Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S–Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 μm, and Nav1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic. Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S–Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 μm, and Nav1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic. According to a Centers for Disease Control and Prevention analysis of the 2016 National Health Interview Survey an estimated 50 million people in the United States experience chronic pain, 19.6 million of which experience pain severe enough to limit their daily activities (1Dahlhamer J. Lucas J. Zelaya C. Nahin R. Mackey S. De Bar L. Kerns R. Von Korff M. Porter L. Helmick C. Prevalence of Chronic Pain and High-Impact Chronic Pain Among Adults—United States, 2016.MMWR Morb. Mortal Wkly. Rep. 2018; 67 (30212442): 1001-100610.15585/mmwr.mm6736a2Crossref PubMed Scopus (962) Google Scholar). Individuals experiencing such severe pain are more likely to have a poor health state and report exhaustion, depression, and anxiety. In addition, these persons have a greater likelihood of needing bed-disability days, accessing medical care, and visiting an emergency room compared with persons experiencing lower pain states (2Nahin R.L. 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Nav1.7 and other voltage-gated sodium channels as drug targets for pain relief.Expert Opin. Ther. Targets. 2016; 20 (26941184): 975-98310.1517/14728222.2016.1162295Crossref PubMed Scopus (129) Google Scholar). Strategies focused on engineering large molecules have emerged as an alternative path for achieving selective Nav1.7 block (18Lee J.H. Park C.K. Chen G. Han Q. Xie R.G. Liu T. Ji R.R. Lee S.Y. A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief.Cell. 2014; 157 (24856969): 1393-140410.1016/j.cell.2014.03.064Abstract Full Text PDF PubMed Scopus (178) Google Scholar). A rich source of good starting points for drug discovery is the venom of animals (19Robinson S.D. Undheim E.A.B. Ueberheide B. King G.F. Venom peptides as therapeutics: advances, challenges and the future of venom-peptide discovery.Expert Rev. Proteomics. 2017; 14 (28879805): 931-93910.1080/14789450.2017.1377613Crossref PubMed Scopus (62) Google Scholar). 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Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena.J. Biol. Chem. 2002; 277 (12228241): 47564-4757110.1074/jbc.M204063200Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We previously identified key structural and functional features of HwTX-IV critical for its activity for both Nav1.7 and Nav1.2 (23Minassian N.A. Gibbs A. Shih A.Y. Liu Y. Neff R.A. Sutton S.W. Mirzadegan T. Connor J. Fellows R. Husovsky M. Nelson S. Hunter M.J. Flinspach M. Wickenden A.D. Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (mu-TRTX-Hh2a).J. Biol. Chem. 2013; 288 (23760503): 22707-2272010.1074/jbc.M113.461392Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Eckert, W. F. 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Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7.Biochim. Biophys. Acta. 2017; 1859 (28115115): 835-84410.1016/j.bbamem.2017.01.020Crossref PubMed Scopus (36) Google Scholar, 27Rahnama S. Deuis J.R. Cardoso F.C. Ramanujam V. Lewis R.J. Rash L.D. King G.F. Vetter I. Mobli M. The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.PLoS One. 2017; 12 (28301520): e017355110.1371/journal.pone.0173551Crossref PubMed Scopus (30) Google Scholar, 28Revell J.D. Lund P.E. Linley J.E. Metcalfe J. Burmeister N. Sridharan S. Jones C. Jermutus L. Bednarek M.A. Potency optimization of huwentoxin-IV on hNav1.7: a neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena.Peptides. 2013; 44 (23523779): 40-4610.1016/j.peptides.2013.03.011Crossref PubMed Scopus (69) Google Scholar). Here, we present our comprehensive engineering efforts focused on the design of potent and Nav1.7-selective variants of HwTX-IV, as well as functional studies describing the activities of these libraries on Nav1.7. In addition, we present the activities of these peptides on Nav1.2 to highlight their selectivity for Nav1.7 over one of the most abundant sodium channel isoforms in the central nervous system (29Westenbroek R.E. Merrick D.K. Catterall W.A. Differential subcellular localization of the RI and RII Na+ channel subtypes in central neurons.Neuron. 