ProTx III

Peptide toxins that adopt the inhibitory cystine knot (ICK) scaffold have very stable three-dimensional structures as a result of the conformational constraints imposed by the configuration of the three disulfide bonds that are the hallmark of this fold. Understanding the oxidative folding pathways of these complex peptides, many of which are important therapeutic leads, is important in order to devise reliable synthetic routes to correctly folded, biologically active peptides. Previous research on the ICK peptide ProTx-II has shown that in the absence of an equilibrating redox buffer, misfolded intermediates form that prevent the formation of the native disulfide bond configuration. In this paper, we used tandem mass spectrometry to examine these misfolded peptides, and identified two non-native singly bridged peptides, one with a Cys(III)-Cys(IV) linkage and one with a Cys(V)-Cys(VI) linkage. Based on these results, we propose that theC-terminus of ProTx-II has an important role in initiating the folding of this peptide. To test this hypothesis, we have also studied the folding pathways of analogs of ProTx-II containing the disulfide-bond directing group penicillamine (Pen) under the same conditions. We find that placing Pen residues at theC-terminus of the ProTx-II analogs directs the folding pathway away from the singly bridged misfolded intermediates that represent a kinetic trap for the native sequence, and allows a fully oxidized final product to be formed with three disulfide bridges. However, multiple two-disulfide peptides were also produced, indicating that further study is required to fully control the folding pathways of this modified scaffold.

Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, β-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether β-subunits also influence ligand interactions, we found that β4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular β4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of β4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of β4. Disrupting this bridge by introducing a β1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts β4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of β-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant β-subunit behavior.

Background and purpose:Naturally occurring dysfunction of voltage-gated sodium (NaV) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaVmodulators, we investigated spider venoms for new inhibitors of NaVchannels.Experimental approach:We used high-throughput screens to identify a NaVmodulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model.Key results:We identified a potent NaVinhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (CaV3) currents but had no activity against the voltage-gated potassium (KV2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaVchannel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaVsubtypes tested (hNaV1.1-1.7). Df1a also slowed fast inactivation of NaV1.1, NaV1.3 and NaV1.5 and modified the voltage-dependence of activation and inactivation of most of the NaVsubtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the NaVactivator OD1.Conclusion and implications:μ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.