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.
Highlights
Toxin peptides derived from venoms have provided key insights into ion channel pharmacology, structure and function in normal and pathophysiological disease states [1]
We report on the in vitro and ex vivo pharmacological characterization of these engineered peptides, including specific block of rodent action potential firing in dorsal root ganglia (DRG) neurons and C-fibers following capsaicin and mechanical stimulation compared to an inactive peptide AM-8374 devoid of these biological activities
Our previous experience with GpTx-1 had shown spider venoms from the Theraphosidae family were enriched in NaV inhibitory peptides [24]
Summary
Toxin peptides derived from venoms have provided key insights into ion channel pharmacology, structure and function in normal and pathophysiological disease states [1]. Voltage-gated sodium channels (NaV), essential for the initiation and propagation of action potentials in excitable cells, represent a rich target family for toxin peptides that both inhibit and activate NaV function [2,3,4,5]. Spider venoms are especially enriched in NaV modulators with one-third of spider toxin peptides targeting Nav channels pharmacologically [6]. NaV1.7 represents a compelling target for the development of both small and large molecule chronic pain clinical candidates due to its exquisite human genetic validation [9,10,11]. The human congenital insensitivity to pain phenotype is mimicked in NaV1.7 knockout mice with cell-type specific knockouts highlighting the involvement of NaV1.7 in both inflammatory and neuropathic pain endpoints [17,18,19]. Known NaV1.7 inhibitory peptides map to voltage-sensor domains and can reduce sensitivity of channel opening to depolarizing voltage stimuli [3]
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