Single Residue Substitutions That Confer Voltage ... - ACS Publications

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Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the NaV1.7 Inhibitory Peptide GpTx‑1 Justin K. Murray,† Jason Long,† Anruo Zou,‡ Joseph Ligutti,‡ Kristin L. Andrews,§ Leszek Poppe,† Kaustav Biswas,† Bryan D. Moyer,‡ Stefan I. McDonough,∥ and Les P. Miranda*,† †

Therapeutic Discovery and ‡Neuroscience, 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

§

S Supporting Information *

ABSTRACT: There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

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and μ-SLPTX-Ssm6a from a centipede.8 The KIIIA, HwTx-IV, and ProTxII peptides display a range of potencies against NaVs and other ion channels, and the possibility of optimizing their selectivity profiles has been investigated through individual SAR campaigns.9,10 The diversity of naturally occurring disulfide-rich peptide toxins has made them valuable tools for the study of voltagegated ion channels.11 Their potency, selectivity, and metabolic stability embody an attractive starting point for the development of ion channel-targeted therapeutic leads.5 While advances in membrane protein crystallography and cryo-electron microscopy continue to improve our understanding of ion channel assembly and structure,12 the extracellular loops that often determine binding are flexible and still remain difficult to exploit with structure-based design. It is especially ineffective to apply structure-based drug design principles to achieve selectivity against naturally occurring close homologues (e.g., the other human voltage-gated sodium ion channels) when no reliable high-resolution structural information is available of either the apoprotein or a ligand-bound cocrystal. Accordingly, there is utility for higher-throughput empirical methodologies to complement homology modeling and docking approaches to develop selective peptide antagonists from novel toxin peptide leads.9 Biological display approaches for peptide optimization are currently not compatible with testing by electrophysiology. A number of combinatorial techniques exist for the elaboration

INTRODUCTION NaV1.7 is one of nine isoforms of human voltage-gated sodium ion channels (NaVs or VGSCs) that play a major role in governing cellular electrical excitability.1 Compelling human genetic evidence has emerged to highlight NaV1.7 in the peripheral nervous system as a promising therapeutic target for pain and itch.2 Gain-of-function variants in the SCN9A gene encoding NaV1.7 result in disorders of spontaneous pain and itch,3 and inactivating mutations in SCN9A produce complete loss of pain perception, with loss of olfaction the only other known abnormality.4 Accordingly, intensive efforts have been underway for on the order of a decade to produce small molecule inhibitors of NaV1.7 that have selectivity significantly greater than existing clinical pan sodium channel inhibitors, such as carbamazepine and lidocaine, to address the large unmet medical need in chronic pain and potentially in neuropathic itch. Engineering selectivity against NaV isoforms that govern function in cardiac tissue (NaV1.5), skeletal muscle (NaV1.4), and the central nervous system into a small molecule with sufficient potency and properties suitable for oral administration apparently has proven challenging, given an increasing repertoire of small molecule inhibitors but limited clinical progress.5,6 Consequently, there is interest in exploring additional modalities for targeting NaV1.7. Recently, a monoclonal antibody was reported to have functional inhibitory activity against NaV1.7, suggesting the potential for a long-lived antagonist.7 Other efforts have identified a variety of NaV1.7 inhibitory peptides from natural sources, including KIIIA from a cone snail, HwTx-IV and ProTxII from tarantula spiders, © 2016 American Chemical Society

Received: December 16, 2015 Published: February 18, 2016 2704

DOI: 10.1021/acs.jmedchem.5b01947 J. Med. Chem. 2016, 59, 2704−2717

Journal of Medicinal Chemistry

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of linear, one- and two-disulfide peptides,13 but synthetic challenges, including parallel oxidative folding, have apparently prevented the application of these methods to three-disulfide peptide toxins. Traditionally, alanine positional scans have been used as a fundamental approach for elucidating structure− activity relationships (SAR) in complex natural peptides and proteins.14 In alanine scanning, individual replacement of each amino acid in a polypeptide chain with alanine produces a series of substitution analogs with a truncated side chain (a methyl group) that can be tested to reveal, usually via a loss in activity, which residues in the native sequence are critical for either target interaction or bioactive conformation. However, peptide− protein interactions occur over a large surface area and are typically the summation of many small interactions. The contribution of a specific residue may not always be discerned simply by exchanging its side chain for a methyl group. In other words, alanine scanning does not always lead to the identification of all positions within the overall peptide and protein sequences that can govern improved potency or selectivity or both. Accordingly, classical peptide medicinal chemistry campaigns can follow a narrow path toward optimizing peptide activity by focusing the analoging at or around only the key binding residues identified predominantly by the initial alanine scan. Moreover, alanine scanning generally overlooks the portions of the peptide that do not directly contact the protein during binding but may affect activity via more subtle conformational effects or via more significant changes than alanine. Here we report our efforts on the systematic brute force whole-molecule analoging of a NaV1.7 inhibitory peptide, GpTx-1.15 We recently reported the screening, deconvolution, electrophysiological characterization, and preliminary analoging of the C-terminally amidated, three-disulfide toxin peptide GpTx-1 from the venom of the Grammostola porteri tarantula (1, Figure 1).15a Wild-type GpTx-1 is potent on NaV1.7

Positional Scan (MAPS) analoging.17 Positional scanning with representative members of different classes of amino acids (i.e., basic, acidic, and hydrophobic) was carried out by sequential incorporation at all non-cysteine positions in the 34-residue, disulfide-rich GpTx-1 peptide toxin and testing each analog for inhibition of NaV1.7. To enable efficient technical work-flow, we have assembled an automated and integrated platform for high-throughput peptide synthesis and electrophysiology to execute this approach and herein report the application of the MAPS analoging methodology to the optimization of the NaV1.7 inhibitory peptide GpTx-1 for potency and NaV subtype selectivity. One round of MAPS analoging generated significantly more insight into the interaction interface with the ion channel than previously available from the traditional approach of Ala scanning plus follow-up structure-guided optimization and produced novel, unanticipated GpTx-1 analogs. Using the aggregate results of this study, we performed docking experiments employing a homology model of the human NaV1.7 protein and our recently reported GpTx-1 NMR structure15a in an attempt to better understand the possible nature of GpTx1peptide-NaV1.7 channel interactions.

