Structure-Function and Therapeutic Potential of Spider Venom-Derived Cysteine Knot Peptides Targeting Sodium Channels

Abstract

Spider venom-derived cysteine knot peptides are a mega-diverse class of molecules that exhibit unique pharmacological properties to modulate key membrane protein targets. Voltage-gated sodium channels (Na_V) are often targeted by these peptides to allosterically promote opening or closing of the channel by binding to structural domains outside the channel pore. These effects can result in modified pain responses, muscle paralysis, cardiac arrest, priapism, and numbness. Although such effects are often deleterious, subtype selective spider venom peptides are showing potential to treat a range of neurological disorders, including chronic pain and epilepsy. This review examines the structure-activity relationships of cysteine knot peptides from spider venoms that modulate Na_V and discusses their potential as leads to novel therapies for neurological disorders.

Keywords: ICK peptides; novel drugs; spider venoms; structure–activity relationship; voltage-gated ion channels.

FIGURE 1

Structure of the voltage-gated sodium channel and the inhibitory cysteine knot (ICK) spider peptide. (A) Three-dimensional structure of the human Na_V1.7 in the presence of the auxiliary subunits β1 and β2 determined by cryo-EM (PDB 6J8J) (Shen et al., 2019). Top and side views are presented. The domain I (DI) is colored in gray, domain II (DII) is colored in green, domain III (DIII) is colored in blue and domain IV (DIV) is colored in yellow. The auxiliary subunits β1 and β2 are colored in salmon and orange, respectively. (B) Representative binding site of a NaSpTx 1 in the DII S3-S4 loop of the hNa_V1.7 channel (PDB 6J8J) (Shen et al., 2019). Domains and auxiliary subunits are colored as in (A), and the NaSpTx1 peptide ProTx-III (PDB 2MXM) (Cardoso et al., 2015) is colored in red. (C) Detailed binding site of the NaSpTx 3 ProTx-II over the hNa_V1.7 voltage–sensor domain 2 (VSD2)-Na_VAb chimeric channel (PBD 6N4I) (Xu et al., 2019). Top view of the hNa_V1.7 VSD2-Na_VAb chimeric channel colored in green showing ProTx-II colored in blue bound to the voltage sensor domain 2 in close proximity with the S3–S4 loop. In the structure on the left, the segments S1 to S4 are colored in orange, red, cyan and magenta, respectively. The loops S1–S2 and S3–S4 are colored in black and the residues in the loop 4 and C-terminal of ProTx-II are represented by green sticks. (D) Typical structure of an inhibitory cysteine knot (ICK) peptide from spider. Primary and three-dimensional structure of ProTx-III (PDB 2MXM) (Cardoso et al., 2015) showing cysteine connectivity (C1 – C4, C2 – C5, and C3 – C6) and loops 1–4 colored in red, green, blue and orange, respectively.

FIGURE 2

Three-dimensional structure of spider ICK peptides displaying key residues involved in the inhibition of Na_V channels and cell membrane binding. (A) Three-dimensional structure of HwTx-IV determined by NMR (PBD 2m4x) (Minassian et al., 2013), (B) HNTX-III determined by NMR (PBD 2jtb), (C) HNTX-IV determined by NMR (PBD 1niy) (Li et al., 2004), (D) ProTx-II determined by X-Ray (PBD 5o0u) (Wright et al., 2017), and (E) JZTX-III determined by NMR (PBD 2i1t). The labeled amino acids residues have key role in potency over Na_V channels and lead to loss in activity as described in the text and Table 1. (F) Structure of ProTx-II determined by X-Ray (PDB 5o0u) (Wright et al., 2017) and (G) Structure of HwTx-IV (PDB 2m4x) (Minassian et al., 2013) determined by NMR. The labeled amino acids residues have key role in cell membrane binding as described in the text and Table 1. Amino acids residues are colored as follow: yellow for hydrophobic, red for acid, blue for basic and green for neutral. All three-dimensional structures were prepared in PyMOL (DeLano, 2002).

