptide sequence of GpTx-1 (1).
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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 analogues.
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,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.
Position 5 Analogues 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 analogues (35−48) was prepared by
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 analogues 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.
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. 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 analogues (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 analogues (56−60, Table 4). The incorporation of
alanine 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 selectivity
similar to [Ala5]GpTx-1 but without the potential oxidative
liability.
Positions 26 and 28 and Combination Analogues 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
analogues 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 Thr26
in GpTx-1 with arginine (62) and
histidine (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).
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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 analogues (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
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 single-cell with manual
patch clamp electrophysiology. Peak inward NaV1.7 currents were
measured at −10 mV every 10 s in the presence of increasing
concentrations of GpTx-1; cells were held at a voltage where channels
were fully noninactivated (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 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.
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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positions 5, 6, 26, and 28 in a final set of analogues (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
similarly 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 analogue 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.
■ 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 analogues. 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 structure. After replacement of the native Met6
to avoid
oxidation and combination with substitutions at positions 26
and 28, we have identified a GpTx-1 analogue (71) that is
nearly 10-fold 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
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 from
Positional Scanning with Alaninea
compd substitution
hNav1.7 IWQ
IC50 (μM)
hNav1.5 IWQ
IC50 (μM)
hNav1.4 IWQ
IC50 (μM)
1 wild type 0.09 ± 0.01 >5 2.7 ± 1.2
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
a
All analogues were C-terminal peptide amides. Samples tested on
IWQ platform (av ± SD, n ≥ 2).
DOI: 10.1021/jm501765v
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2306

the power of structure-guided design together with systematic
analoguing to improve upon a natural scaffold. GpTx-1 and
related analogues 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-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 Scientific); 1-cyano-2-ethoxy-2-
oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexa-
fluorophosphate (COMU, Matrix Innovation, Montreal, Canada);
HPLC-quality water and acetonitrile (Burdick and Jackson); and 1.0
M 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 indicated
position of GpTx-1 (SD, n ≥ 2). Peak concentration tested was 5 μM.
Figure 10. Dose−response curve of [Ala5]GpTx-1 (10) against
human 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-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.
DOI: 10.1021/jm501765v
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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 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, eluted with
80% 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 dissolved
in 0.1% TFA to approximately 1 mg venom/mL, separated by C18 RP
HPLC chromatography, and collected into approximately 1 min 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
−30 °C.
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 residues: Asn(Trt), Asp(Ot
Bu), Arg(Pbf), Cys(Trt),
Gln(Trt), Glu(Ot
Bu), His(Trt), Lys(Nϵ
-Boc), Ser(Ot
Bu), Thr(Ot
Bu),
Trp(Boc), and Tyr(Ot
Bu). 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 mequiv/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 mol equiv, 120 μL, 0.5 M in DMF)
were preactivated (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 M
in dichloromethane (DCM)). Preactivated amino acids were trans-
ferred to the appropriate well. Resins were incubated for 30 min and
drained, and the cycle was repeated. Following the second amino acid
incubation, the plates were drained and washed with DMF eight times
(3 mL per column of 8 wells). The Fmoc groups were then removed
by two sequential incubations in 500 μL of a 20% piperidine in DMF
solution. The first incubation was 5 min. The resin was drained, and
the second incubation was for 20 min. The resin was drained and
washed with DMF 10 times (3 mL per column of eight wells). After
removal of the final Fmoc protecting group, the resin was washed with
DCM 5 times (3 mL per column of eight 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 pipet. To the resin in each well was added TFA
(1 mL) using a Dispensette Organic dispenser. 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 pipet with wide bore tips. A white precipitate formed.
The mixture was agitated with the pipet 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 pipet 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
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.
DOI: 10.1021/jm501765v
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2308

