Functional studies and molecular dynamics reveal BPP-BrachyNH-induced relaxation in male rat small mesenteric arteries via binding to argininosuccinate synthase.

Previous studies have reported the involvement of argininosuccinate synthase (AsS) as a putative target for the cardiovascular effects of proline-rich oligopeptides. The present study investigated the mechanisms underlying the vasorelaxant effect of BPP-BrachyNH in rat small mesenteric arteries and applied in silico molecular dynamics studies to explore the interaction between AsS and BPP-BrachyNH. Segments of male rat mesenteric arteries mounted in microvascular myographs were treated with 10 μg/mL lipopolysaccharides (LPS), and then, BPP-BrachyNH was cumulatively added (10 - 3 × 10 M) to noradrenalin-contracted (1-10 μM) preparations. Molecular Dynamics calculations were performed between AsS enzyme (RCSB Protein Data Bank ID: 2NZ2) as the target and both BPP-BrachyNH and L-citrulline (PubChem CID 9750 code) as ligands. Immunohistochemistry showed expression of AsS and endothelial nitric oxide (NO) synthase (eNOS) in mesenteric arteries and of inducible NO synthase (iNOS) in segments exposed to LPS. The vasorelaxant effect of BPP-BrachyNH was abolished in the presence of 100 μM L-NNA (L-N-nitroarginine) and 3 μM ODQ ([1H-[1,2,4]-oxadiazolo-[4,3-a] quinoxalin-1-one]), and attenuated in the presence of 10 μM 1400W (N-(3-(aminomethyl)-benzyl)-acetamidine) and 1 mM MDLA (α-methyl-D, L-aspartic acid), inhibitors of iNOS and AsS, respectively. The AsS_BPP-BrachyNH complex showed increased binding energy, inhibition constant, and the number of interactions with amino acids when compared with the AsS_L-citrulline complex. These results suggest that the positive interaction of BPP-BrachyNH with AsS leads to L-citrulline recycling and increases L-arginine bioavailability, thereby improving the mechanism for the vasorelaxant effect. Our findings open new perspectives for potential therapeutic applications of proline-rich oligopeptides in vascular dysfunction.