1989; 3 (2561976): 695-70410.1016/0896-6273(89)90238-9Abstract Full Text PDF PubMed Scopus (379) Google Scholar). We also describe the broader selectivity of some of the peptides with the most favorable modifications by measuring peptide activity against other sodium channel members. These data are a road map for further optimization of HwTX-IV and ultimately may provide a basis for the discovery of a novel, peptide-based nonopiate analgesic. As described previously, we produced recombinant huwentoxin (rHwTx-IV) in a mammalian expression system (23Minassian N.A. Gibbs A. Shih A.Y. Liu Y. Neff R.A. Sutton S.W. Mirzadegan T. Connor J. Fellows R. Husovsky M. Nelson S. Hunter M.J. Flinspach M. Wickenden A.D. Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (mu-TRTX-Hh2a).J. Biol. Chem. 2013; 288 (23760503): 22707-2272010.1074/jbc.M113.461392Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This peptide differs from native HwTx-IV in three capacities. 1) Because this peptide was produced as a fusion protein to optimize expression levels, it retains a glycine and proline on its N terminus (remains of an HRV3C cleavage site). 2) This peptide contains two additional residues—a Gly at position 36 and a Lys at position 37 on its C terminus. 3) The C terminus is not amidated (Fig. 1A). This peptide was screened for activity against Nav1.7 and Nav1.2 in a FRET-based FLIPR assay. In this assay, the mean pIC50 values for rHwTx-IV on Nav1.7 and Nav1.2 were 7.1 ± 0.07 and 6.6 ± 0.12, respectively. As reported previously, these values are right-shifted relative to potencies observed when testing synthetically produced, amidated huwentoxin (Peptides International) in this assay (Nav1.7, 7.4 ± 0.04, and Nav1.2, 7.4 ± 0.03, Fig. 1, B and C) (23Minassian N.A. Gibbs A. Shih A.Y. Liu Y. Neff R.A. Sutton S.W. Mirzadegan T. Connor J. Fellows R. Husovsky M. Nelson S. Hunter M.J. Flinspach M. Wickenden A.D. Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (mu-TRTX-Hh2a).J. Biol. Chem. 2013; 288 (23760503): 22707-2272010.1074/jbc.M113.461392Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To define the impact of alterations in HwTx-IV sequence on its ability to impair sodium channel gating, we systematically substituted amino acids (except cysteine and methionine) at noncysteine positions and tested them for activity in FLIPR on hNav1.7 and hNav1.2. Mean Nav1.7 and Nav1.2 pIC50 values for 373 mutant peptides are presented in Figs. S1 and S2, respectively. To better understand general trends in HwTX-IV's SAR, we first interrogated the dataset to determine the mean impact of all mutations in each of HwTX-IVs loops (as well as the N and C termini) on Nav1.7 and Nav1.2 activity. Except for the N-terminal mutations in Nav1.2, changes in HwTX-IV decreased activity on both Nav1.2 and Nav.17 with the most robust effects resulting from mutations in HwTX-IV's loop 4 and the C terminus (Fig. 2A). Next, we looked at the mean impact of the substitution of amino acids with similar general properties in each HwTX-IV segment (Fig. 2, B–F). This property-focused loop analysis showed differential effects on HwTX-IV's Nav1.7 and Nav1.2 activity that were not detected by the original loop analysis. Of note, introduction of basic and nonpolar amino acids on HwTX-IV's N terminus (position Glu-1) significantly increased potency on Nav1.2 (Fig. 2B). In loop 2, substitution of acidic and basic functionalities significantly diminished Nav1.7 activity (Fig. 2C). Introduction of acidic amino acids in loop 3 significantly decreased both Nav1.7 and Nav1.2 potency (Fig. 2D). Loop 4 was the least-tolerant segment, with acidic and uncharged polar substitutions decreasing both Nav1.7 and Nav1.2 potency and nonpolar insertions reducing Nav1.7 potency (Fig. 2F). In the C terminus, acidic functionalities selectively decreased HwTX-IV Nav1.7 potency, and amino acids with uncharged polar properties decreased potency on both isoforms (Fig. 2F). No significant changes in activity were seen with the property-based analysis in loop 1 (data not shown). To further explore HwTX-IV's SAR, we also examined the mean change in pIC50 caused by all mutations at each HwTX-IV amino acid position for both Nav1.7 and Nav1.2. In this analysis, no significant increases in mean potency for either Nav isoform were observed at any position. Mutations at positions 5, 6, 11, 14, 25, 27, 28, 30, 32, 33, and 35 significantly decreased Nav1.7 potency. Likewise, mutation of positions 6, 14, 26, 27, 30, and 32, 33, and 35 significantly decreased Nav1.2 