Dual-Action Tetrapeptide Analogue of Psychrophilic Fungal Origin: Potent Inhibitor of Human Nicotinic Acetylcholine Receptors with Antinociceptive and Muscle-Relaxant Activity

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating synaptic transmission at neuromuscular junctions and within both the central and peripheral nervous systems. The psychrophilic fungal tetrapeptide WvVf-OCH₃ exhibits ∼55% inhibition of human muscle-type nAChR (hα1β1εδ) at 10 μM, suggesting potential for optimization. Structure-activity studies identified analogues, WrFr-OCH₃ and WrFk-OCH₃, with significantly increased potency (75-90% inhibition at 1 μM) against hα1β1εδ nAChRs and, notably, the hα9α10 subtype. These analogues exhibit high-affinity binding through stable π-π stacking with the principal face and electrostatic interactions with the complementary face of the receptor binding site. Their unique alternating L/D chirality enhances proteolytic stability. <i>In vivo</i>, intravenous application of WrFr-OCH₃ alleviated oxaliplatin-induced cold allodynia, while intramuscular injection reduced forelimb grip strength, consistent with muscle relaxant activity. Together, these findings identify WrFr-OCH₃ as the shortest high-activity peptide ligand of human nAChRs reported to date, with therapeutic potential for both inflammatory pain management and muscle relaxation.