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RESULTS AND DISCUSSION Here we present the results of MAPS analoging aimed at the identification of GpTx-1 analogs with >500-fold selectivity against NaV1.4 and NaV1.5 and low single digit nanomolar activity on NaV1.7. We selected a set of modifications that scan the effect of the introduction of acidicity, basicity, and steric bulk/ hydrophobicity attributes individually at each wild-type position (Figure 2A). In total, five series of single substitution GpTx-1 analogs incorporating the canonical lysine, arginine, glutamic acid, and tryptophan residues or the unnatural 1-naphthylalanine (1-Nal) residue at each non-cysteine amino acid position were prepared and screened. This strategy requires the integration of high-throughput peptide chemistry capable of the parallel preparation of individual disulfide-rich peptides in high purity and recent advances in high-throughput electrophysiology methods using population patch clamp available on the IonWorks Quattro (IWQ) system (Molecular Devices, LLC, Sunnyvale, CA). A requirement of the MAPS analoging approach is the efficient preparation of each of usually >100 discrete disulfide-rich peptide analogs in sufficient quantity and purity for evaluation. To enable this, first the linear peptide sequence, often >30 amino acids, requires highly efficient Fmoc solid-phase peptide synthesis (SPPS) chemistry to produce a crude linear product in workable purity (>70%). Second, the multi-cysteine containing peptide must be oxidized into the correct disulfide bond architecture. This technical challenge is exacerbated in parallel peptide synthesis because the folding reaction is preferentially carried out at high dilution to minimize the intermolecular interactions that lead to multimer formation or aggregation. Following successful oxidation, each compound must then be isolated in >95% purity to ensure confidence in screening results. Finally, peptide preparation must interface with an equally high-throughput electrophysiology platform for timely and robust functional testing. An electrophysiology instrument measures direct inhibition of the channel mechanism and so determines the functional potency of the peptide−channel interaction, and assay conditions can also be tuned to examine inhibition of different gating states of the channel. In terms of the efficiency of analog preparation, we found that the GpTx-1 scaffold was reasonably robust toward single amino acid substitution encompassing the designed variety of attributes, affording an overall 76% success rate (131/172) for

Figure 1. GpTx-1 toxin peptide amino acid sequence with six cysteines engaged in three disulfide bonds (C2C17, C9C23, and C16C30 or C1C4, C2C5, and C3C6).

(IC50 = 10 nM) and selective against the NaV1.5 isoform important for cardiac function (IC50 > 10 μM), and alanine positional scanning of the 34-amino acid polypeptide was initially employed with the objective of improving its modest 20-fold selectivity against NaV1.4, which is involved in skeletal muscle function.15a In that published work, the substitution of alanine for phenyalanine at position 5 was found to increase the NaV1.4 selectivity to >300-fold with less than a 3-fold loss in NaV1.7 potency relative to the wild-type peptide. Six additional rounds of structure-guided analoging eventually yielded combination analogs with single digit nanomolar NaV1.7 inhibitory activity and 1000-fold NaV1.4 and NaV1.5 selectivity.16 In this report, we applied a brute force whole-molecule analoging strategy to broaden and deepen our understanding of the SAR of GpTx-1 for selective NaV1.7 inhibition and identify additional scaffolds for the pharmacological optimization of GpTx-1. Our laboratory has established a predominantly automated platform to help facilitate the comprehensive investigation of SAR of discrete disulfide-rich peptides by Multi Attribute 2705



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Figure 2. MAPS analoging of GpTx-1. (A) Example positional scan of GpTx-1 sequence and amino acid substitutions generating six discrete analogs at each non-cysteine position for a theoretical total of 172 peptides. (B) Successful preparation of GpTx-1 MAPS analogs by series. Residues highlighted in yellow indicate that the MAPS analog was not isolated from folding of the crude linear peptide. Gray indicates that the substitution residue is identical to the native sequence. Cysteine residues were not substituted; disulfide connectivities are omitted for clarity.

distinct physicochemical properties at positions where activity was unchanged by alanine substitution.17 Similarly, the MAPS analoging of GpTx-1 exposed key aspects of the peptide’s SAR that were not evident from our initial alanine scanning investigation (Table 1 and Figure 3). Positional scanning of glutamic acid in the basic GpTx-1 sequence (three arginine and four lysine residues in the wild-type peptide sequence, calculated pI = 8.9) yielded the series with the largest number of analogs having disrupted NaV1.7 activity. It indicated the importance of positions Phe5, Met6, Ser24, His27, Trp29, Lys31, Tyr32, Phe34, and the C-terminus for NaV1.7 inhibition by GpTx-1, since their replacement with glutamate resulted in a >40-fold loss in potency (red or dark orange in Figure 3). According to the NMR solution structure,15a these amino acid residues appear to cluster together in three-dimensional space to form the putative NaV1.7-binding face of GpTx-1 (Figure 4). By comparison, only GpTx-1 amino acid positions 29 and 31 had been highlighted as critical for activity by the alanine scan with moderate impact for alanine substitution at position 34 (orange in Figure 3). These three are likely the most important residues for strong interaction of GpTx-1 with the NaV1.7 channel because substitution with any other residue, even 1-Nal for Trp29 or Arg for Lys31, caused a significant loss of activity.

