Peptide-Membrane Interactions Affect the Inhibitory Potency and

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Peptide-membrane interactions affect the inhibitory potency and selectivity of spider toxins ProTx-II and GpTx-1 Nicole Lawrence, Bin Wu, Joseph Ligutti, Olivier Cheneval, Akello Joanna Agwa, Aurelie H Benfield, Kaustav Biswas, David J Craik, Les P Miranda, Sonia Troeira Henriques, and Christina I Schroeder ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00989 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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TITLE Peptide-membrane interactions affect the inhibitory potency and selectivity of spider toxins ProTx-II and GpTx-1

AUTHORS Nicole Lawrence,† Bin Wu,§ Joseph Ligutti,¶ Olivier Cheneval,† Akello Joanna Agwa,† Aurélie H. Benfield,† Kaustav Biswas,§ David J. Craik,† Les P. Miranda,§ Sónia Troeira Henriques,†#* and Christina I. Schroeder.†*

†

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072,

Australia §

Therapeutic Discovery and ¶ Neuroscience, Amgen Research, Thousand Oaks, California 91320,

USA # current address: School of Biomedical Sciences, Faculty of Health, Institute of Health & Biomedical Innovation, Queensland University of Technology, Princess Alexandra Hospital, Translational Research Institute, Brisbane, QLD 4102, Australia

*

To whom correspondence should be addressed:

Sónia Troeira Henriques Tel. +61 7 3443 7342, E-mail: [email protected] Christina I. Schroeder Tel. +61 7 3346 2021, E-mail: [email protected]


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ABSTRACT: Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaV that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaV subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues, and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes, and that altering these residues can have dramatic effects on NaV inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting NaV1.7, and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaV residues, thereby affecting peptide-channel interactions. Our detailed structure activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.


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INTRODUCTION Many spider toxins are being considered for therapeutic drug development because of their ability to inhibit nerve signal propagation by voltage gated sodium channels (NaV).1-3 Of the nine widely distributed human NaV subtypes (NaV1.1–1.9),4 NaV1.7 has received the most attention as a therapeutic target5 due to the genetic link between NaV1.7-mutations and pain disorders, including complete insensitivity to pain.6 NaV are comprised of four domains (DI–DIV), each with six transmembrane segments (S1– S6), that assemble to form a central pore flanked by four membrane-associated voltage sensor domains (VSDs)7, 8 (Figure 1a). Gating of sodium through the pore is controlled by movement of a voltage sensitive segment (S4) within the cell membrane,9, 10 and a subset of spider toxins, classified as gating modifier toxins (GMTs), can inhibit sodium gating by binding to VSDs and affecting the movement of the S4 segment. Several spider GMTs that inhibit NaV1.7 have now been described, including GpTx-1,11,

12

ProTx-I and ProTx-II,13,

14

HnTx-IV,15 HwTx-IV,16,

17

CcoTx1, CcoTx2 and CcoTx3,18 PaurTx3,18 Pn3a19 and JzTx-V,20 and the inhibitory activity of ProTx-I, ProTx-II,21-23 HwTx-IV,23 and HnTx-IV24 has been shown to result from their interaction with the S3–S4 extracellular loop of domain II (DII) of NaV1.7 (Figure 1a–b). Many GMTs that interact with VSDs can also bind to lipid bilayers.25 This dual capacity is explained by their amphipathic surface profile, whereby hydrophobic amino acid residues are arranged into a patch and surrounded by a ring of charged amino acid residues (Figure 1b). Whereas membrane binding is not required for the activity of all GMTs, we previously showed that it is requisite for the inhibitory activity of ProTx-II against NaV1.7,

14

and the potency of

HwTx-IV could also be increased through improving its membrane binding properties.17


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Binding of GMTs to surface-exposed VSD regions, for example the NaV1.7 DII S3–S4 loop, is promoted by ionic interactions between positively charged GMTs and negatively charged VSD loops (Figure 1b–c). However, anionic amino acid residues are displayed on exposed VSD (DI–DIV) loops across all NaV subtypes, and also on exposed regions of voltage gated calcium26 and potassium27 channels. The similarity between exposed regions across these channels can result in non-specific inhibition of several voltage-gated channels, which has been observed for many GMTs.2,

28

Lack of subtype specific inhibition is undesirable when developing GMT-based

therapeutic molecules, therefore, potency toward a specific NaV subtype and selectivity over other channels should be considered together. To gain a better understanding of the relationship between membrane binding, and potency and selectivity toward the NaV1.7 subtype, we have undertaken a detailed study with ProTx-II and GpTx-1. Both GMTs are potent inhibitors of NaV1.7; however, GpTx-1 is more selective for NaV1.7 over NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) subtypes.11 Both peptides have an amphipathic surface profile, however, the number and type of hydrophobic residues contributing to the membrane-binding patch differs (Figure 1c), which correlates with differences in their membrane-binding affinities

2, 14

(Figure 1d). The peptides also differ in the location,

positioning and arrangement of charged residues, including the cationic residues that are more closely associated with hydrophobic patch residues. These differences probably result in different access to and interaction with channel loop residues, including anionic residues D816 and E818 (Figure 1e, boxed), that have been shown through channel mutation studies to be important for inhibition by ProTx-II, HwTx-IV,23 and HnTx-IV.24 In the current study, we investigated the role of hydrophobic and charged amino acid residues of ProTx-II and GpTx-1 analogues by comparing membrane-binding properties, relative


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to NaV potency and selectivity. Here we show that changes to hydrophobic residues in both ProTxII and GpTx-1 result in altered membrane-binding properties, NaV1.7 potency and selectivity over NaV1.4. Also, that substitution of residues within the hydrophobic patch, especially those that change the overall peptide hydrophobicity, induce changes in potency and selectivity. Changes to charged residues were less likely to alter membrane binding, but did modulate NaV1.7 selectivity. Examination of the three-dimensional (3D) structure of analogues and comparison with the parent GMT revealed that point mutations of hydrophobic and charged residues could induce localized structural changes. These changes are likely to alter peptide orientation relative to the membrane surface and to exposed VSD loops, thereby affecting NaV1.7 inhibitory activity and selectivity.

