Yoko Yamaguchi

Synthesis and characterization of amino acid deletion analogs of κ-hefutoxin 1, a scorpion toxin on potassium channels

Nine analogs of scorpion toxin peptide κ-hefutoxin 1 were synthesized by stepwise deletion of its amino acid residues. Disulfide bond pairings of the synthetic analogs were confirmed by enzymatic digestion followed by MALDI-TOF-MS measurements. Functional characterization shows that analogs in which N-terminal residues were deleted retained biological activity, whereas deletion of middle part residues resulted in loss of activity. Furthermore, κ-hefutoxin 1 and analogs were subjected to a screening on voltage-gated potassium channels in order to determine their subtype selectivity. It is shown that κ-hefutoxin 1 is suitable as template for peptidomimetics in order to design small peptide-based therapeutic compounds.

Expanding the pharmacological profile of κ-hefutoxin 1 and analogues: A focus on the inhibitory effect on the oncogenic channel Kv10.1.

HIGHLIGHTS&kgr;‐Hefutoxin 1 is a novel inhibitor of the oncogenic potassium channel Kv10.1.This scorpion toxin is the first peptide toxin described that is able to inhibit Kv10.1.The structure‐activity relationship was investigated by testing eight peptide variants.Several important residues were identified, including the functional dyad residues. ABSTRACT Peptide toxins, such as scorpion peptides, are interesting lead compounds in the search for novel drugs. In this paper, the focus is on the scorpion peptide &kgr;‐hefutoxin 1. This peptide displays a cysteine‐stabilized helix‐loop‐helix fold (CS&agr;/&agr;) and is known to be a weak Kv1.x inhibitor. Due to the low affinity of &kgr;‐hefutoxin 1 for these channels, it is assumed that the main target(s) of &kgr;‐hefutoxin 1 remain(s) unknown. In order to identify novel targets, electrophysiological measurements and antifungal assays were performed. The effect of &kgr;‐hefutoxin 1 was previously evaluated on a panel of 11 different voltage‐gated potassium channels. Here, we extended this target screening with the oncogenic potassium channel Kv10.1. &kgr;‐Hefutoxin 1 was able to inhibit this channel in a dose‐dependent manner (IC50 ˜ 26 &mgr;M). Although the affinity is rather low, this is the first peptide toxin ever described to be a Kv10.1 inhibitor. The structure‐activity relationship of &kgr;‐hefutoxin 1 on Kv10.1 was investigated by testing eight &kgr;‐hefutoxin 1 variants using the two‐electrode voltage clamp technique. Several important amino acid residues were identified; the functional dyad residues (Tyr5 and Lys19), N‐terminal residues (Gly1 and His2) and the amidated C‐terminal residue (Cys22). Since the CS&agr;/&agr; fold is also found in a class of antifungal plant peptides, the &agr;‐hairpinines, we investigated the antifungal activity of &kgr;‐hefutoxin 1. &kgr;‐Hefutoxin 1 showed low activity against the plant pathogen Fusarium culmorum and no activity against three other yeast and fungal species, even at high concentrations (˜100 &mgr;M).

Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning

Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

Roles of Basic Amino Acid Residues in the Activity of μ‐Conotoxin GIIIA and GIIIB, Peptide Blockers of Muscle Sodium Channels

To study in detail the roles of basic amino acid residues in the activity of μ‐conotoxin GIIIA (μ‐GIIIA) and GIIIB (μ‐GIIIB), specific blockers of muscle sodium channels, seven analogs of μ‐GIIIA, and two analogs of μ‐GIIIB were synthesized. μ‐GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ‐GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ‐GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ‐GIIIB showed higher activity than native μ‐GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ‐GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.

Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom

Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer, is a K+ channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/β scaffold that is cross-linked by four disulfide bridges (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7, and Cys4-Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4-Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1-Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3-Cys4 and Cys7-Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1-Cys5 and Cys2-Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.

Effects of deletion and insertion of amino acids on the activity of HelaTx1, a scorpion toxin on potassium channels

Four analogs of HelaTx1, a 25-mer peptide from scorpion venom, were synthesized by deleting its C-terminal hexapeptide fragment and N-terminal Ser residue and by inserting an amino acid in the middle part of the molecule. CD spectrum of HelaTx1(1-19) was almost superimposable to that of native HelaTx1. Functional characterization showed that HelaTx1(1-19) retained its inhibitory activity on Kv1.1 channel although 3 times less potent than HelaTx1, indicating that C-terminal part of HelaTx1 was not essential for its conformation and activity. Further deletion of N-terminal Ser residue and insertion of Ala in the middle part of the molecule affected the CD spectra and resulted in the decrease of activity.