analog preparation via folding directly from crude linear peptide (Figure 2B). In pursuit of completeness of the alanine scan series, three alanine analogs required purification in their linear form before subjection to folding for successful isolation.15a However, the aspartate residue at position 14 was found to be essential for GpTx-1 folding because none of the six substitution analogs at this position, not even the closely related [Glu14]GpTx-1, yielded an isolable product. Only two of the possible six analogs were obtained for each of the positions at Pro19, Leu21, and Ser24, indicating their importance presumably for the peptide folding pathway. Substitution of 1-Nal proved to be detrimental to folding at the largest number of positions in GpTx-1 under our standard oxidation conditions, such that only about half of the possible compounds in this series were isolated in acceptable purity. In general, incorporation of hydrophobic amino acids, tryptophan and 1-Nal, was poorly tolerated at a large majority of the basic positions in the native peptide sequence. Other amino acid substitutions were fairly well tolerated, providing adequate material after the first preparation attempt for obtaining a comprehensive NaV activity data set. Previous work with a different natural peptide scaffold and target in our lab showed that ion channel selectivity can be achieved through incorporation of amino acid residues with 2706


2707

Asp Leu Gly Phe Met Arg Lys IIe

GpTx-1 residue

wild-type

Asp Leu Gly Phe Met Arg Lys IIe Pro Asp Asn Lys Arg Pro Asn Leu Val Ser Arg Thr His Lys Trp Lys Tyr Val Phe

GpTx-1 residue

81 82 83 84

N-term. 1 3 4 5 6 7 8 10

87

85 86

no.

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 1

N-term. 1 3 4 5 6 7 8 10 11 12 13 15 18 19 20 21 22 24 25 26 27 28 29 31 32 33 34 C-term

position

no.

position

0.12 0.15 0.12 0.21 b >5 0.54 c 0.23 ± 0.01

± 0.06

0.01 0.02 0.05 0.04

>5 >5 >5 >5 b >5 >5 c >5

lysine

± ± ± ±

0.24 0.02 0.14 0.03 0.01

± ± ± ± ±

>5 >5 >5 >5 27 ± 10 >5 >5 >5 >5 >5 >5 >5 >5 >20 >20 >5 >20 >5 >4.7 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 NaV1.5 IC50 (μM)

0.02 0.01 0.13 0.13 0.12 0.05 0.09 0.01 0 0.07 0.05 0.04 0.05 0.02 0.01 0 0.02 0.14 0.15 0.16 0.05 0.18 0.05

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

NaV1.5 IC50 (μM)

alanine

NaV1.7 IC50 (μM)

0.37 0.10 0.43 0.27 0.63 0.43 0.94 0.50 0.21 0.17 0.12 0.19 0.23 0.13 0.09 0.69 0.19 0.38 0.47 0.33 0.26 0.97 0.47 >5 >5 0.80 0.17 1.20 0.41 0.09

NaV1.7 IC50 (μM)

2.4 1.5 1.9 >5 b >5 >4.0 c 2.6 ± 0.6

± 0.3 ±0 ± 0.4

NaV1.4 IC50 (μM)

>4.8 1.8 ± 0.1 >3.0 3.2 ± 0.4 45 ± 3 3.8 ± 0.5 >4.6 >3.9 3.0 ± 0.1 3.0 ± 0.5 0.9 ± 0.1 2.7 ± 0.2 4.7 ± 0.3 3.0 ± 0.6 1.5 ± 0.3 >5 3.0 ± 0.7 3.7 ± 0.3 1.9 ± 0.6 >5 3.2 ± 0.1 4.1 ± 0.4 >4.9 >4.5 >5 >5 4.5 ± 0.5 >5 >5 2.7 ± 1.2

NaV1.4 IC50 (μM)

Table 1. NaV Inhibitory Activity from MAPS Analoging of GpTx-1a

107

104 105 106

102 103

no.

46 47 48 49 50 51 52 53 54 55 56 57

45

31 32 33 34 35 36 37 38 39 40 41 42 43 44

no.

0.71 0.34 0.57 0.06 0.12 0.05 0.03 0 0.01

± ± ± ± ± ± ± ± ±

± 0.64

± 0.08

± 0.08

± 0.04 ± 0.06

± 0.57

±0

0.03 0.05 0.04 0.21

± ± ± ±

0.20 0.07 b 0.05 0.20 0.03 b b 0.13

±0

± 0.01 ± 0.08 ± 0.02

>5 >5 b >5 >5 3.4 ± 0.6 b b >5

NaV1.5 IC50 (μM)

tryptophan

>5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 b >5 b >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5

NaV1.5 IC50 (μM)

glutamic acid

± 0.06 ± 0.02

NaV1.7 IC50 (μM)

0.27 0.22 0.67 1.75 >5 3.50 0.86 1.70 0.09 0.22 0.28 0.28 0.59 0.22 b 1.40 b 1.40 >5 0.72 0.69 >5 0.34 >5 >5 >5 0.56 >5 4.45

NaV1.7 IC50 (μM)

± 0.1 ± 0.3

± 0.2

± 0.1

3.9 2.6 b 0.6 2.3 0.2 b b 4.4

± 0.1

± 0.2 ± 0.1 ± 0.1

± 0.2 ± 1.3

NaV1.4 IC50 (μM)

>2.2 3.5 >5 >5 >5 >5 >5 >5 4.5 >5 2.3 4.4 >5 >4.8 b >5 b >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5

NaV1.4 IC50 (μM)

126

123 124 125

122

no.