RESULTS AND DISCUSSION In our previous studies, we identified key residues from both the hydrophobic face – hereafter referred to as the binding face – and solvent-accessible regions of ProTx-II and GpTx-1, that were important for activity against NaV1.7. These studies yielded valuable information on the membrane-binding properties of ProTx-II analogues14 and NaV1.7 potency and selectivity of GpTx-1 analogues,29 and provided an excellent base for our current study where we investigate the relationship between membrane binding, NaV1.7 potency and selectivity over NaV1.4, for both peptides. GpTx-1 and ProTx-II analogue selection and design. GpTx-1 has low binding affinity for membranes, compared to ProTx-II, but has better selectivity toward NaV1.7.2, 11 Thus, we were interested in determining whether GpTx-1 analogues shown in our previous studies to be more potent and/or selective (with substitutions in the binding face) had improved membrane-binding affinity, compared to the parent toxin. This was addressed by including the binding-face analogues


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[F5A]GpTx-1, [M6F]GpTx-1, [F5A,M6F,T26L,K28R]GpTx-1 ([AFLR]GpTx-1),11 and related dimer peptides with improved potency (PEG45-bis[M6F, N13Atz]GpTx-1 (([M6F]GpTx-1)2PEG), PEG45-bis[AFLR, N13Atz]GpTx-1 (([AFLR]GpTx-1)2PEG)30 (see Table 1). Conversely, ProTx-II has a high affinity for membranes but is less selective for NaV1.7, and we were interested in determining whether membrane binding of ProTx-II could be reduced by including residues/portions from the binding face of GpTx-1. We proposed that selective binding for NaV1.7 might be improved by reducing the inherent high membrane-binding affinity of ProTx-II. To achieve this aim, we separately investigated the effect of engineering the N- or the C-terminal regions of the binding-face of GpTx into ProTx-II, as shown in Table 1. [K4G,W5A,M6F,W7R]ProTx-II ([N-patch_G]ProTx-II) was designed to incorporate the residues GAFR into the N-terminal patch of ProTx-II. With the [AFLR]GpTx-1 analogue it was shown that the GAFR sequence contributed to increased selectivity for NaV1.7,11 and we were interested in determining whether the KWMKàGAFR substitution in ProTx-II could similarly improve selectivity. C-terminal ProTx-II residues were replaced with GpTx-1 residues (ProTx-II L23R and K27K28L29àYVF) to produce [K27Y,K28V,L29F]ProTx-II(1-29) ([C-patch_G]ProTx-II) and [L23R,K27Y,K28V, L29F]ProTx-II(1-29) ([L23R,C-patch_G]ProTx-II). To examine the combined effect of including both N- and C-termini of GpTx-1 into ProTx-II, we produced [K4G,W5A,M6F,W7R,L23R,K27Y,K28V,L29F]ProTx-II(1-29) ([(N_C)-patch_G,L23R]ProTxII). To further examine our hypothesis that reducing the membrane-binding affinity of ProTxII might improve selectivity toward NaV1.7, we included a double Trp mutant, [W7Q,W30L]ProTx-II,31 reported to be more potent and selective than ProTx-II. We also included


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a similar analogue with an E17K substitution to produce [W7Q,E17K,W30L]ProTx-II, as we have previously shown that the E17K substitution can improve ProTx-II membrane-binding affinity.14 To examine whether solvent-accessible charged residues of GpTx-1 alter membranebinding characteristics we studied [I10E]GpTx-1 and [R18K]GpTx-1 analogues with substitutions on the solvent-accessible face and improved potency toward NaV1.7.29 To investigate the importance of the location of charged residues and determine whether ProTx-II potency or selectivity could be improved through substitution of residues on the solvent-accessible face (see Table 1) we designed analogues in which ProTx-II residues in positions 8, 10 and 14 were mutated to produce analogues with charge distributions more similar to that of GpTx-1. Lys residues were included to introduce positive charge, negatively-charged residues were replaced with Asn, and Glu residues were used to introduce a bulkier negatively charged side chain. The resultant ProTxII analogues with changes in their overall charge were: [T8K,D10N,E17N]ProTx-II, [T8K,K14N,E17K]ProTx-II and [D10E,E17K]-Endo-Glu10a-ProTx-II ([D10EE,E17K]ProTx-II). [E17K]ProTx-II14 was also included to compare the effect of the Lys substitution at ProTx-II position 17 in different charge distribution settings. Synthesis and overall structure of GpTx-1 and ProTx-II analogues. GpTx-1 and ProTx-II analogues (Table 1) were successfully synthesized and folded, and their mass and purity (>95%) confirmed by ESI-MS (Table S1) and analytical RP-HPLC. GMTs contain six cysteine residues arranged in a CI-CIV, CII-CV and CIII-CVI framework giving rise to the inhibitory cystine knot motif.32 This framework results in a rigid structure, that is extremely stable at pH 2–12, and against chaotropic (6 M guanidine HCl), proteolytic (human serum) and thermal (100 ºC) assault.33 Nevertheless, to allow direct comparison of analogue structures, NMR experiments herein were conducted under identical concentration, pH and


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temperature conditions, as previously optimized for the parent peptides.14 One-dimensional (1D) 1

H NMR spectra confirmed a well dispersed amide region indicative of native disulfide bond

arrangement, and a conserved overall structure of the analogues compared to their parent toxin (Figure S1). More localized structural characteristics were compared by examining deviations in Ha chemical shifts derived from two-dimensional (2D) NMR spectra from random coil values34 (Figure 2). Consistent with the 1D spectra, the same global fold was observed between analogue and parent toxin, suggesting identical disulfide connectivity and conservation of Cys-Cys loop structure. Localized structural deviations were observed for GpTx-1 and ProTx-II analogues with changes to hydrophobic residues within the binding face. For GpTx-1, removal of the aromatic ring in position 5 by substituting the Phe with an Ala in [F5A]GpTx-1 and [AFLR]GpTx-1, induced local structural change at position 5 (F5A) and also local changes at Lys31 and Tyr32 (Figure 2a). It is unlikely that T26L and K28R substitutions contributed to this change, as Ha deviation was not observed locally at these positions. The ProTx-II analogue [N-patch_G]ProTxII, had local structural change at positions 5 and 6 (W5A, M6F), and also at Thr8, Met19, Lys26, Lys27 and Lys28 (Figure 2c). C-terminal residue substitution in [C-patch_G]ProTx-II and [L23R,C-patch_G]ProTx-II analogues induced local structural changes at positions 28 and 29 (K28F, Leu29) and at Trp5 and Met19 (Figure 2d). By contrast, substitutions with charged residues in GpTx-1 analogues [I10E]GpTx-1 and [R18K]GpTx-1 (Figure 2b), and ProTx-II analogues [E17K]ProTx-II, [T8K, D10N, E17N]ProTxII and [T8K,K14N,E17K]ProTx-II (Figure 2e), did not show alterations in the backbone Ha secondary chemical shifts, suggesting no changes in the overall 3D structure.