potency (Fig. 3, A and B). Both loop and position-focused analyses of the differential impact of mutations on Nav1.7 and Nav1.2 activity were also performed using a linear regression where the location of amino acid replacement was used as a predictor variable for estimating the change in Nav1.2 pIC50 values. The corresponding change in Nav1.7 pIC50 values that result from the replacement was also included as a predictor variable in the model. This allowed us to identify when the changes in Nav1.2 pIC50 values were greater than expected given the observed change in Nav1.7 pIC50 values that resulted from the amino acid replacement. When applied to HwTX-IV's loops, the above analysis showed that alterations in the N terminus and loop 1 shifted huwentoxin's activity preference toward Nav1.2 (Fig. 4A). These trends are also reflected in the position-based analysis as seen in of all noncysteine positions through Ser-12, which show significant shifts of huwentoxin's activity toward Nav1.2. The remaining positions show either nominal impact or increased selectivity for Nav1.7. The most robust of these was Arg-26, which improved selectivity for Nav1.7 by more than 3-fold (Fig. 4B). Examination of the effect of individual mutations, however, revealed unique behaviors at each position that were not predicted by the more general analyses. These data allowed us to narrow our focus to a smaller subset of mutants that contain an attractive combination of two specific properties: potency toward Nav1.7 and selectivity against other Nav1.2. Specifically, we identified 17 point mutations that produced 3-fold or better improvements on HwTX's Nav1.7 potency compared with recombinant HwTx-IV (Table 1). In addition, we found multiple point mutations that caused differential effects on Nav1.7 and Nav1.2 activity with 11 mutant peptides exhibiting 10-fold or greater selectivity for Nav1.7 over Nav1.2 (Table 2).Table 1Individual mutations that improve HwTX-IV potency versus Nav1.7 and selectivity versus Nav1.2Peptide1.7 pIC50nrHwTX-IV7.0 ± 0.146E1N7.6 ± 0.14E4R7.9 ± 0.063E4N7.6 ± 0.031E4Q7.5 ± 0.101A8R7.7 ± 0.021S19N7.7 ± 0.042S19P7.5 ± 0.092S19Q7.4 ± 0.221S20N7.5 ± 0.052K21R7.9 ± 0.132S25I7.7 ± 0.072Y33W7.7 ± 0.033Q34S7.5 ± 0.072Q34F7.7 ± 0.182Q34L7.5 ± 0.042K37R7.7 ± 0.031 Open table in a new tab Table 2Individual mutations that improve HwTX-IV potency versus Nav1.7 and selectivity versus Nav1.2Peptide1.7 pIC50n1.2 pIC50nSelectivityN13G6.8 ± 0.0715.8 ± 0.4311.0D14P6.6 ± 0.0315.5 ± 0.5121.1K18F6.5 ± 0.0325.0 ± 2.621.5S19Q7.6 ± 0.2116.4 ± 1.111.2R26K6.8 ± 0.2215.8 ± 0.1311.1R26G6.6 ± 0.062>5.42>1.2K27W7.2 ± 0.0615.8 ± 0.3121.4W30K6.9 ± 0.121>5.521.4W30Y5.7 ± 0.082>4.51>1.2Y33T7.2 ± 0.0716.2 ± 0.0511.0G36I7.0 ± 0.0725.4 ± 0.521.4 Open table in a new tab Although the FRET-based FLIPR assay is a powerful screening tool, it measures changes in membrane potential, an event initiated by sodium channel gating but easily confounded by the subsequent activation of other voltage-sensitive conductances. In addition, a lack of voltage control in this assay limits its ability to accurately determine the potency of voltage-dependent compounds. Therefore, we verified the activity of recombinant HwTX-IV and six peptides with key individual mutations on sodium channel gating using the QPatch HT automated patch-clamp system. For both Nav1.7 and Nav1.2, we saw good agreement between FLIPR and QPatch data for most peptides with a linear fit yielding a R2 = 0.80, m = 1.25, and b = −1.97 for Nav1.7 and R2 = 0.84, m = 0.52, and b = 3.79 for Nav1.2 (Fig. S3) with the discrepancies largely accounted for by assay variability. FLIPR did, however, underestimate the potency of peptides with a lower Nav1.2 potency, underscoring the need for verification of potency in QPatch for compound selectivity. The full amino acid scan uncovered a wide range of position-dependent activity relationships throughout the parent peptide. Thus, we introduced multiple individual mutations with favorable effects to look for additive or synergistic effects on Nav1.7 potency and/or selectivity over Nav1.2. To this end, we produced a small library of HwTX-IV–based peptides that contained varied combinations of E1N, E4D, Q34S, R26K, Y33W, and G36I. These peptides were tested in FLIPR against Nav1.7 and Nav1.2 (see Table S1 for full table of FLIPR activities), and we identified a peptide with improved Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08, and Nav1.2 pIC50 = 7.8 ± 0.09) and a peptide that showed selectivity for Nav1.7 over Nav1.2 (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18). The activity of these peptides was verified in QPatch with good agreement (E1N, E4D, Y33W, Q34S, Nav1.7 pIC50 = 8.3 ± 0.26, and Nav1.2 pIC50 = 7.8 ± 0.06, E1N, R26K, Q34S, G36I, and Nav1.7 pIC50 = 7.4 ± 0.10, Nav1.2 pIC50 = 5.9 ± 0.14, Fig. 5, A and B, respectively). To further characterize the selectivity profiles of these peptides, we tested them in QPatch against Nav1.1, 1.3, 1.4, 1.5, and 1.6. Compared with its Nav1.7 activity, E1N, E4D, Y33W, and Q34S remained active on Nav1.1 (pIC50 = 8.1 ± 0.10), 1.3 (pIC50 = 8.2 ± 0.14), and 1.6 (pIC50 = 7.5 ± 0.08) but showed diminished activity on Nav1.4 (pIC50 = 6.0 ± 0.12) and Nav1.5 (62% inhibition at 10 μm). In contrast, relative to its Nav1.7 activity, E1N, R26K, Q34S, G36I only showed strong Nav1.1 (pIC50 = 7.3 ± 0.15) and 1.6 activity (pIC50 = 8.1 ± 0.65) and reduced or no Nav1.3 (pIC50 = 6.4 ± 1.0), 1.4 (inactive at 3 μm), or 1.5 (inactive at 10 μm) activities (Fig. 6A). These mutations also translated to improvements in selectivity for Nav1.7 over some Nav isoforms compared with WT HwTX-IV. For E1N, E4D, Y33W, and Q34S, the most substantial gains were over Nav1.2 (increased from 0.4- to ∼2-fold) and Nav1.6 (equipotent to 4-fold) with selectivity over other isoforms largely unchanged. E1N, R26K, Q34S, and G36I also showed the most robust improvement in selectivity for Nav1.7 over Nav1.2 (increased from 0.4- to 30-fold) but also showed meaningful improvements in selectivity over Nav1.3 (increased from equipotent 10-fold) and Nav1.6 (increased from equipotent to 3-fold) (Fig. 6B). To further improve potency and selectivity of the two best-combinatorial peptides, we grafted additional point mutations into the two scaffolds. These peptides were tested in QPatch against Nav1.7. Most of the substitutions we introduced, however, significantly decreased Nav17 potency (Table S2). Significant improvements in the E1N, E4D, Y33W, and Q34S's Nav1.7 potency were observed with the following mutations: K37Q, R26T, K37S, R26W, R29N, and R29H. In contrast, no changes to E1N, R26K, Q34S, and G36I resulted in significant improvements in potency. Select peptides were subsequently tested against Nav1.2. This identified R26T as a favorable addition to E1N, E4D, Y33W, and Q34S (QPatch: Nav1.7 pIC50 = 8.9 ± 0.11, and Nav1.2 pIC50 = 7.7 ± 0.11, see Fig. 7). HwTx-IV was docked onto a Nav1.7 homology model generated using a composite method with a cryo-EM structure of Nav1.7 (PDB 6J8G) serving as the template for most of the structure except for the domain II voltage sensor (VSD2), which used as a template the deactivated voltage sensor (PDB 6N4R). Once the WT Nav1.7 model was generated, hwentoxin-IV (PDB 1MB6) was manually docked to the Nav1.7 homology model using the protoxin-II–binding site from the Nav1.7 VSD2–NavAb channel chimera protein structure in complex with protoxin-II (PDB 6N4I) as a guide. HwTx-IV was initially structurally aligned to the protoxin-II and then manually adjusted to generate a docking pose. The entire system was further minimized and refined using molecular dynamics. The final docked and refined model of HwTX-IV to Nav1.7 shows key interactions involving residues that impact HwTX-IV potency and/or selectivity over Nav1.2. Lys-27 forms a salt bridge with Glu-818 (with proximity to Asp-816), and Lys-32 forms a salt bridge with Glu-811 (Fig. 8A); this area is key to VSD2-gating movement. Trp-30 sits in a hydrophobic pocket formed by Ala-766, Ile-767, Leu-770, and Leu-812 (Fig. 8B). The indole nitrogen from the Trp-30 also forms a hydrogen-bonding interaction with the backbone carbonyl of Leu-812. Finally, Arg-26 makes a salt-bridge interaction with Glu-760, whereas Arg-29 primarily makes an interaction with the polar headgroups of the lipid membrane, although within the molecular dynamic's simulations, 30% of the time Arg-29 rotated and also made a salt-bridge interaction with Glu-760 (Fig. 8C). We also selected positions 6, 19, 20, 21, 27, 30, and 32 in which to substitute noncoded amino acid analogs into E1N, K21F, R26K, Q34S, and G36I. (Nav1.7 pIC50 = 7.2 ± 0.2 and Nav1.2 pIC50 = 6.1 ± 0.1). Selection was based upon apparent participation in a Nav channel epitope, phospholipid interaction, and/or their location in regions of potential metabolic liability (i.e. unstructured secondary structure). It should be noted that t