Functional studies and molecular dynamics reveal BPP-BrachyNH2-induced relaxation in male rat small mesenteric arteries via binding to argininosuccinate synthase

Daniel Dias Rufino Arcanjo; Isadora Basílio Meneses Bezerra; Simón Gabriel Comerma-Steffensen; Judit Prat-Duran; Daniel Carneiro Moreira; Andreanne Gomes Vasconcelos; Alexandra Plácido; Cátia Teixeira; Paula Gomes; Ana Carolina Cardoso-Teixeira; Andrelina Noronha Coelho-de-Souza; Marcelo Bemquerer; Birgitte Mønster Christensen; Reinhard Wimmer; Aldeidia Pereira de Oliveira; Ricardo Martins Ramos; Ulf Simonsen; José Roberto de Souza de Almeida Leite

doi:10.1016/j.ejphar.2025.177857

Functional studies and molecular dynamics reveal BPP-BrachyNH₂-induced relaxation in male rat small mesenteric arteries via binding to argininosuccinate synthase

Eur J Pharmacol. 2025 Jun 19:1002:177857. doi: 10.1016/j.ejphar.2025.177857. Online ahead of print.

Authors

Daniel Dias Rufino Arcanjo¹, Isadora Basílio Meneses Bezerra², Simón Gabriel Comerma-Steffensen³, Judit Prat-Duran⁴, Daniel Carneiro Moreira⁵, Andreanne Gomes Vasconcelos⁵, Alexandra Plácido⁶, Cátia Teixeira⁶, Paula Gomes⁶, Ana Carolina Cardoso-Teixeira⁷, Andrelina Noronha Coelho-de-Souza⁷, Marcelo Bemquerer⁸, Birgitte Mønster Christensen⁴, Reinhard Wimmer⁹, Aldeidia Pereira de Oliveira¹⁰, Ricardo Martins Ramos¹¹, Ulf Simonsen¹², José Roberto de Souza de Almeida Leite¹³

Affiliations

¹ Pulmonary and Cardiovascular Pharmacology, Department of Biomedicine, Aarhus University, Aarhus, Denmark; Department of Biophysics and Physiology, Federal University of Piaui, Ministro Petrônio Portella Campus, Teresina, Brazil.
² Department of Biophysics and Physiology, Federal University of Piaui, Ministro Petrônio Portella Campus, Teresina, Brazil; Information Systems Research Laboratory, Department of Information, Environment, Health and Food Production, Federal Institute of Piaui, IFPI, Teresina, Brazil.
³ Pulmonary and Cardiovascular Pharmacology, Department of Biomedicine, Aarhus University, Aarhus, Denmark; Animal Physiology, Department of Biomedical Sciences, Faculty of Veterinary, Central University of Venezuela, Venezuela.
⁴ Pulmonary and Cardiovascular Pharmacology, Department of Biomedicine, Aarhus University, Aarhus, Denmark.
⁵ Research Group in Morphology and Applied Immunology, NuPMIA, Faculty of Medicine, University of Brasilia, Brasilia, Brazil.
⁶ LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal.
⁷ Institute of Biomedical Sciences, State University of Ceará, Fortaleza, Brazil.
⁸ Embrapa Genetic Resources and Biotechnology, Brasilia, Brazil.
⁹ Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.
¹⁰ Department of Biophysics and Physiology, Federal University of Piaui, Ministro Petrônio Portella Campus, Teresina, Brazil.
¹¹ Information Systems Research Laboratory, Department of Information, Environment, Health and Food Production, Federal Institute of Piaui, IFPI, Teresina, Brazil.
¹² Pulmonary and Cardiovascular Pharmacology, Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address: us@biomed.au.dk.
¹³ Research Group in Morphology and Applied Immunology, NuPMIA, Faculty of Medicine, University of Brasilia, Brasilia, Brazil; LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal. Electronic address: jrsaleite@gmail.com.

PMID: 40543742
DOI: 10.1016/j.ejphar.2025.177857

Abstract

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-BrachyNH₂ in rat small mesenteric arteries and applied in silico molecular dynamics studies to explore the interaction between AsS and BPP-BrachyNH₂. Segments of male rat mesenteric arteries mounted in microvascular myographs were treated with 10 μg/mL lipopolysaccharides (LPS), and then, BPP-BrachyNH₂ was cumulatively added (10^-9 - 3 × 10^-5 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-BrachyNH₂ 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-BrachyNH₂ was abolished in the presence of 100 μM L-NNA (L-N^G-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-BrachyNH₂ 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-BrachyNH₂ 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.

Keywords: Brachycephalus ephippium; Citrulline; Endotoxin; Hypertension; Proline-rich oligopeptide; Vasorelaxation.