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The NaV1.7 selective [W7Q,W30L]ProTx-II analogue31 had a similar overall structure to ProTx-II; with anticipated structural deviations at positions 7 and 30 (W7Q, W30L), but also local changes at Lys4 and Met6 within the binding face (Figure 2f). Increasing the overall charge for the analogue [W7Q,E17K,W30L]ProTx-II did not induce further structural change. To examine changes in the overall structure of ProTx-II and GpTx-1 in the presence of lipid-like systems, their NMR spectra (Figure S2) were compared at different temperatures in the absence and presence of deuterated dodecylphosphocholine (DPC) micelles at a saturating concentration (100 mM). Peptide structure was unaltered between 283–308K (Figure S2a, c). Addition of DPC micelles resulted in peak broadening, which precluded complete assignment of secondary structure (Figure S2b, d), however, dispersion of peaks within the 1D NMR amide region was similar in the absence and presence of DPC, suggesting that changes in overall structure did not occur. This observation is in agreement with studies conducted with other cystine knotted peptides,35, 36 including the GMT w-Agatoxin IVA,36 where the overall 3D structure was unaltered in the presence of micelles. ProTx-II showed increased line broadening compared to GpTx-1 in the presence of DPC consistent with the higher affinity interaction of ProTx-II with phosphatidylcholine (PC)-headgroups compared to GpTx-1 (examined below, Figure S4a). In addition, the Trp protons in ProTx-II all experienced altered chemical shifts in the presence of DPC, consistent with the requisite involvement of these residues for peptide-membrane interaction.14 Comparison of 3D structure of [E17K]ProTx-II, [W7Q,W30L]ProTx-II and [AFLR]GpTx-1. Although ProTx-II and GpTx-1 share overall structural similarities with their respective analogues, we were interested in comparing the 3D structure of more potent and/or more selective analogues with their parent toxins. To achieve this, we determined 3D NMR solution


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structures for [E17K]ProTx-II and [AFLR]GpTx-1, and compared these against existing structures for ProTx-II (PDB 2N9T), [W7Q,W30L]ProTx-II (PDB 5TCZ) and GpTx-111 (Figure 3). Through our 3D structural investigations, we were especially interested in identifying changes in the relative orientation/location of binding-face residues, and also in identifying specific localized changes that might affect peptide-binding affinity for exposed NaV VSD loop residues. Comparison of the lowest energy structures of ProTx-II and [E17K]ProTx-II (Figure 3a, b) revealed that substitution of Glu17 with Lys disrupted favourable packing between Glu17 and Lys27 in ProTx-II. A clash between Lys17 and Lys27 in [E17K]ProTx-II appeared to be introduced, causing Lys17 to “flip out” and become much more surface exposed compared to Glu17 in the parent ProTx-II. This change in orientation was evidenced by altered f +60 to –60, and j 0 to +120 for ProTx-II and [E17K]ProTx-II, respectively. A follow-on effect from this release of Lys27 by the removal of Glu17, was an altered positioning of the important Trp5 residue, also evidenced by altered f +60 to –60 and j –130 to –20 angles for ProTx-II and [E17K]ProTxII, respectively. This detailed analysis provided a structural basis for the altered activity of [E17K]ProTx-II, that was not detectable by comparing Ha chemical shifts alone (Figure 2e), whereby movement of Lys17 away from the hydrophobic patch results in the increased affinity for membranes that we have previously reported.14 The NaV1.7 selective [W7Q,W30L]ProTx-II analogue31 (Figure 3c) has two fewer Trp residues than ProTx-II, resulting in a significantly reduced hydrophobic patch. Although the overall structure, disulfide connectivity and loop conformations are very similar, backbone overlay of [W7Q,W30L]ProTx-II and ProTx-II revealed that the positioning, or plane of the remaining hydrophobic residues, relative to solvent-accessible residues was shifted (Figure 3d). We have previously shown that ProTx-II W5 and W24 are essential for membrane binding and activity


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against NaV1.7;14 therefore, we make the assumption that these residues, on the surface of [W7Q,W30L]ProTx-II, also bind to the membrane. Because the relative positioning of the hydrophobic patch of [W7Q,W30L]ProTx-II is shifted compared to the parent toxin, we expect that solvent-accessible residues would also be spatially shifted thus changing the way that the peptide interacts with NaV loop residues. This hypothesis is consistent with the observed decrease in membrane-binding affinity and altered NaV channel selectivity of [W7Q,W30L]ProTx-II. It is also possible that localized structural changes observed for [W7Q,W30L]ProTx-II residues K4, M6, W7, and L30 (Figure 2f), reduced steric hindrance between the analogue and NaV loop residues that contribute to improved potency and selectivity for NaV1.7. ProTx-II and GpTx-1 both have four hydrophobic residues that contribute to a hydrophobic patch (Figure 1c, Figure 3a, 3f); however, backbone overlay of the two structures shows that the plane of hydrophobic residues relative to solvent-accessible residues, is markedly different (Figure 3e). This difference may affect the orientation of the peptides at the membrane-NaV VSD interface, and explain the differences in membrane-binding affinity and NaV subtype selectivity that we previously observed for GpTx-1 and ProTx-II.2, 11, 14 The relative positioning of the hydrophobic patch appears to be maintained for [AFLR]GpTx-1 compared to GpTx-1, despite the location of substituted residues within the binding face (see Table 1). A detailed analysis of interactions between hydrophobic residues from the N- and C-terminus of [AFLR]GpTx-1 and GpTx-1 (Figure 3f–h) revealed that the localized changes observed from 2D Ha shift analysis (Figure 2a) resulted from the disruption of a π-π event between Phe5 and Tyr32 that is present for GpTx-1 (Figure S3). The introduction of a Phe in position 6 of [AFLR]GpTx-1 appeared to recapitulate this π-π stacking (Figure S3).


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Overall hydrophobicity of GpTx-1 and ProTx-II analogues. To examine the effect of peptide hydrophobicity on membrane binding and NaV activity, we compared the overall hydrophobicity of the peptide analogues by recording their retention time (RT) using analytical RP-HPLC (Table 2). GpTx-1 analogue with additional aromatic side chain, [M6F]GpTx-1, and dimeric peptides with a PEG linker ([M6F]GpTx-1)2PEG and ([AFLR]GpTx-1)2PEG, showed increased RT compared to parent GpTx-1. The remaining analogues had very similar RT to GpTx1, consistent with their minor changes in polarity or hydrophobicity of side chains. GpTx-1 eluted earlier than ProTx-II, in agreement with GpTx-1 possessing less bulky aromatic side chains: GpTx-1 has one Trp, two Phe, one Tyr, whereas ProTx-II has four Trp residues. [C-patch_G]ProTx-II is more hydrophobic than the parent peptide, as shown with longer RT and consistent with this peptide having two anionic residues and one nonpolar residue (KKL) replaced with three hydrophobic residues (YVF). Analogues with substitutions that reduced hydrophobicity ([N-patch_G]ProTx-II, [W7Q,W30L]ProTx-II, [W7Q,E17K,W30L]ProTx-II) all had shorter RTs. Increasing the overall charge of analogues by replacing nonpolar residues also resulted

in

a

shorter

RT

([L23R,C-patch_G]ProTx-II,

[T8K,D10N,E17N]ProTx-II,

[T8K,K14N,E17K]ProTx-II). In contrast, substitution of a cationic residue with an anionic residue in the [E17K]ProTx-II analogue did not affect the RT. Peptide-lipid binding models. To determine whether substitutions that change the overall hydrophobicity or charge of GpTx-1 and ProTx-II analogues have an effect on their membranebinding properties, we examined their ability to bind to model lipid bilayers using surface plasmon resonance (SPR) as previously described.14, 17 The set of analogues was first compared for their ability to bind to membranes composed of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mixed with palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-serine (POPS) at a molar ratio


