Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V

Abstract

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

“…Some of them, mainly isolated from spider venoms, showed promising selectivity for Na V 1.7 channels . Very recently, the Jingzhaotoxin-V analogue AM-8145 was reported to display a more than 100-fold higher potency on Na V 1.7 compared to all Na V channels (Moyer et al, 2018). These positively charged toxins are gating-modifier peptides that bind to the receptor sites 3 (on Domain IV) and/or 4 (on Domain II) of Na V channels, inducing variable pharmacological effects in in vitro tests and in vivo pain models (Saez et al, 2010).…”

“…Interestingly, the in vivo safety margin of this toxin is increased for the mutant peptide JNJ 63955918, due to an improved selectivity against Na V 1.1, 1.2, and 1.6 channels (Flinspach et al, 2017). Very recently, the Jingzhaotoxin-V analogue AM-8145 was reported to display a more than 100-fold higher potency on Na V 1.7 compared to all Na V channels (Moyer et al, 2018).…”

Discovery of a Selective, State-Independent Inhibitor of NaV1.7 by Modification of Guanidinium Toxins

“…(11,12) Similarly, a class of small molecule aryl and acyl sulfonamide compounds bind to the activated conformation of VSD IV ( Figure 1b). (13-16) By contrast, cationic guanidinium toxins and peptide cone snail toxins inhibit ion conduction by sterically occluding the extracellular vestibule of the 25 channel pore (Site 1). The former are a unique collection of natural products exemplified by saxitoxin and tetrodotoxin -high affinity, state independent blockers against six of nine NaV subtypes ( Figure 1c).…”

“…The half maximal inhibitory concentration (IC50) value for saxitoxin (STX) is markedly altered (250-fold change) depending on the presence or absence of the 1398T/1399I variant. Against rNaV1.4, the IC50 of STX is 2.8 ± 0.1 nM compared to hNaV1.7 25 for which the IC50 = 702 ± 53 nM. (31) Introduction of the alternative domain III pore loop sequence by mutagenesis restores potency (hNaV1.7 T1398M/I1399D IC50 = 2.3 ± 0.2 nM).…”

“…While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target 25 sodium channel isoforms, NaV1.1-1.6 and NaV1. 8.…”