72 73 74 75 76 77 78 79 80

71

69 70

66 67 68

64 65

58 59 60 61 62 63

no. 0.06 0.03 0 0.06 0.28

± ± ± ± ±

>5 >5 >5 >5 >5 >5 c >5 >5 b >4.8 >5 >5 c >5 >5 b >5 b c 3.5 ± 0.5 >5 >5 >5 >5 >5 >5 >5 >5

NaV1.5 IC50 (μM)

arginine

b 0.10 ± 0.02 b 0.04 ± 0.05 0.08 ± 0.01 0.007 ± 0 b b 0.16 ± 0.02

b >5 b >4.0 >5 2.3 ± 0.8 b b >5

NaV1.5 IC50 (μM)

1-naphthylalanine

0.08 0.24 0.01 0.64 0.01

± 0.01 ± 0.06 ± 0.03

± 0.01

± 0.02 ± 0.01

± 0.01 ± 0.03 ± 0.14

± 0.05 ± 0.11

± ± ± ± ±

NaV1.7 IC50 (μM)

0.17 0.11 0.03 0.23 3.60 >4.9 c 0.13 0.17 b 0.11 0.14 0.22 c 0.07 0.17 b 0.06 b c 0.04 0.45 0.09 >5 0.95 1.03 0.08 2.35 0.13

NaV1.7 IC50 (μM)

± 0.8

± 0.2 ± 0.3 ± 0.1

± 0.2

± 0.424 ± 0.1

± 0.2

± 0.2

±0 ± 0.1

± 0.1 ±01 ± 0.1

b 2.0 b 0.5 2.3 0.3 b b 3.0

± 1.1

±0 ± 0.7 ± 0.1

±0

NaV1.4 IC50 (μM)

2.6 1.5 0.9 >3.7 >5 >5 c 1.8 0.9 b 0.6 >4.1 2.3 c 1.4 1.4 b 0.7 b c 0.6 2.6 2.8 >5 >5 >5 1.5 >5 >5

NaV1.4 IC50 (μM)

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2708

11 12 13 15 18 19 20 21 22 24 25 26 27 28 29 31 32 33 34 C-term

position

98 99 100 101

97

94 95 96

93

90 91 92

88 89

no.

NaV1.5 IC50 (μM) b >4.7 >5 c >5 >5 >5 b >5 b >5 >5 >5 c >5 c >5 >5 >5 >5

b 0.12 ± 0.01 0.21 ± 0.01 c 0.36 ± 0.09 0.12 ± 0.01 0.63 ± 0.16 b 0.20 ± 0.12 b 0.38 ± 0.23 0.04 ± 0.01 1.5 ± 0.28 c >5 c >4.9 0.05 ± 0.02 >5 0.59 ± 0.04

lysine NaV1.7 IC50 (μM) b 0.7 2.4 c 3.8 >3.4 3.7 b 2.0 b 3.9 0.7 >4.9 c >5 c >5 1.2 >5 >4.6 112 113 114

± 0.3

± 0.2

± 0.1 ± 0.2

117 118 119 120 121

115 116

111

± 0.1

± 0.4

108 109 110

no.

± 0.1 ±0

NaV1.4 IC50 (μM)

± ± ± ± 0.02 0.01 0.04 0.02

± 0.02 ±0

± 0.03 ± 0.03 ± 0.03

± 0.04

>5 >5 >5 b >5 b >5 >5 >5 b b 3.6 ± 1.1 >5 b c >5 >5 >5 >5 >5

±0 ± 0.01 ± 0.01

0.08 0.06 0.08 b 0.18 b 0.07 0.11 0.06 b b 0.05 0.32 b c >4.9 0.09 0.05 0.27 0.05

NaV1.5 IC50 (μM)

tryptophan NaV1.7 IC50 (μM) 1.5 0.4 2.0 b 3.7 b 0.7 2.5 3.4 b b 0.7 2.6 b c >5 >4.7 0.5 >5 2.7 ± 0.8

± 0.1

± 0.2 ± 1.2

± 0.2 ± 0.4 ± 1.0

± 0.4

± 0.3 ± 0.1 ± 0.7

NaV1.4 IC50 (μM)

133 134 135

132

131

129 130

127 128

no. b 0.10 ± 0.07 0.13 ± 0.02 b b b 0.08 ± 0 0.09 ± 0.02 b b b b 0.25 ± 0.09 b 0.40 ± 0.11 b 0.009 ± 0.01 0.07 ± 0.01 0.04 ± 0.02 b

b >5 >5 b b b >5 >5 b b b b 0.9 ± 0.1 b >5 b 3.5 ± 0.8 >5 >5 b

NaV1.5 IC50 (μM)

1-naphthylalanine NaV1.7 IC50 (μM)

b 0.5 2.3 b b b 0.4 2.4 b b b b 0.6 b >5 b 0.2 0.6 0.8 b

±0 ±0 ± 0.4

± 0.1

± 0.2 ± 1.5

± 0.2 ± 0.7

NaV1.4 IC50 (μM)

a

b

All analogs were C-terminal peptide amides. Samples tested on IWQ platform (avg ± SD, n ≥ 2). Not determined because folded peptide analog was not isolated. cSubstitution corresponds to wild-type sequence.

Pro Asp Asn Lys Arg Pro Asn Leu Val Ser Arg Thr His Lys Trp Lys Tyr Val Phe

GpTx-1 residue

Table 1. continued

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Figure 3. Heat map showing inhibition of NaV1.7, NaV1.5, and NaV1.4 for each GpTx-1 analog from MAPS analoging. Samples were tested on the IWQ platform. All values are avg ± SD, n ≥ 2 (see Table 1). Colors indicate IC50 values in each assay, with green indicating highly potent, yellow meaning potent, light orange indicating moderately potent, dark orange indicating weakly potent, and red signifying not potent; see legend for IC50 ranges. Gray indicates no data because the folded peptide analog was not isolated. Data for the wild-type sequence (GpTx-1 (1) NaV1.7 IC50 = 0.09 ± 0.01 μM, NaV1.5 IC50 > 5 μM, and NaV1.4 IC50 = 2.7 ± 1.2 μM) has been included wherever the indicated substitution is the same as the native residue (Arg7, Arg18, Arg25, Lys8, Lys15, Lys28, Lys31, and Trp29) and marked with a black rectangle.