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of 4:1. This lipid mixture was used to represent the PC-rich and mainly fluid membranes of mammalian cells, and although PS-headgroups are restricted to the inner leaflet of healthy mammalian cells, POPS was included here to represent negatively charged moieties, including charged extracellular loops of channel proteins, that are present at the cell surface. We have previously validated this model by showing correlation between peptide binding to POPC/POPS (4:1) bilayers, binding to neuronal cell membranes and to NaV inhibitory activity in vitro.14 We have also demonstrated the involvement of ionic interactions for peptide-binding to POPC/POPS (4:1) for GMTs including GpTx-1, whereby peptide-binding affinity was reduced in low ionic strength buffers (50 mM NaCl) and increased affinity in high ionic strength buffers (300 mM NaCl), compared to physiological salt concentration (150 mM).2 To infer on the contribution of hydrophobic interactions to peptide-lipid binding, we also examined the binding characteristics for a selected set of analogues with neutral bilayers (see Figure S4). POPC bilayers were included to represent the predominant phospholipid in mammalian membranes, a mixture of POPC/1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) (POPC/POPE 4:1) was included to examine peptide-binding to an alternative zwitterionic headgroup, while a mixture of POPC with cholesterol (Chol) and sphingomyelin (SM) (POPC/Chol/SM; 2.7:3.3:4) was included to examine whether the analogues bound differently to model membranes with properties akin to the (more-ordered) raft domains that surround NaV channels.37 Differences between the affinity of peptide-binding to neutral versus anionic bilayers provide additional indication of the involvement of ionic interactions. Binding of GpTx-1 analogues with anionic lipid bilayers. SPR sensorgrams were obtained by injecting peptide over the POPC/POPS (4:1) bilayers and representative sensorgrams (32 µM peptide) and dose-response curves for 8–32 µM peptide are shown in Figure 4. Peptide-


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lipid binding affinity was determined by calculating the amount of peptide bound to the membrane at the end of association phase following injection of 32 µM peptide, and normalized to peptideto-lipid ratio (mol/mol; P/L, Table 2). The rate of dissociation of peptides from the membrane (kd) was also determined, by fitting the dissociation phase considering Langmuir kinetics (Table 2). P/L at 32 µM petide allows quantification and comparison of the relative amount of peptide bound to the model membranes, and kd gives information on how tight the peptide-lipid association is. Overall, GpTx1 and its analogues had low affinity for POPC/POPS (4:1) bilayers, but [M6F]GpTx-1 showed an increased peptide-membrane binding affinity compared to GpTx-1, consistent with its increased overall hydrophobicity (see RT, Table 2). By contrast, the dimeric peptides ([M6F]GpTx-1)2PEG and ([AFLR]GpTx-1)2PEG showed weak membrane-binding, similar to the parent GpTx-1 (Figure 4a–b), which indicates that dimerization, or the presence of the PEG linker, did not affect membrane-binding affinity. In contrast, [M6F]GpTx-1, [AFLR]GpTx-1, ([M6F]GpTx-1)2PEG and ([AFLR]GpTx-1)2PEG showed decreased dissociation rates compared to GpTx-1 (Figure 4a, Table 2). GpTx-1 analogues with charge substitutions, [I10E]GpTx-1 and [R18K]GpTx-1, had similar overall affinity for POPC/POPS (4:1) bilayers, but decreased dissociation rates compared to GpTx-1, as shown by the P/L obtained with 32 µM peptide (Figure 4a–b, Table 2). It is possible that alterations in the ionic interactions with charged moieties at the membrane surface are important for increasing the potency of [AFLR]GpTx-1, [I10E]GpTx-1 and [R18K]GpTx-1 by prolonging peptide binding, which could translate to prolonged NaV inhibition at the cell surface. Binding of GpTx-1 analogues with neutral lipid bilayers. GpTx-1 analogues with increased binding affinity for POPC/POPS (4:1) bilayers were additionally tested for their affinity to POPC, POPC/POPE (4:1) and POPC/Chol/SM (2.7:3.3:4) to examine the relative importance


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of hydrophobic versus ionic interactions. Whereas the amount of peptide bound to neutral bilayers (P/L) was reduced for [M6F]GpTx-1 and ([M6F]GpTx-1)2PEG, when compared to POPC/POPS (4:1) bilayers (Figure S4), the trend in the affinity observed was the same. This suggests that peptide-membrane binding involves hydrophobic interactions; however, increased affinity for negatively charged membranes indicates the involvement of ionic interactions. The membranebinding affinity for POPC/Chol/SM was only slightly reduced compared to the POPC and POPC/POPE (4:1) bilayers, suggesting that differences in membrane fluidity are less important than overall hydrophobic and ionic attractions between peptides and the membrane surface. Binding of ProTx-II analogues with anionic lipid bilayers. ProTx-II displays higher affinity to and a slower dissociation from POPC/POPS (4:1) lipid bilayers than GpTx-1 (Figure 4a–d, Table 2). To examine whether the reduced membrane-binding affinity of GpTx-1 could be transferred onto peptides with a ProTx-II backbone, analogues with N- and C-terminal patch substitutions of GpTx-1 residues; [N-patch_G]ProTx-II, [C-patch_G]ProTx-II, [L23R,Cpatch_G]ProTx-II, [(N_C)-patch_G,L23R]ProTx-II, were tested for their ability to bind POPC/POPS (4:1) bilayers. The lower peptide-binding affinity observed (Figure 4c–d; P/L and kd values Table 2), correlated with the reduced hydrophobicity of these analogues, and confirmed the importance of hydrophobic interactions for peptide-lipid binding. The localized structural changes of N- and C-terminal regions that contribute to the ProTx-II binding face, as observed above (Figure 2c–d), may have also reduced the ability of these analogues to bind to the membrane by altering how hydrophobic residues interact with the membrane surface. [N-patch_G]ProTx-II, [Cpatch_G]ProTx-II, and [(N_C)-patch_G,L23R]ProTx-II maintained the slow membrane dissociation of ProTx-II (Table 2). However, additional inclusion of the LeuàArg substitution in [L23R,C-patch_G]ProTx-II increased the membrane dissociation rate, compared to ProTx-II,


