BackgroundThe need of agonists and antagonists of β2 adrenoceptor (β2AR) is warranted in various human disease conditions including cancer, cardiovascular and other metabolic disorders. However, the sources of agonists of β2AR are diverse in nature. Interestingly, there is a complete gap in the exploration of agonists of β2AR from serum that is a well-known component of culture media which supports growth and proliferation of normal and cancer cells in vitro.MethodsIn this paper, we employed a novel vertical tube gel electrophoresis (VTGE)-assisted purification of intracellular metabolites of MCF-7 cells grown in vitro in complete media with fetal bovine serum (FBS). Intracellular metabolites of MCF-7 cells were then analyzed by LC-HRMS. Identified intracellular tripeptides of FBS origin were evaluated for their molecular interactions with various extracellular and intracellular receptors including β2AR (PDB ID: 2RH1) by employing molecular docking and molecular dynamics simulations (MDS). A known agonist of β2AR, isoproterenol was used as a positive control in molecular docking and MDS analysesResultsWe report here identification of a few novel intracellular tripeptides, namely Arg-His-Trp, (PubChem CID-145453842), Pro-Ile-Glu, (PubChem CID-145457492), Cys-Gln-Gln, (PubChem CID-71471965), Glu-Glu-Lys, (PubChem CID-11441068) and Gly-Cys-Leu (PubChem CID145455600) of FBS origin in MCF-7 cells. Molecular docking and MDS analyses revealed that among these molecules, the tripeptide Arg-His-Trp shows a favorable binding affinity with β2AR (−9.8 Kcal/mol). Furthermore, agonistic effect of this tripeptide, Arg-His-Trp is significant and comparable with that of a known agonist of β2AR, isoproterenol.ConclusionIn conclusion, we identified a unique Arg-His-Trp tripeptide of FBS origin in MCF-7 cells by employing a novel approach. This unique tripeptide Arg-His-Trp is suggested to be a potential agonist of β2AR and it may have applications in the context of various human diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD).NOVELTY & IMPACT STATEMENTSThis paper reports on a novel vertical tube gel electrophoresis (VTGE) system that assisted in the purification and identification of a few intracellular tripeptides in the in vitro grown breast cancer cells, MCF-7ls.Molecular docking and molecular dynamics simulation analyses strongly suggest that the tripeptide Arg-His-Trp among others forms the most stable ligand-protein complex with β2 adrenoceptor (β2AR). Its binding affinity and the nature of molecular interactions are comparable or even better than the known agonists of β2AR.This tripeptide Arg-His-Trp is predicted to show manyfold less cytotoxicity, mutagenicity, cardiotoxicity, drug-drug interactions, microsomal stability, and drug-induced liver injury over the other known agonists of β2AR.The tripeptide Arg-His-Trp is therefore suggested as an effective agonist of β2AR and this may be validated in future, in preclinical and clinical models.

Background: The need of agonists and antagonists of β2 adrenoceptor (β2AR) is warranted in various human disease conditions including cancer, cardiovascular and other metabolic disorders. However, the sources of agonists of β2AR are diverse in nature. Interestingly, there is a complete gap in the exploration of agonists of β2AR from serum that is a well-known component of culture media which supports growth and proliferation of normal and cancer cells in vitro. Methods: In this paper, we employed a novel vertical tube gel electrophoresis (VTGE)-assisted purification of intracellular metabolites of MCF-7 cells grown in vitro in complete media with fetal bovine serum (FBS). Intracellular metabolites of MCF-7 cells were then analyzed by LC-HRMS. Identified intracellular tripeptides of FBS origin were evaluated for their molecular interactions with various extracellular and intracellular receptors including β2AR (PDB ID: 2RH1) by employing molecular docking and molecular dynamics simulations (MDS). A known agonist of β2AR, isoproterenol was used as a positive control in molecular docking and MDS analyses. Results : We report here identification of a few novel intracellular tripeptides, namely Arg-His-Trp, (PubChem CID-145453842), Pro-Ile-Glu, (PubChem CID-145457492), Cys-Gln-Gln, (PubChem CID-71471965), Glu-Glu-Lys, (PubChem CID-11441068) and Gly-Cys-Leu (PubChem CID-145455600) of FBS origin in MCF-7 cells. Molecular docking and MDS analyses revealed that among these molecules, the tripeptide Arg-His-Trp shows a favorable binding affinity with β2AR (-9.8 Kcal/mol). The agonistic effect of Arg-His-Trp is significant and comparable with that of a known agonist of β2AR, isoproterenol. Conclusion: In conclusion, we identified a unique Arg-His-Trp tripeptide of FBS origin in MCF-7 cells by employing a novel approach. This unique tripeptide Arg-His-Trp is suggested to be a potential agonist of β2AR and it may have applications in the context of various human diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD).