Abstract
Human voltage-gated sodium channel Nav1.7 (hNav1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav1.7. Site-directed mutagenesis analysis indicated key residues on hNav1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 μM) and >1000-fold selectivity against Nav1.4 and Nav1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia.
Highlights
Voltage-gated sodium channels (VGSCs or Navs) are essential for the initiation and propagation of action potentials in excitable tissues such as nerve, muscle, and other excitable cells [1, 2]
HNav1.7 has emerged as a validated pain target based on clinical genetic studies, as gain-of-function mutations in the SCN9A gene encoding hNav1.7 result in disorders of spontaneous pain and itch, including erythromelalgia, paroxysmal extreme pain disorder, and small fiber neuropathy [8,9,10,11], and loss-of-function mutations of hNav1.7 produce complete insensitivity to pain [12]
There exist challenges in developing subtype-selective voltagegated sodium channel (VGSC) inhibitors since VGSCs share the same domain structure with a high amino acid sequence similarity between different subtypes [16], especially attaining sufficient selectivity against Nav1.5 expressed in cardiac tissue and Nav1.4 in skeletal muscle so as not to impair normal cardiac and skeletal muscle function [17]
Summary
Voltage-gated sodium channels (VGSCs or Navs) are essential for the initiation and propagation of action potentials in excitable tissues such as nerve, muscle, and other excitable cells [1, 2]. Unremitting efforts have been underway to produce analgesics with higher efficacy and better selectivity to address the large unmet medical need in chronic pain, as the existing clinical broad VGSC antagonists, such as lidocaine, carbamazepine, and phenytoin, have been reported to be effective on alleviating pain in humans and animal models but may cause side effects because of the lack of Nav subtype specificity [14, 15]. Engineering of HNTX-III mutant against Nav1.7 attention as potential lead molecules for pharmaceutical development owing to their extremely high specificity and potency for targets [18, 19] These disulfide-rich venom peptides typically bind to the less conserved voltage-sensing domain, and they often achieve much better subtype selectivity than small molecules that bind to the pore region of the channel [18, 20]. The resolution of the spatial structure of hNav1.7 greatly promoted the development of drug design and modification based on structure–activity relationship [30, 31]
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