15

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suggesting that replacement of the nonpolar Leu with charged anionic Arg further reduced the ability to bind to membranes. The membrane-binding properties of the NaV1.7-selective analogue [W7Q,W30L]ProTxII31 were also examined by SPR with POPC/POPS (4:1) membranes. The 10-fold reduction in peptide-binding affinity compared to ProTx-II (Figure 4c–d, Table 2) is consistent with studies on [W7Y]ProTx-II and [W30Y]ProTx-II14 showing that these Trp residues are important for membrane binding. Including the E17K substitution for [W7Q,E17K,W30L]ProTx-II provided a small increase in affinity for POPC/POPS (4:1) bilayers; however, both analogues showed a faster dissociation from the lipid bilayer compared to ProTx-II (Table 2). ProTx-II analogues with substitutions that altered the overall charge, or distribution of charged solvent-accessible residues, were also tested for their ability to bind to POPC/POPS (4:1) lipid bilayers. The 1.7-fold increase in POPC/POPS (4:1) binding affinity observed for [E17K]ProTx-II (Figure 4e–f, Table 2) was consistent with our previous observations,14 which could be explained by movement of the charged side chain at ProTx-II position 17 away from the membrane surface, as above (Figure 3b). A further increase in POPC/POPS (4:1) binding affinity was observed for [D10EE,E17K]ProTx-II compared to ProTx-II (2.7-fold) (Figure 4e, Table 2). This increased binding is not explained by increased peptide hydrophobicity (see RT, Table 2); nor by changes in overall charge, as an extra anionic residue would be expected to decrease binding to anionic phospholipid headgroups at the POPC/POPS (4:1) bilayer surface. It is possible that localized structural changes may account for the change in membrane-binding affinity through displacement of charged residues away from the binding face, as was observed for the [E17K]ProTx-II analogue.


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[T8K,D10N,E17N]ProTx-II and [T8K,K14N,E17K]ProTx-II have an increased overall charge and different location of charged residues at the surface of the molecule compared to the parent ProTx-II. Both analogues showed decreased affinity for POPC/POPS (4:1) lipid bilayers and increased rates of dissociation compared to ProTx-II (Figure 4e, Table 2). The ThràLys substitution at ProTx-II position 8 could be responsible for the reduced affinity, possibly through interfering with Trp7 binding, as both analogues contain this mutation. Notably, including the E17K substitution for [T8K,K14N,E17K]ProTx-II did not result in the increased affinity observed for other E17K-containing analogues. Binding of ProTx-II analogues with neutral lipid bilayers. ProTx-II analogues with high membrane-binding affinity and/or high NaV1.7 inhibitory potency, [D10EE,E17K]ProTx-II, [W7Q,W30L]ProTx-II and [W7Q,E17K,W30L]ProTx-II, were also examined for their relative ability

to

bind

neutral

model

membranes

(Figure S4). The binding affinity

of

[D10EE,E17K]ProTx-II for POPC/Chol/SM (2.7:3.3:4) and POPE/POPE (4:1) was similar to that observed with ProTx-II for neutral membranes, suggesting that the increased membrane-binding observed with [D10EE,E17K]ProTx-II for POPC/POPS (4:1) was due to increased ionic interactions. The affinity for all lipid bilayers was low for [W7Q,W30L]ProTx-II, irrespective of phospholipid composition, whereas [W7Q,E17K,W30L]ProTx-II had a slightly higher affinity for POPC/POPS (4:1) than to neutral lipid bilayers, probably due to the increase in overall positive charge due to the E17K mutation. Shallow location of peptides in model membranes. To further investigate the in-depth location and the involvement of hydrophobic Trp residues for interaction of GpTx-1 and ProTx-II analogues with lipid membranes, Trp fluorescence emission of analogues with higher affinity for


17

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membranes (see Figure 4a, 4e), was recorded upon titration with increasing amounts of POPC/POPS (4:1) vesicles as previously described.14 The fluorescence emission spectrum of peptides can inform whether Trp residues are solvent exposed. Comparison of the fluorescence emission spectra of ProTx-II and [D10EE,E17K]ProTx-II in aqueous solution revealed a maximum at 355 nm, similar to that of LTrp amino acid, suggesting that at least one of the four Trp residues was exposed (Figure S5a). In contrast, the blue shift of 7 nm observed for GpTx-1, [M6F]GpTx-1 and [R18K]GpTx-1 indicated that the single Trp residue was not fully exposed (Figure S5a). This reduced surface availability of the single Trp residue of GpTx-1 provides further explanation for the lower membrane-binding affinity of GpTx-1 and analogues compared to ProTx-II. Using Trp fluorescence emission and molecular modeling studies with model membranes, we have previously demonstrated that ProTx-II and ProTx-II[E17K], despite their high affinity for membranes, do not insert deeply into POPC/POPS (4:1) vesicles.14 The results for the analogues in our current study are similar, as none of the ProTx-II or GpTx-1 peptides tested displayed a change in quantum yield or a shift in the maximum fluorescence emission upon addition of POPC/POPS (4:1) vesicles up to 3 mM of lipid, suggesting that the Trp residues do not insert deeply into the membrane (Figure S5b–f). This observation suggests that these GMTs can only access VSD residues at or above the membrane surface. Peptide cytotoxicity toward red blood cells (RBCs). An important consideration when developing therapeutic molecules is their toxicity toward healthy cells. Ideally, cytotoxic concentrations should be >100-fold higher than active concentrations. Therefore, to examine whether the stronger membrane interactions observed for [M6F]GpTx-1, [R18K]GpTx-1, [E17K]ProTx-II and [D10EE,E17K]ProTx-II analogues alter cytotoxicity, the peptides were


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incubated with RBCs (0.25% v/v) at concentrations up to 32 µM, and release of hemoglobin due to cell lysis was monitored. ProTx-II, [E17K]ProTx-II , [D10EE,E17K]ProTx-II and GpTx-1 induced hemolysis at the highest concentration tested (32 µM), but minimal hemolysis was detected for [M6F]GpTx-1 or [R18K]GpTx-1 (Figure S6). By comparison, melittin,38 a peptide known to be highly hemolytic, lysed 100% of RBCs at ~3 µM. Notably, the peptide concentrations required to induce RBC lysis were 1000-fold greater than the reported inhibitory concentrations of GpTx-1 or ProTx-II.11, 39, 40 GpTx-1 and ProTx-II analogue inhibitory potency and selectivity toward NaV channels. To compare membrane-binding affinity with peptide inhibitory activity against NaV, we determined the inhibitory potencies of GpTx-1 and ProTx-II analogues against the therapeutically desirable NaV1.7 subtype and the NaV1.4 (skeletal muscle) subtype. Patch clamp electrophysiology of HEK293 cells stably expressing the alpha subunits of hNaV1.7 and hNaV1.4 was used to determine pIC50 values (see Table 2) from dose response curves. Cells were initially held at -125 mV, then currents were recorded from partially inactivated channels with a voltage that yielded ∼20% channel inactivation (calculated automatically for individual cells), and a test potential of 10 mV. These conditions, previously optimized to compare GpTx-1 inhibition11 and consistent with conditions used to compare GMTs including ProTx-II,21 were selected to allow direct comparison between all GpTx-1 and ProTx-II analogues. Comparison of the NaV1.7 potency and selectivity of the GpTx-1 analogues, from our previous studies,11, 29, 30 alongside hydrophobicity and membrane-binding characteristics (presented above), revealed that the most potent and selective GpTx-1 analogues displayed similar low membrane-binding, and that analogues with increased membrane binding were less potent against


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NaV1.7. Therefore, in contrast to our previous observations with ProTx-II14 and HwTx-IV,17 improving the membrane-binding affinity of GpTx-1 did not improve its NaV inhibitory potency. A similar comparison for ProTx-II analogues revealed that reducing membrane-binding affinity, through substitution of GpTx-1 residues into the N- or C-terminal patch of the ProTx-II binding face for [N-patch_G]ProTx-II, [C-patch_G]ProTx-II, [L23R,C-patch_G]ProTx-II and [(N_C)-patch_G,L23R]ProTx-II, resulted in >100-fold loss of potency against NaV1.7, with only the [C-patch_G]ProTx-II analogue displaying an improved selectivity for NaV1.7 over NaV1.4. Although this is disappointing from the perspective of therapeutic development, it is consistent with our previous reports of the importance of individual Trp residues within the ProTx-II hydrophobic binding face for NaV1.7 potency.14 Together these results show that reducing the membrane binding of ProTx-II analogues by substituting residues within the hydrophobic patch, reduced their inhibitory potency against NaV1.7, but by replacing C-terminal residues KKLW, for example with YVF, the selectivity over NaV1.4 could be improved. Substitutions to ProTx-II solvent-accessible residues that altered the overall charge distribution were better tolerated for [T8K,D10N,E17N]ProTx-II and [T8K,K14N,E17K]ProTxII analogues than the patch-substition analogues above, with only five-fold loss of potency against NaV1.7; however, decreased selectivity over NaV1.4 was also observed. We previously showed the improved binding and moderate increase in NaV1.7 potency for [E17K]ProTx-II;14 however, combining the E17K substitution in ProTx-II with replacement of Asp10 with two Glu residues (improved potency this position for GpTx-1 analogues11) abolished activity against NaV1.7 for the [D10EE,E17K]ProTx-II analogue. The ability of peptides to inhibit NaV in different states (closed/resting, inactivated, open) was not examined in the current study; however, GpTx-1 and ProTx-II parent toxins, and their


20


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high membrane-binding analogues, are probably restricted to accessing VSD residues at or above the membrane surface (see above, Figure S5). Also, the parent toxins share the same NaV1.7 binding site as JzTx-V, which causes state-independent inhibition (between -140 and -80 mV) via interaction with NaV1.7 in a closed state.20

CONCLUSIONS This detailed structure activity relationship study of GpTx-1 and ProTx-II demonstrates that both hydrophobic and ionic interactions are important for activity against NaV channels. Residue substitutions that reduce the hydrophobicity of ProTx-II have the most dramatic effect on affinity for membranes, NaV1.7 potency and selectivity over off-target NaV1.4. We propose that the most important role of hydrophobic interactions between peptide and membrane is to correctly position and orient the peptide relative to exposed residues of NaV VSDs. This proposal is supported by our observations from this study whereby the most potent and selective analogues, [F5A,M6F,T26L,K28R]GpTx-1 and [W7Q,W30L]ProTx-II, displayed low affinity for lipid bilayers; and also from earlier observations that strong membrane binding is not requisite for GMT activity against NaV channels.2, 25 Further to this observation, we compared the structures of strong membrane-binding ProTx-II to low-binding GpTx-1 and [W7Q,W30L]ProTx-II, and showed that whereas localized changes to residues within the (hydrophobic) binding face do not change the overall structure, they do induce changes to the positioning of the hydrophobic patch relative to solvent-accessible charged residues. For future development of GMT-based therapeutics, GMTs with reduced membrane binding may be the preferred candidates for developing potent and selective NaV inhibitors, and candidate GMTs with this criterion include GpTx-1, HwTx-IV, HnTx-IV, PaurTx-III (see ref 2 for


21

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comparison of membrane binding vs NaV potency/selectivity of these GMTs). It is also possible that GMTs with stronger membrane-binding characteristics, for example ProTx-II, CcoTx-I and CcoTx-II, could be modulated to reduce membrane binding as this has been successfully achieved for the potent and selective [W7Q,W30L]ProTx-II.31 Maximizing the potency of interaction between GMT residues and specific exposed NaV loops, while minimizing cross-reactivity with other channels, including other NaV subtypes, as well as CaV and KV channels, are both important for developing GMT-based NaV inhibitors. Thus, examining peptide-membrane binding characteristics is essential when developing new analgesic drugs, as strong peptide-membrane interactions may worsen non-specific binding across multiple channels. We have demonstrated here the challenges associated with rational design to improve NaV subtype-specific inhibitory potency of GMT-based peptides, considering both potency and selectivity, and have shown through structural analysis of two peptide analogues how localized changes of single residues can induce changes that affect more global peptide characteristics. Given the unforeseeable consequences of rationally designed modifications, it is most likely that future improvements to GMTs will need to be achieved through positional scanning of multiple simultaneous residues and/or wider-scale peptide library screening.

METHODS Peptide synthesis and characterization. GpTx-1 analogs were assembled using Fmoc solid-phase peptide synthesis methodologies as described previously.11 The peptides were synthesized on rink amide MBHA resin using an automated peptide synthesizer (Intavis Multipep).


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Parallel peptide cleavage from the resin and purification of the refolded peptides were also performed as previously described.11 ProTx-II and analogues were synthesized on 2-chlorotrityl resin using standard Fmoc solidphase chemistry on an automated Symphony peptide synthesizer (Gyros Protein Technologies Inc) Peptides were folded and purified as previously described.14 Peptide mass and purity was confirmed by RP-HPLC and MS. Overall hydrophobicity was compared by determining retention time (RT) from analytical RP-HPLC with a 2% min-1 gradient of solvent B (90% v/v acetonitrile, 0.1% v/v trifluoroacetic acid against solvent A (0.1% v/v trifluoroacetic acid). Peptides were quantified by absorbance at 280 nm using the extinction coefficient (Table S1). NMR spectroscopy. All peptides were dissolved to a concentration of ~1 mg mL-1 in 90/10% H2O/D2O at pH 3.5–4. NMR spectra (1D, TOCSY and NOESY) were collected on a Bruker Avance III 600 MHz spectrometer equipped with a cryoprobe (Bruker Biospin, Callerica, MA, USA), processed using TopSpin 3.5 (Bruker) and resonances were assigned using CCPNMR Analysis 2.4.1 (CCPN, University of Cambridge, Cambridge, UK). Native disulfide connectivity was inferred from the dispersion of peaks in the 1D NMR spectra. Global structural comparisons were performed by comparing chemical shifts of backbone resonances by analysis of 2D TOCSY and NOESY spectra. Secondary Ha chemical shifts were calculated as the difference between the observed Ha chemical shifts and that of the corresponding residues in a random coil peptide.34 The overall structure of ProTx-II and GpTx-1 (~1 mg mL-1) was also examined in the absence/presence of 100 mM deuterated dodecylphosphocholine (DPC, Cambridge Isotopes) micelles from 283–308 K as previously described.35 A high concentration of DPC was added to ensure saturation of peptide-lipid binding and an elevated temperature was used to minimise line


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broadening induced by DPC. The micelles were expected to be in a fluid phase at and above 298 K. Additional spectra (1H-15N HSQC, 1H-13C HSQC (samples in 90/10% H2O/D2O), E.COSY and 1

H-13C HSQC (samples in 100% D2O)) were aquired for 3D solution structure determination of

[E17K]ProTx-II and [AFLR]GpTx-1. Backbone dihedral angles were derived using TALOS_N41 and temperature coefficients, D2O exchange measurements and side chain dihedral angles were determined42 and structures were calculated as previously described42 using a combination of the AUTO and ANNEAL functions in CYANA43 and further refined in a watershell using CNS.44 Hydrogen bonds derived from temperature coefficient and D2O exchange experiments were included in the later stage of the structure refinements. An ensemble containing 20 structures was chosen based on lowest energy and the best MolProbity statistics (Table S2). Preparation of lipid vesicles. Synthetic POPC, POPS, POPE, POPG, and C18:1 oleoyl SM (Avanti Polar Lipids distributor) and synthetic cholesterol (Chol, Sigma) were used in this study to prepare large unilamellar vesicles (diameter 100 nm) or small unilamellar vesicles (diameter 50 nm) in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) by freeze-thaw and sized by extrusion as previously described.45 Peptide binding to model membranes using SPR. Measurements were conducted using an L1 sensor chip on a BIAcore 3000 instrument (GE Healthcare) at 25 °C as described previously.14 Peptide samples were prepared in HEPES buffer from 8-64 µM. Suspensions of small unilamellar vesicles composed of POPC, POPC/POPS (4:1), POPC/POPE (4:1), or POPC/Chol/SM (2.7:3.3:4) with 0.5 mM lipid concentration were injected onto an L1 chip with a flow rate of 2 µL min-1 for 40 min, to achieve saturation of deposited lipid bilayers. Association of peptides was followed by injecting peptide over the lipid bilayers (flow rate 5 µL min-1, 180 s),


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and peptide-lipid dissociation was followed for 600 s. Response units (RU) were converted into mol/mol (assuming 1 RU = 1 pg (mm2)-1 of lipid and peptide) as before.14 Peptide binding at 32 µM was selected for comparing peptide-lipid binding characteristics across different lipid systems, and peptide maximum binding P/L (mol/mol) was determined for each lipid system at the range of peptide concentrations tested. Tryptophan fluorescence to measure peptide in-depth on lipid vesicles. The intrinsic Trp fluorescence emission of GpTx-1 and ProTx-II analogues with higher affinity for membranes was examined in HEPES buffer and upon titration with large unilamellar vesicles up to 3 mM POPC/POPS (4:1), as described previously.14 GpTx-1, [M6F]GpTx-1 and [R18K]GpTx-1 were tested at 25 µM; ProTx-II, [E17K]ProTx-II and [D10EE, E17K]ProTx-II at 6.25 µM; and L-Trp control at 12.5 µM with identical fluorescence emission intensity. Fluorescence emission spectra were corrected by subtracting the blank; to inspect the existence of a shift in the fluorescence emission, each spectrum was normalized to the maximum fluorescence. RBC hemolysis. GpTx-1, ProTx-II and higher-binding analogues were tested for lysis of human RBCs as previously described.46 Briefly, GpTx-1, [M6F]GpTx-1, [R18K]GpTx-1, ProTxII, [E17K]ProTx-II and [D10EE, E17K]ProTx-II, with concentrations in the range 0.25–32 µM, were incubated with 0.25% (v/v) RBC for 1 h at 37 ºC. Melittin was included as a positive control for lysis and tested in the range 0.25–16 µM. The experiment was conducted in triplicate using blood from human donors. NaV channel inhibition using PatchXpress electrophysiology. Electrophysiology experiments were conducted in HEK293 cells stably expressing the alpha subunits of hNaV1.7 and hNaV1.4 as previously described.11 Cells were voltage clamped using the whole-cell patch clamp configuration (∼22 °C) at a holding potential of −125 mV. Currents were recorded from partially


25

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inactivated channels with a voltage that yielded ∼20% channel inactivation (calculated automatically for individual cells), and a test potential of -10 mV. Peptide dilutions were applied individually. Cells were washed out and the current was allowed to recover before resetting the holding voltage between each individual concentration. pIC50 values were determined from at least n = 10 different cells, with two to three data points per concentration. Dose response curves were prepared using a Hill (4 parameter logistic) fit in DataXpress 2.0 software.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge via the internet at http://pubs.acs.org. This material includes: peptide characteristics of GpTx-1 and ProTx-II analogues; structural statistics for the family of 20 lowest-energy structure of [E17K]ProTx-II and [AFLR]GpTx-1; 1D NMR spectra of GpTx-1 and ProTx-II analogues used to infer overall fold; 1D NMR spectra of ProTx-II and GpTx-1 from 283–308K and in the presence or absence of lipid micelles; 3D structure showing p–p interactions between hydrophobic residues of GpTx-1 and [AFLR]GpTx1; SPR sensorgrams for GpTx-1 and ProTx-II analogue binding to POPC, POPC/POPE (4:1) and POPC/Chol/SM (2.7:3.3:4); Trp fluorescence emission spectra for GpTx-1 and ProTx-II analogues in the presence of POPC/POPS (4:1) vesicles; hemolytic activity of GpTx-1 and ProTx-II analogues.

ACKNOWLEDGEMENTS Funding. This work was supported by an Australian National Health and Medical Research Center (NHMRC) Project Grant to C.I.S. and S.T.H (APP1080405). C.I.S. and S.T.H


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were supported by Australian Research Council (ARC) Future Fellowships (FT160100055 and FT150100398, respectively). D.J.C. is supported by an ARC Australian Laureate Fellowship (FL150100146). C.I.S. is an Institute for Molecular Bioscience (IMB) Industry Fellow, and S.T.H, is an IMB Fellow. Salaries for N.L., O.C. and A.H.B. were supported from NHMRC Project Grant APP1080405. A.J.A. was supported by a University of Queensland International Postgraduate Research Scholarship. Financial support for this research was partly provided by Amgen, including salaries for B.W., J.L, K.B., and L.P.M. The authors thank C. Wang and P. Harvey for valuable discussion related to NMR experiments and J. Murray for editorial assistance with analogue nomenclature.