Substitution of glutamate for GpTx-1 residues Leu3, Gly4, Arg7, Lys8, Asn20, Val22, Arg25, Thr26, and Val33 brought about a 5−20× reduction in NaV1.7 potency, and each may contribute to the peptide’s interaction with the channel through direct contact during binding or indirectly by changing the peptide’s binding conformation. Other GpTx-1 amino acid positions were more tolerant toward substitution with glutamic acid, including the N-terminus, Asp1, Ile10, Pro11, Asp12, Asn13, Lys15, and Arg18. These residues assemble on one side of the folded GpTx-1 structure but opposite to that of the putative binding face and are likely exposed to solvent during the GpTx-1/ NaV1.7 binding interaction.18 Our objective was to identify peptide analogs with improved NaV1.4 selectivity relative to the native GpTx-1 sequence. Select compounds with greater than the ∼30-fold selectivity for NaV1.7 over NaV1.4 displayed by wild-type GpTx-1 in the initial screening on the high-throughput IWQ platform were subsequently tested using the automated whole-cell planar patch clamp PatchXpress (PX) system (Molecular Devices, LLC, Sunnyvale, CA). Although there is a general shift in the IC50 values between these two electrophysiology platforms, the IWQ system has been useful for rapidly identifying potential hits and establishing trends in the data, while the PX system has shown good agreement with results from traditional manual whole cell patch clamp electrophysiology.15a Interestingly, the most promising compounds from the glutamate scan series resulted from substitution at two positions, Ile10 and Pro11, that were unaffected by alanine incorporation. These results were surprising given that the residues are on the putative solventexposed face of GpTx-1 (Figure 5). [Glu10]GpTx-1 (39)

maintained equivalent NaV1.7 potency to native GpTx-1, but its selectivity against NaV1.4 was increased from 30× to 50×. [Glu11]GpTx-1 (40) was about 2-fold less potent against NaV1.7 and NaV1.4 in the IWQ assays. Testing these two glutamate-containing GpTx-1 peptide analogs and others on the PX platform (Table 2) revealed that 39 and 40 were single digit nanomolar potent NaV1.7 inhibitors with >500× selectivity against NaV1.4, thus accomplishing twice within a single substitution series what had required numerous rounds of iterative analoging following the initial alanine scan. The positional scanning of GpTx-1 with the basic amino acid residues arginine and lysine corroborated data from the glutamate and alanine scans (e.g., positions critical for activity) but also gave unique insights into the SAR of other amino acid positions. Arginine or lysine substitution at native GpTx-1 residues Phe5, Met6, Trp29, Tyr32, and Phe34 again led to large losses in NaV1.7 potency. More moderate effects (5−25× reduction in NaV1.7 activity) were seen with lysine substitution at Arg7, Asn20, His27, and the C-terminus. Interestingly, the critical binding residue Lys31 in GpTx-1 could not be replaced with arginine without a 10-fold decrease in activity (76). Although both can be generally classified as basic amino acids and conservative changes, we have observed the distinct effects of arginine and lysine residues in other cases.17 Moreover, we found incorporating a lysine residue in place of the native arginine at GpTx-1 position 18 on the putative solvent-exposed face produced an analog (90) with a 1.6 nM NaV1.7 PX IC50 value and >300-fold selectivity against NaV1.4 (Table 2). A few other analogs appeared to have slightly increased NaV1.7 potency while retaining some NaV1.4 selectivity (10−30×), 2709



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Figure 4. Comparison of alanine scanning to MAPS analoging for elucidation of the GpTx-1 binding face. (A) NaV1.7 functional block (IWQ) from alanine scan and average of alanine, glutamate, arginine, and lysine scans at each GpTx-1 amino acid position. (B) Surface rendering of NMR structure of GpTx-1 with residues colored according to their potency (range of IC50 values with green being very potent to red for inactive) upon substitution with alanine. (C) GpTx-1 surface rendered with colors resulting from average MAPS analoging data in panel A. Figures generated using PyMOL (The PyMOL Molecular Graphics System, Version 1.7.2 Schrödinger, LLC.).

namely, [Arg3]GpTx-1 (60), [Arg22]GpTx-1 (71), [Arg26]GpTx-1 (72), [Lys26]GpTx-1 (95), and [Lys33]GpTx-1 (99). The modest selectivity improvement at GpTx-1 position 26 for 72 and 95 was also observed for [Trp26]GpTx-1 (115), and its consistency inspired the further analoging that we reported previously around the native threonine residue to increase NaV1.7 potency without reducing NaV1.4 selectivity.15a

Noteworthy, incorporation of the residues tryptophan and 1-Nal in most cases had the opposite effect on GpTx-1 activity relative to the alanine, glutamate, arginine, and lysine scanning analogs. In general, increasing hydrophobicity at the amino acid positions within the peptide that are postulated to be directly interacting with the channel either maintained or increased NaV1.7 potency. Only the substitution of tryptophan for the 2710



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Figure 5. Surface rendering of the putative solvent-exposed face of the GpTx-1 NMR structure with (A) basic residues colored blue and acidic residues colored red and (B) residues whose substitution improves selectivity against NaV1.4 without a loss in NaV1.7 potency colored yellow. (C) Model of GpTx-1 incorporating selectivity-enhancing substitutions with coloring of basic (blue) and acidic (red) residues. Figures generated using PyMOL.

