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bioRxiv · 10.1101/2020.12.28.424618

An intracellular tripeptide Arg-His-Trp of serum origin detected in MCF-7 cells is a possible agonist to β2 adrenoceptor

Chandore, H.·Raj, A. K.·Lokhande, K. B.·Swamy, K. V.·Pal, J. K.·Sharma, N. K.
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Abstract

BackgroundThe need of agonists and antagonists of {beta}2 adrenoceptor ({beta}2AR) is warranted in various human disease conditions including cancer, cardiovascular and other metabolic disorders. However, the sources of agonists of {beta}2AR are diverse in nature. Interestingly, there is a complete gap in the exploration of agonists of {beta}2AR 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 {beta}2AR (PDB ID: 2RH1) by employing molecular docking and molecular dynamics simulations (MDS). A known agonist of {beta}2AR, isoproterenol was used as a positive control in molecular docking and MDS analyses ResultsWe 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 {beta}2AR (-9.8 Kcal/mol). Furthermore, agonistic effect of this tripeptide, Arg-His-Trp is significant and comparable with that of a known agonist of {beta}2AR, 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 {beta}2AR and it may have applications in the context of various human diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD). NOVELTY & IMPACT STATEMENTSO_LIThis 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. C_LIO_LIMolecular docking and molecular dynamics simulation analyses strongly suggest that the tripeptide Arg-His-Trp among others forms the most stable ligand-protein complex with {beta}2 adrenoceptor ({beta}2AR). Its binding affinity and the nature of molecular interactions are comparable or even better than the known agonists of {beta}2AR. C_LIO_LIThis 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 {beta}2AR. C_LIO_LIThe tripeptide Arg-His-Trp is therefore suggested as an effective agonist of {beta}2AR and this may be validated in future, in preclinical and clinical models. C_LI

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Chandore, H., Raj, A. K., Lokhande, K. B., Swamy, K. V., Pal, J. K., Sharma, N. K.. 2020-12-28. An intracellular tripeptide Arg-His-Trp of serum origin detected in MCF-7 cells is a possible agonist to β2 adrenoceptor. https://doi.org/10.1101/2020.12.28.424618

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