pipet. 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 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 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 pipet. Using a Tecan
automated 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 the
peptide 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 manually
with 2 × 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 5μ C18 300 Å, 100 mm ×
10 mm 5 μm 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
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 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.
DOI: 10.1021/jm501765v
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fractions were pooled, concentrated, and reformatted into vials on a
Tecan automated liquid handler. Final QC (Phenomenex Jupiter 20
mm × 2 mm, 100 Å, 5 μm column eluted with a 10 to 60% B over 10
min gradient (A, water; B, acetonitrile, 0.1% TFA in each) at a 0.750
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 mm × 50 mm column eluted with a 1−100% B over 1.5
min gradient (A, water; B, 2-propanol, 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 analogues).
Ion-Works Quattro Population Patch Clamp Electrophysiol-
ogy. Adherent cells were isolated from tissue culture flasks using
0.25% trypsin−EDTA treatment for 10 min and were resuspended in
external solution consisting of 140 mM NaCl, 5.0 mM KCl, 10 mM
HEPES, 2 mM CaCl2, 1 mM MgCl2, and 10 mM glucose, pH 7.4.
Internal solution consisted of 70 mM KCl, 70 mM KF, 10 mM
HEPES, and 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 nonspecific 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 min and then were incubated in complete
culture medium containing 10% fetal bovine serum for at least 15 min
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 nonspecific 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 data set 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 whole-cell 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 uncompen-
sated. 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
Table 3. NaV Inhibitory Activity of Position 5 Analogues of
GpTx-1a
compd
Phe5
substitution
hNav1.7 PX
IC50 (μM)
hNav1.5 PX
IC50 (μM)
hNav1.4 PX
IC50 (μM)
10 Ala 0.027 >10 8.5
35 Gly 0.009 >10 11
36 Abu 0.079 >10 >10
37 Nva 0.039 >10 4.6
38 Val 0.019 >10 4.1
39 Met 0.002 >10 0.9
40 Ile 0.009 >10 2.0
41 Leu 0.005 >10 0.9
42 NMe-Leu 0.025 >10 7.7
43 Tle 0.024 >10 4.7
44 Cha 0.004 3.5 0.1
45 Cpg 0.005 >10 1.7
46 Chg 0.006 >10 1.5
47 4-I-Phe 0.236 >10 1.1
48 Bip 0.086 2.1 0.1
a
Abu, 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.
GpTx-1a
substitution
compd Phe5 Met6
hNav1.7 PX
IC50 (μM)
hNav1.5 PX
IC50 (μM)
hNav1.4 PX
IC50 (μM)
1 0.010 >10 0.20
10 Ala 0.027 >10 8.5
49 Nle 0.008 1.1 0.07
50 Leu 0.024 >10 0.41
51 Cha 0.004 2.8 0.09
52 Phe 0.019 3.0 0.14
53 Tyr 0.063 >10 3.4
54 Trp 0.023 3.4 0.24
55 1-Nal 0.004 0.4 0.11
56 Ala Nle 0.010 >10 2.6
57 Ala Leu 0.028 >10 9.9
58 Ala Phe 0.013 >10 0.74
59 Ala Trp 0.059 >10 6.1
60 Ala 1-Nal 0.003 3.6 0.50
a
Cha, L-cyclohexylalanine; 1-Nal, L-1-naphthylalanine; Nle, L-norleu-
cine.
GpTx-1
substitution
compd Phe5 Met6 Thr26 Lys28
hNav1.7
PX IC50
(μM)
hNav1.5
PX IC50
(μM)
hNav1.4
PX IC50
(μM)
1 0.010 >10 0.20
10 Ala 0.027 >10 8.5
61 Leu 0.004 4.6 0.53
62 Arg 0.005 0.5 0.11
63 Glu 0.034 >10 4.9
64 His 0.004 4.5 0.24
65 Arg 0.008 8.8 0.80
66 Glu 0.029 >10 14
67 Ala Leu 0.054 >10 >10
68 Ala Phe Leu 0.004 >10 1.2
69 Ala Phe Arg 0.011 >10 3.3
70 Ala Leu Arg 0.008 >10 6.6
71 Ala Phe Leu Arg 0.0016 >10 1.9
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positioned directly in front of a micropipette connected to a solution
exchange manifold for compound perfusion. To record from
noninactivated channels, 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. Then 10
ms pulses were delivered every 10 s and peak inward currents were
recorded before and after compound addition. Compound dilutions
contained 0.1% bovine serum albumin to minimize nonspecific
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 data set with a Hill (4-parameter logistic) fit
in Origin Pro 8 software.
DRG Neuron Isolation and Manual Patch Clamp Electro-
physiology. Adult male and female C57BL/6 mice (Harlan
Laboratories, Indianapolis, IN) were euthanized with sodium
pentobarbital (Nembutal, 80 mg/kg, ip, 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), 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 Pasteur
pipet 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 (21−24 °C) 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 ms every 10 s. 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 Pro 8
(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 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 α protons (Supporting Information). The
structure was calculated from 500 NOE constraints (216 long-range),
45 dihedral angle constraints, 11 hydrogen-bond constraints, and three
disulfide-bond constraints using Cyana 2.1 software. The final RMSD
for the backbone atoms was 0.1 ± 0.05 and 0.74 ± 0.12 Å for all heavy
atoms. The disulfide connectivity was confirmed by the PADLOC27
calculations, which gave a 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 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 in
HPLC grade water. The peptide working solutions were stored in a
refrigerator at 2−8 °C prior to use. Stability samples were prepared by
adding 225 μL of plasma into the vials containing 25 μL of 20 μg/mL
peptide working solution and incubating at 37 °C. 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 h) 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 analogue 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
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.37
DOI: 10.1021/jm501765v
J. Med. Chem. 2015, 58, 2299−2314
2311