FIGURE 3

Three-dimensional structure of spider ICK peptides displaying key residues involved in the enhancement of activity for Na_V channels, or enhancement of selectivity for Na_V channels off-targets. (A) Structures of HwTx-IV (PDB 2m4x) (Minassian et al., 2013) and gHwTx-IV (PDB 5tlr) (Agwa et al., 2017) determined by NMR. (B) Structures of CcoTx1 determined by NMR (PDB 6br0) (Agwa et al., 2018) and the derived analog 2670 determined by X-ray (PDB 5epm) (Shcherbatko et al., 2016). (C) Structures of HNTX-I (PDB 2mqf) and the derived analog G7W/N24S (PDB 2mxo) determined by NMR (Klint et al., 2015a). (D) Structures of ProTx-II determined by X-Ray (PDB 5o0u) (Wright et al., 2017) and derived analog JNJ63955918 determined by NMR (PDB 5tcz) (Flinspach et al., 2017). (E) Structure of analogs Pra-1 JZTX-V (PDB 6chc) and AM-8145 (6cgw) determined by NMR (Moyer et al., 2018). (F) Structure of JZTX-III determined by NMR (PDB 2i1t) (Liao et al., 2006) and modeling of the derived analog R13A using SWISS-MODEL (Arnold et al., 2006). Amino acids residues are colored as follow: yellow for hydrophobic, red for acid, blue for basic and green for neutral. Amino acids substitutions or additions labeled in the respective analogs are involved in the improvement of Na_V inhibitory activity or selectivity as described in the text and Table 1. All three-dimensional structures alignments were prepared in PyMOL (DeLano, 2002).

FIGURE 4

SAR integration of spider ICK peptides. Primary and three-dimensional structures comparison of spider ICK peptides belonging to NaSpTx families 1, 3, and 7. (A) Primary structure alignment of wild-type peptides and respective optimized analogs showing enhancement of activity and/or selectivity of Na_V channels. The amino acids residues are highlighted as follow: yellow for hydrophobic, red for acid, blue for basic, green for neutral, and gray for the cysteines. (B) Alignment of the three-dimensional structures of HwTx-IV (PDB 2m4x) (Minassian et al., 2013) colored in gray and gHwTx-IV (PDB 5tlr) (Agwa et al., 2017) colored in cyan. Structures are represented by cartoon and surface, and substitutions E1G, E4G and Y32W are represented by sticks colored in green, red, and blue, respectively. (C) Alignment of the three-dimensional structures of Pra-1 JZTX-V (PDB 6chc) colored in gray and AM-8145 (6cgw) colored in cyan (Moyer et al., 2018). Structures are represented by cartoon and surface, and the Pra-1 addition and substitution I28E are represented by sticks colored in green and red, respectively. (D) Alignment of the three-dimensional structures of HNTX-I (PDB 2mqf) colored in gray and the derived analog G7W/N24S (PDB 2mxo) colored in cyan (Klint et al., 2015a). Structures are represented by cartoon and surface, and substitutions G7W and N24S are represented by sticks colored in green and red, respectively. All three-dimensional structures alignments were performed in PyMOL (DeLano, 2002). Asterisk (^∗) denotes C-terminal amidation.

FIGURE 5

Primary amino acids sequence alignment and phylogenetic analysis of NaSpTx peptides with described Na_V1.7 modulatory activity. (A) Primary sequences alignment of members of NaSpTx1, 2, 3, and 7, with EC₅₀ values represented in nM, Loops, 1, 2, 3, 4, and C-terminal shaded in yellow, green, pink, (blue and gray, respectively, and cysteines colored in red. (B) Phylogenetic analysis of the full primary sequences of NaSpTx NaSpTx1, 2, 3, and 7, followed by Loop 1 (C), Loop 2 (D), Loop 3 (E), Loop 4 (F), and C-terminals (G). For the phylogenetic trees, the peptides names are followed by their respective NaSpTx families represented by F1, F2, F3, and F7, and their Na_V1.7 potency represented by I (EC₅₀ > 1 μM), II (EC₅₀ between 1 and 0.1 μM), and III (EC₅₀ < 0.1 μM). These analyses were performed using Clustal Omega (Sievers et al., 2011) and Simple Phylogeny (Saitou and Nei, 1987). The loops were flanked by cysteine for these analyses.)