Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7

Abstract

Improper function of voltage-gated sodium channels (Na V s), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na V s hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na V through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na V 1.7 (hNa V 1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na V inhibitors.sodium channel | guanidinium toxin | mutant cycle analysis M odulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na V s) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na V 1.1-Na V 1.9, Na x ) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na V s have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1-4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na V isoforms with nanomolar affinity (5-7). Guanidinium toxins inhibit Na + influx through Na V s by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8-10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11-16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5, 6, 21-23)....

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7

“…To our knowledge, an analogous study has not been reported to date for a Nav channel binding to an ICK peptide. Parsons and Du Bois synthesized maleimide crosslinkers based on smallmolecule Nav pore-blocker saxitoxin; however, identification of the sites of interaction in their subsequent work was indirect, and based on mutagenesis (Parsons and Du Bois, 2013;Thomas-Tran and Du Bois, 2016). Whilst elegant work by Catterall and colleagues determined photocrossinglinking sites for a scorpion toxin using sequence-specific antibodies (Tejedor and Catterall, 1988), the closest comparable example of identification of a photocrosslinking site by mass spectrometry was reported for a simple sevoflurane analog on a voltage-gated potassium channel Kv1.2 (Woll et al, 2017).…”

The evolutionary advantage of toxin production among cyanobacteria, the oldest known organisms on Earth

“…Furthermore, it should be noted that most secondary metabolites produced via NRPS/PKS are modular compounds, and it is currently recognized that variability in one or more substituents leads to different target specificity. For example, the affinity for genetic variants of the same ion channel differs across saxitoxin analogs 31 , whereas the specificity for serine proteases with trypsin- and thrombin-like activity differs among aeruginosins 32 . Thus, the grouping of biosynthetic gene clusters according to the chemical structure of the secondary metabolite likely reflects the bioactivity of the metabolite rather than its specific chemical structure.…”

Synthetic Approaches to Zetekitoxin AB, a Potent Voltage-Gated Sodium Channel Inhibitor

“…They found that the carbamoyl group at C13 in STX (1) interacts with Gly1407 and Thr1409 in domain III, the two guanidines interact with Glu364 in domain I and Glu930 in domain II, and the geminal diol interacts with Asp1701 in domain IV. Interestingly, residues 1409 and 1410, located in the P2 loop of domain III in Na V 1.7, were mutated to Thr and Ile from Met and Asp, respectively, which may explain why STX (1) has a weaker affinity for Na V 1.7 compared with other subtypes (Met1409 and Asp1410 are conserved in the other subtypes of TTX-s) [46,47].…”

“…Synthetic studies of STX (1) and its analogs have been extensive, and several total syntheses have been achieved [52][53][54][55][56][57][58][59][60][61], as recently reviewed by Du Bois [62]. Approaches for developing of subtype-selective modulators based on the STX structure have also been explored [25,47,[63][64][65]. However, in this review, we focus on recent synthetic work related to ZTX (8).…”