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 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 h time
point.
■ ASSOCIATED CONTENT
*S Supporting Information
Characterization of synthetic GpTx-1 and comparison to native
peptide, NMR chemical shifts of GpTx-1, analytical character-
ization of peptide analogues, 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.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: 1-805-447-9397. Fax: 1-805-480-3015. E-mail: lesm@
amgen.com.
Notes
The authors declare no competing ﬁnancial interest.
■ ACKNOWLEDGMENTS
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, 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; t
Bu, 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 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, 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 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
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. 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 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 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, 4630−4634. (e) Hoyt, S. B.; London, C.; Ok, H.;
Gonzalez, E.; Duffy, J. L.; Abbadie, C.; Dean, B.; Felix, J. P.; Garcia, M.
DOI: 10.1021/jm501765v
J. Med. Chem. 2015, 58, 2299−2314
2312

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 (Suppl1), S25−S32. (d) Backonja, M. M. Use of
anticonvulsants 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 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.; 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 phase peptide synthesis of the NaV1.7-selective
peptide−protoxin II. J. Pept. Sci. 2012, 18, 442−448.
(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. 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. 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, 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 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. 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 NaV
channel blockers. Toxicon 2013, 77, 58−67.
(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, 265−276.
(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.
(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 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 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,
DOI: 10.1021/jm501765v
J. Med. Chem. 2015, 58, 2299−2314
2313

H. D. Automated Edman degradation: the protein sequenator.
Methods 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, P.; Stöcklin, R. The venom of the snake
genus Atheris contains a new class of peptides with clusters of histidine
and glycine residues. Rapid Commun. Mass Spectrom. 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 fish-
hunting 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: Mississauga, Ontario, 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.
(35) Molecular Operating Environment (MOE), 2013.08; Chemical
Computing Group Inc.: 1010 Sherbooke Street West, Suite #910,
Montreal, QC, Canada, H3A 2R7, 2014.
(36) Wildman, S. A.; Crippen, G. M. Prediction of physiochemical
parameters by atomic contributions. J. Chem. Inf. Comput. Sci. 1999,
39, 868−873.
(37) The PyMOL Molecular Graphics System, version 1.7.2;
Schrödinger, LLC: New York, 2014.
DOI: 10.1021/jm501765v
J. Med. Chem. 2015, 58, 2299−2314
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