Abstract:Simple Summary:& nbsp;This study presents in silico models of neurokinin B receptor tiTac3Ra (tilapia Tachykinin 3 receptor a) and its potential binding sites, as well as docking native tilapia Neurokinin F (tiNKF) and tilapia Neurokinin B (tiNKB) to theses orthosteric binding sites. For a better understanding of the binding confirmation and interaction of the structures, we compared the conformation between peptide docking and induced fit docking results. We have tried to analyze the affinity of binding and binding site interactions parallel to in-vitro results of receptor activity. NKB antagonists inhibit male tilapia gonadal development and gonadotropin release. We further verified the in-vivo effect of the antagonists on gonadal development in males. Studying the receptor activity of variants with alanine mutations at Phe251(6.44) and Met289(7.43) respectively, we found that, while both variants completely underperformed, there was no direct interaction with the ligand in the binding process indicating their role in post binding receptor activation rather than the binding process itself.
NKB (Neurokinin B) is already known to play a crucial role in fish reproduction, but little is known about the structure and function of NKB receptors. Based on an in silico model of the tilapia NKB receptor Tachykinin 3 receptor a (tiTac3Ra) found in the current study, we determined the key residues involved in binding to tilapia NKB and its functional homologue NKF (Neurokinin F). Despite studies in humans suggesting the crucial role of F251(6.44) and M289(7.43) in NKB binding, no direct peptide interaction was observed in tilapia homologs. In-silico, Ala mutations on residues F251(6.44) and M289(7.43) did not influence binding affinity, but significantly affected the stability of tiTac3Ra. Moreover, in vitro studies indicated them to be critical to tiNKB/tiNKF-induced receptor activity. The binding of NKB antagonists to tiTac3Ra both in-vitro and in vivo inhibits FSH (follicle stimulating hormone) and LH (luteinizing hormone) release and sperm production in mature tilapia males. Non-peptide NKB antagonist SB-222200 had a strong inhibitory effect on the Tac3Ra activation. SB-222200 also decreased LH plasma levels; two hours post intraperitoneal injection, changed sperm volume and the ratios of the different stages along the spermatogenesis in tilapia testes.