Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the Na_V1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH₂, Ac-RER-NH₂, Ac-RAAR-NH₂, Ac-REAR-NH₂, Ac-RERR-NH₂, Ac-REAAR-NH₂, Ac-PRERRA-NH₂, and Ac-PRARRA-NH₂ has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand–receptor ion–ion bonds between the attacking peptide molecules and the Na_V1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH₂, and in the range of 9–12 Å in the active peptide molecules.

Two short arginine-containing tripeptides, H-Arg-Arg-Arg-OH (TP1) and Ac-Arg-Arg-Arg-NH₂ (TP2), have been shown by the patch-clamp method to modulate the Na_V1.8 channels of DRG primary sensory neurons, which are responsible for the generation of nociceptive signals. Conformational analysis of the tripeptides indicates that the key role in the ligand-receptor binding of TP1 and TP2 to the Na_V1.8 channel is played by two positively charged guanidinium groups of the arginine side chains located at the characteristic distance of ~9 Å from each other. The tripeptide effect on the Na_V1.8 channel activation gating device has been retained when the N- and C-terminal groups of TP1 were structurally modified to TP2 to protect the attacking peptide from proteolytic cleavage by exopeptidases during its delivery to the molecular target, the Na_V1.8 channel. As demonstrated by the organotypic tissue culture method, the agents do not affect the DRG neurite growth, which makes it possible to expect the absence of adverse side effects at the tissue level upon administration of TP1 and TP2. The data obtained indicate that both tripeptides can have great therapeutic potential as novel analgesic medicinal substances.

The present work continues our recent series of articles that aim to elucidate the ligand–receptor binding mechanism of short cationic peptides to the Na_V1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the Na_V1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH₂ has been shown to effectively modulate the Na_V1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH₂ molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11–12 Å. According to the previously suggested mechanism of ligand–receptor binding of short peptides to the Na_V1.8 channel molecule, Ac-KEKK-NH₂ should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH₂ (Ac-KKK-NH₂) to the Na_V1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH₂ application at 100 nM, a significant decrease in the effective charge carried by the Na_V1.8 channel activation gating system Z_eff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH₂ analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH₂ molecule upon binding to the Na_V1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the Na_V1.8 channel has made it possible to locate the Ac-KKK-NH₂ binding site on the extracellular side of the voltage-sensing domain VSD_I. The Ac-KKK-NH₂ amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Z_eff value. According to the results presented, Ac-KKK-NH₂ is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans.

Full-length ACTH molecule and ACTH-related hexapeptide H-PKKRRP-OH are demonstrated by the patch-clamp method to decrease the Na_V1.8 channel activation gating system effective charge in the nociceptive neuron membrane, while ACTH-related tetrapeptide Ac-KKRR-NH₂ has no effect. ACTH(1–24), a fully functional ACTH mimetic, and H-PKKRRP-OH show analgesic effects in the formalin test in vivo. All peptides contain the cationic KKRR motif, but only H-PKKRRP-OH and ACTH(1–24) relieve acute pain, targeting the Na_V1.8 channel as a receptor. This seemingly controversial result is explained by application of conformational analysis and blind docking. Though conformational analysis indicates that both H-PKKRRP-OH and Ac-KKRR-NH₂ contain the cationic functional groups at the earlier suggested characteristic distance of 9–12 Å, Ac-KKRR-NH₂ does not interact with the S4_I voltage sensor of the Na_V1.8 channel activation gating system. The docking demonstrates that an extensive network of ligand–receptor ionic and hydrogen bonds involving D151, E157, R218, and R221 VSD_I residues, essential for the analgesic tripeptide Ac-KKK-NH₂ binding, is formed upon the H-PKKRRP-OH binding. Particularly important are the ionic bonds between the H-PKKRRP-OH C-terminal carboxylate anion and the S4_I R218 and R221 guanidinium groups. The described mechanism of Na_V1.8 channel modulation is fundamentally different from the effect of channel blockers.