Table 2. NaV Inhibitory Activity of Select GpTx-1 MAPS Analogs on the PatchXpress (PX) Platform

a

cmpd

substitution

hNaV1.7 PX IC50 (μM)



hNaV1.4 PX IC50/hNaV1.7 PX IC50

1 5 39 40 44 61 74 84 90 91 111 114 118 121 125 126 128 132 133 135

wild-type Phe5Ala Ile10Glu Pro11Glu Arg18Glu Gly4Arg Lys28Arg Gly4Lys Arg18Lys Pro19Lys Arg18Trp Val22Trp Tyr32Trp GpTx-1(1−34)-Trp Met6[1-Nal] Ile10[1-Nal] Asn13[1-Nal] Trp29[1-Nal] Tyr32[1-Nal] Phe34[1-Nal]

0.010 0.027 0.0021 0.0025 0.017 0.003 0.008 0.006 0.0016 0.0023 0.018 0.042 0.012 0.010 0.004 0.006 0.0020 0.033 0.004 0.005

>10 >10 >10 >10 >10 4.5 8.8 10.1 15.9 >10 >10 3.2 a 2.7 0.4 13.0 7.8 >10 1.0 1.8

0.20 8.5 1.3 1.4 1.7 0.16 0.80 0.29 0.51 0.21 1.3 0.75 1.77 0.65 0.11 0.42 0.28 5.6 0.07 0.17

20 315 593 574 100 48 100 47 321 92 71 18 146 67 29 74 167 166 16 38

Not determined.

native histidine at position 27 in GpTx-1. Compound 131 was a pan-NaV blocker (50×. The three most potent NaV1.7 inhibitory peptides identified by the MAPS analoging, [1-Nal6]GpTx-1 (125), [1-Nal12]GpTx-1 (127), and [1-Nal32]GpTx-1 (133), all had NaV1.7 IWQ IC50 values ≤10 nM and maintained or improved NaV1.4 selectivity from 20- to 50-fold. However, these peptide analogs containing tryptophan and 1-Nal did not show increased selectivity or greatly increased potency when tested on the PX system (Table 2). From these series, only compound 128, incorporating a 1-Nal residue onto the solvent-exposed face of GpTx-1 at position 13, had increased NaV1.7 potency and selectivity in the PX assay format (Table 2).

critical lysine at position 31 (117) resulted in a 40-fold loss in NaV1.7 activity relative to wild-type GpTx-1, confirming the importance of the electrostatics for this residue’s interaction. Other compounds with decreased activity from these series demonstrate that even conservative changes can impact key amino acid positions: swapping tryptophan for Phe34 (120) or 1-Nal for Trp29 (132), each caused a ≥3-fold loss in NaV1.7 potency. The increase in NaV1.7 potency for the hydrophobic substitution analogs of GpTx-1 was typically accompanied by a disproportionate increase in potency against the other NaV isoforms. This manifested as a decrease in overall NaV1.4 selectivity (300-fold selectivity against cardiac NaV1.5, which is a tetrodotoxin-resistant NaV channel and less homologous to NaV1.7 than NaV1.4. The exception was peptide analog 131, which contained a 1-Nal residue in place of the 2711



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Table 3. NaV Inhibitory Activity of GpTx-1 Analogs on IonWorks Quattro (IWQ) Platforma cmpd

substitution

1 wild-type Ile10 Substitution 39 Glu 136 Asn 137 Asp 138 Phe 139 His 140 Leu 141 Met 142 Ser 143 Thr 144 Val 145 Tyr 146 4-CO2−F 147 2-Pal Arg18 Substitution 90 Lys 148 Cit 149 2-Pal 150 SDMA 151 Lys(Me2) Lys28 Substitution 74 Arg 152 Asp 153 Gln 154 SDMA 155 Lys(Me2) N-Terminal Modification 156 Ac-GpTx-1(1−34) 157 GpTx-1(2−34) C-Terminal Modification 158 GpTx-1(1−34)-CO2H 159 GpTx-1(1−33)-NH2 160 GpTx-1(1−32)-NH2 161 GpTx-1(1−31)-NH2 a

hNaV1.7 IWQ IC50 (μM) 0.09 ± 0.01

hNaV1.5 IWQ IC50 (μM) >5

hNaV1.4 IWQ IC50 (μM) 2.7 ± 1.2

0.09 0.33 0.22 0.11 0.05 0.14 0.09 0.05 0.10 0.10 0.14 0.28 0.19

± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.23 0.01 0.01 0.01 0.02 0 0 0.01 0.04 0.05 0.03 0.04

>5 >5 >5 >5 >5 >5 3.4 ± 0.1 >2.7 >5 >5 >5 >4.8 >5

4.5 >5 >5 1.1 0.7 2.5 3.1 2.0 1.1 1.4 1.0 4.0 2.9

0.36 0.18 0.30 0.14 0.21

± ± ± ± ±

0.09 0.01 0.12 0.02 0.11

>5 >5 >5 >5 >5

3.8 ± 0.1 >4.4 >4.9 3.4 ± 1.3 >5

0.09 0.73 0.20 0.18 0.26

± ± ± ± ±

0.03 0.14 0.12 0.06 0.08

>5 37.9 ± 8.6 22.7 ± 6.8 >5 >5

0.21 ± 0.12 0.07 ± 0.01

>5 >5

2.00 ± 0.23 2.35 ± 0.21 1.50 ± 0.42 >5

>5 >5 >5 >5

± 0.2

± ± ± ± ± ± ± ± ± ±

0.2 0.2 1.0 1.0 0.9 0.2 0.5 0.5 0.1 0.6

2.8 ± 0.1 >42.4 >50 >3.8 >5 3.4 ± 0.9 1.4 ± 0.6 >5 >5 >5 >5

Cit, citrulline; 4-CO2-F, 4-carboxy-phenylalanine; Lys(Me2), dimethyllysine; 2-Pal, 2-pyridylalanine; SDMA, symmetrical dimethyl-arginine