Conflict of interest. N.L, O.C, A.A, D.J.C, S.T.H and C.I.S declare no conflict of interest with the contents of this article. B.W., J.L., K.B., and L.P.M. declare the following competing financial interest(s): the authors were Amgen full-time employees at the time when this study was performed.


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TABLES Table 1. Amino acid sequence of GpTx-1 and ProTx-II analogues used in this studya Peptide

Sequence

GpTx-1 + potent/selectb hydroph obic chg

[F5A]GpTx-1 [M6F]GpTx-1 [AFLR]GpTx-1 ([M6F]GpTx-1)2PEG ([AFLR]GpTx-1)2PEG [I10E]GpTx-1 [R18K]GpTx-1

ProTx-II + bind/selectc

hydrophobic charge

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

[N-patch_G]ProTx-II [C-patch_G]ProTx-II [L23R,C-patch_G]ProTx-II [(N_C)-patch_G,L23R]ProTx-II [W7Q,W30L]ProTx-II [W7Q,E17K,W30L]ProTx-II [T8K,D10N,E17N]ProTx-II [T8K,K14N,E17K]ProTx-II [D10EE,E17K]ProTx-II [E17K]ProTx-II

10 20 30 DCLGFMRKCIPDNDKCCRPNLVCSRTHKWCKYVF AF EE K L R DCLGAMRKCIPDNDKCCRPNLVCSRTHKWCKYVF DCLGFFRKCIPDNDKCCRPNLVCSRTHKWCKYVF DCLGAFRKCIPDNDKCCRPNLVCSRLHRWCKYVF DCLGFFRKCIPDpDKCCRPNLVCSRTHKWCKYVF DCLGAFRKCIPDpDKCCRPNLVCSRLHRWCKYVF DCLGFMRKCEPDNDKCCRPNLVCSRTHKWCKYVF DCLGFMRKCIPDNDKCCKPNLVCSRTHKWCKYVF 10 20 30 YCQKWMWTCD-SERKCCEG-MVC-R--LWCKKKLW Q K L YCQGAFRTCD-SERKCCEG-MVC-R--LWCKKKLW YCQKWMWTCD-SERKCCEG-MVC-R--LWCKYVF YCQKWMWTCD-SERKCCEG-MVC-R--RWCKYVF YCQGAFRTCD-SERKCCEG-MVC-R--RWCKYVF YCQKWMQTCD-SERKCCEG-MVC-R--LWCKKKLL YCQKWMQTCD-SERKCCKG-MVC-R--LWCKKKLL YCQKWMWKCN-SERKCCNG-MVC-R--LWCKKKLW YCQKWMWKCD-SERNCCKG-MVC-R--LWCKKKLW YCQKWMWTCEESERKCCKG-MVC-R--LWCKKKLW YCQKWMWTCD-SERKCCKG-MVC-R--LWCKKKLW

Cysteines are shown in bold; anionic residues in red; cationic residues in blue; shaded residues contribute to the binding face;14, 29 amino acid mutations in the analogues are underlined; orange residues, GpTx-1 sequence (-patch_G) inserted into ProTx-II backbone; grey residues, change to neutral charge. Analogue abbreviations: [AFLR]GpTx-1 is [F5A,M6F,T26L,K28R]GpTx-1, ([M6F]GpTx-1)2PEG is PEG45-bis[M6F, N13Atz]GpTx-1, ([AFLR]GpTx-1)2PEG is PEG45bis[AFLR, N13Atz]GpTx-1, [N-patch_G]ProTx-II is [K4G,W5A,M6F,W7R]ProTx-II, [Cpatch_G]ProTx-II is [K27Y,K28V,L29F]ProTx-II (1-29), [(N_C)-patch_G,L23R]ProTx-II is [K4G,W5A,M6F,W7R,L23R,K27Y,K28V,L29F]ProTx-II (1-29), [D10EE,E17K]ProTx-II is [D10E,E17K]-Endo-Glu10a-ProTx-II b GpTx-1 mutations previously shown to improve potency or selectivity for NaV1.7: [AFLR]GpTx1;11 PEGylated dimers ([M6F]GpTx-1)2PEG and ([AFLR]GpTx-1)2PEG;30 [I10E]GpTx-1, [P11E]GpTx-1, [R18K]GpTx-129 d ProTx-II mutations previously shown to improve NaV1.7 selectivity: [W7Q, W30L]ProTx-II;31 or membrane binding [E17K]ProTx-II14 a


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Table 2. Comparison of peptide hydrophobicity and overall charge relative to membrane binding, NaV1.7 potency and relative 1.7/1.4 selectivity of GpTx-1 and ProTx-II analoguesa Peptide Chgc

GpTx-1 [F5A]GpTx-1 [M6F]GpTx-1 [AFLR]GpTx-1 ([M6F]GpTx-1)2PEG ([AFLR]GpTx-1)2PEG [I10E]GpTx-1 [R18K]GpTx-1

RTb (min) 22.9 22.9 24.0 23.0 25.5 25.1 23.0 23.0

ProTx-II [N-patch_G]ProTx-II [C-patch_G]ProTx-II [L23R,C-patch_G]ProTx-II [(N_C)-patch_G,L23R]ProTx-II [W7Q/W30L]ProTx-II [W7Q/E17K/W30L]ProTx-II [T8K,D10N,E17N]ProTx-II [T8K,K14N,E17K]ProTx-II [D10EE,E17K]ProTx-II [E17K]ProTx-II

25.7 24.4 26.4 24.9 23.7 24.0 23.9 24.4 24.5 23.9 25.7

+4 +4 +2 +3 +3 +4 +6 +7 +6 +5 +6

+4 +4 +4 +4 +8 +8 +3 +4

Model lipid binding P/Ld kd (mol/mol) (s-1) 0.03 0.11 0.03 0.08 0.06 0.02 0.02 0.03 0.04 0.06 0.04 0.02 0.02 0.03 0.04 0.02

0.1 0.02 0.03 0.02 0.02 0.01 0.03 0.05 0.05 0.27 0.17

0.03 0.03 0.04 0.14 0.03 0.31 0.22 0.06 0.07 0.01 0.02

pIC50 PXe NaV1.7 NaV1.4 8.0 6.7 7.6 5.1 7.7 8.8 5.7 8.0 6.6 7.0 5.7 8.7 5.9 8.8 6.3 pIC50 PXh NaV1.7 NaV1.4 8.5 7.5 5.5

Peptide-Membrane Interactions Affect the Inhibitory Potency and

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