and >1000-fold selectivity against NaV1.4 and NaV1.5 (Table 4). The expanded compound sets at these three positions underscore the richness and general unpredictability of GpTx-1 SAR revealed by MAPS analoging but not evident by alanine scanning alone. The results with Asn10, Cit18, and Gln28 suggest that a neutral hydrophilic residue would be a useful addition to future MAPS analoging campaigns. Additional structural studies of these peptide analogs may reveal some insight into the effects of conformation on inhibitory activity. A final set of peptide analogs (156−161) demonstrated very different effects from modification of the N- and C-termini of GpTx-1 (Tables 3 and 4). The N-terminus of GpTx-1 was tolerant toward acetylation (156), truncation (157), or extension. However, conversion of the GpTx-1 C-terminal amide to a free acid (158) or C-terminal truncation resulted in greatly reduced NaV1.7 potency. Peptides isolated as components from animal venom have been described with specificity for a wide range of ion channels, including voltage-gated potassium, calcium, and sodium channels; ligand-gated channels; TRP family channels; and even stretchactivated channels.19 In many cases, the physiological inhibition results from biophysical alteration of channel gating states, especially for voltage-gated ion channels as described here.

The identification of single substitutions on the proposed solvent-exposed face of GpTx-1 that resulted in increased NaV1.4 selectivity with retention or improvement of NaV1.7 potency was intriguing because it suggests the importance of peptide conformation or an additional potential interaction interface with the channel. Further investigation of the SAR was conducted via focused analoging at positions 10, 18, and 28 (Table 3). Variation of the amino acid at position 10 had little effect on potent NaV1.7 inhibitory activity, but the Asn10 and Asp10 analogs had NaV1.4 IC50 values >5 μM in the IWQ assay (>15−20× NaV1.4 selectivity). Testing in the PX assay format showed that [Asn10]GpTx-1 (136) and [Asp10]GpTx-1 (137) were similar to 39, both with single digit nanomolar potency against NaV1.7 and with 350−600× selectivity against NaV1.4 (Table 4). The impact of substitution of the native arginine at position 18 was explored with related amino acid analogs in compounds 148−151. Incorporation of citrulline (148) or 2-pyridylalanine (149) produced peptides with 400-fold NaV1.4 selectivity and NaV1.7 IC50 values 10

0.20

20

0.0021 0.0019 0.004

>10 >10 >10

1.3 0.73 2.2

593 389 635

0.0016 0.0019 0.004 0.006 0.005

15.9 >10 13.1 >10 >10

0.51 0.75 1.5 0.55 0.72

321 402 419 100 141

0.008 0.016 0.004

8.8 >10 >10

0.80 13.0 4.4

100 786 1063

0.012 0.008

>10 5.0

2.1 0.24

176 31

a Cit, citrulline; Lys(Me2), dimethyllysine; 2-Pal, 2-pyridylalanine; SDMA, symmetrical dimethyl-arginine.

Figure 6. Homology model of hNaV1.7 docked with NMR structure of GpTx-1. NaV1.7 domain II residues shown in blue; domain III helices S5 and S6 shown in purple. GpTx-1 residue colors represent the average IC50 value for substitution at each position from select MAPS analoging data with green being very potent ranging to red for inactive as described in Figure 3. Top view of model rendered as ribbon (A) and surface (B). Side view of model rendered as ribbon (C) and surface (D). (E) Side view of interface focused on proximity of GpTx-1 residue Phe5 with NaV1.7 residue Ile767. (F) Side view of interface focused on proximity of GpTx-1 residue Lys31 with NaV1.7 residue Glu811. 2713



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additional peptides having asparagine or aspartic acid at position 10 (136 and 137), citrulline or 2-pyridylalanine at position 18 (148 and 149), or glutamine at position 18 (153) with 2.5−5× improved NaV1.7 potency and 20−50× increased NaV1.4 selectivity relative to wild-type GpTx-1. The comprehensive SAR facilitated docking studies with a NaV1.7 homology model and peptide NMR structure in an attempt to rationalize the key potency and selectivity enhancing modifications. These findings deepen our understanding of peptide antagonists of NaV1.7.

S1−S2 and S3−S4 transmembrane helices. All of the GpTx-1 residues characterized as important for functional activity by MAPS analoging, not just the two or three identified by the alanine scan, make close contact with S2, S3, and the linker between S3 and S4 in the NaV1.7 model. Specifically, Lys31 is in close proximity with NaV1.7 residue Glu811 at the bottom of the binding pocket.21 There is potential for other electrostatic interactions between Arg25 on the peptide and Asp816 or Glu818 on the ion channel and Arg7 with Glu759, similar to the proposed interaction model of the HwTx-IV peptide with the domain II voltage sensor region of NaV1.7.9,21 The side chain of position 5 in GpTx-1, which was found to confer selectivity through substitution of the native phenylalanine with alanine, points toward residue differences between NaV1.7 and NaV1.4 (Ile767 in NaV1.7 is the smaller Val850 in NaV1.4). The neighboring residue in GpTx-1, Met6, is also in close proximity to an amino acid that varies between the isoforms (Ala766 in NaV1.7 is Thr849 in NaV1.4); this may help to explain the interplay in the SAR for the combination analogs at these two positions.15a The docked model suggests that GpTx-1 residues Thr26 and His27 may be oriented toward the bottom of the binding pocket, which could rationalize why an increase in hydrophobicity through substitution at these positions improves potency. A homologous hydrophobic pocket may also exist on NaV1.5, which could be occupied to a greater extent by the nonselective analog [1-Nal27]GpTx-1 (131). Tyr32, Val33, and Phe34 are found orientated away from the ion channel, perhaps toward the surrounding phospholipid bilayer. This could be a possible factor in larger aromatic/hydrophobic groups and C-terminal extension increasing the NaV1.7 activity, whereas conversely, polar/charged groups and C-terminal truncation, including the free acid, are not tolerated. As a point of critique of the model, it appears that the peptide could potentially sit deeper in the presumed hydrophobic pocket at the bottom of the cleft between S1−S4 if the transmembrane helices were to splay slightly wider, but such a manipulation of the protein could only be justified with increased ion channel structural information. By the same token, our rigid docking methodology does not account for a possible induced fit in the peptide conformation. As described, the model is useful for generating potential explanations of key GpTx-1/ NaV interaction sites consistent with experimental potency and selectivity data and may be able to guide future peptide analoging efforts.

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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-dioxa1,8-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-gated sodium (NaV) channels (HEK293-hNaV1.4, HEK293-hNaV1.5, and HEK293hNaV1.7) were used for experiments. 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(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 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 COMU (5 mol equiv, 170 μL, 0.35 M in DMF) and DIEA (7.5 mol equiv, 70 μL, 1.25 M in DCM). Preactivated amino acids were transferred 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 8 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 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 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

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CONCLUSION Engineering ion channel selectivity into peptide toxins through medicinal chemistry remains challenging for important therapeutic targets like NaV1.7. The NaV subtype specificity of GpTx-1 has now been increased. Traditional alanine scanning was used to identify key interacting residues followed by multiple rounds of iterative analoging at those positions to find a combination for maintaining NaV1.7 potency and increasing selectivity against NaV1.4. As described in this work, the alternate strategy of MAPS analoging that integrates highthroughput peptide preparation and electrophysiology screening of multiple positional scanning series in a single work-flow resulted in a number of novel low single digit nanomolar potent and ∼500-fold selective GpTx-1 analogs (39, 40, and 90) and a more complete understanding of the peptide’s SAR in a matter of weeks. Substitutions that significantly impacted NaV potency and selectivity included glutamic acid at positions 10 or 11 (39 and 40) and lysine at position 18 (90). Follow-up analoging at these positions validated the initial findings and produced 2714



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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. 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 and filtered. 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. A total of 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 semipreparative HPLC (Agilent 1100/LEAP, Phenomenex Jupiter 5u 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 fractions were pooled 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 10−60% B over 10 min gradient (A = water and 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 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. Methods used as described previously in ref 15a. PatchXpress 7000A Electrophysiology. Methods used as described previously in ref 15a. 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, fitting the resulting data set with a Hill (four-parameter logistic) fit in DataXpress 2.0 software to produce a single IC50 curve.22 NaV1.7 Homology Model and Docking with GpTx-1. A homology model of the hNaV1.7 domain II voltage sensor domain (VSD) was constructed using the published model (S4 down resting

state) of Kv1.2 by Pathak et al.20 as a template using the modeling package MOE.23 Helices S5 and S6 from the adjacent domain III pore subunit were included in the homology model to account for the possible influence of the adjacent pore domain on peptide binding. Extracellular loops were deleted, and a minimum channel scaffold was used for the domain III pore domain (residues 1305−1335, 1383− 1447); however, the complete domain II VSD was modeled. The S1−S2 (EHHPMTEEFKN) and S3−S4 (ELFLADVEGLSVL) loops were iteratively optimized using the Loop Search algorithm in MOE. Both de novo and PDB templates were considered, and a diverse set of three VSD conformations were used in the docking studies. The three possible channel models and five diverse members from the previoulsy reported GpTx-1 NMR ensemble15a were prepared and minimized using the CHARMM force field in Discovery Studio 4.1.24 Each GpTx-1 structure was docked to the set of channel models using the ZDOCK program25 as implemented in the Discovery Studio 4.1 modeling package. An angular step size of 15° was used in the docking, and a maximum of 2000 poses were saved. To limit the explored poses to the extracellular side of the domain II VSD, channel residues 721−734, 781−797, 830−958, 1305−1324, and 1383−1447 were blocked from the binding interface. The resulting docked poses were subsequently filtered such that at least one atom from channel residues 750, 753, 811, 816, and 818 was required to be within 5 Å of any atom on the docked peptide.21 Likewise, peptide residues 5, 6, 29, 31, 32, and 34 were required to be within 5 Å of any receptor atom in accordance with the MAPS analoging results. These filters reduced the number of returned docking poses to approximately 10−50 for each docking run. The resulting poses were visually examined with respect to the available SAR. The hydrophobic face and GpTx-1 residues Lys29 and His27 were required to make contacts with the channel, and residues tolerant to conjugation were required to be exposed to solvent.18 The most consistent model was further optimized through iterative molecular mechanics minimizations; first keeping the peptide fixed and allowing the channel homology model to relax, followed by all-atom optimization using the AMBER10EHT force field with generalized Born implicit solvation as implemented in MOE.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01947. Analytical characterization of synthetic GpTx-1 MAPS analogs (PDF) Machine-readable structure representations (SMILES) (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1.805.447.9397. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Jennifer Aral, Stephanie Diamond, Ryan Holder, and Jingwen Zhang for peptide synthesis support and Lei Jia for preparation of data heat map.

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ABBREVIATIONS USED Boc, tert-butoxycarbonyl; Cit, citrulline; 4-CO2-F, 4-carboxyphenylalanine; Fmoc, Nα-9-fluorenylmethoxycarbonyl; IWQ, IonWorks Quattro; Lys(Me2), dimethyllysine; 2-Pal, 2pyridylalanine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran5-sulfonyl; PX, PatchXpress; SDMA, symmetrical dimethylarginine; tBu, tert-butyl; Tle, L-tert-butylglycine; Trt, trityl 2715



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