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In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents

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Abstract

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca2+ antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 μM (FRA77, GDE6, GDE19) and >10 μM (verapamil) in EA.hy926 cells, or >10 μM (FRA77, GDE6, GDE19) and at 100 μM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K+ (K60)-induced contraction at concentrations ≥1 and ≥3 μM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 μM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

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Acknowledgments

This work was supported by MIUR (Futuro in Ricerca 2012, RBFR12SOQ1_001). The authors wish to thank Prof. D. Ghigo (University of Torino, Italy) and Prof. G. Valacchi (University of Ferrara, Italy) for providing A7R5 and EA.hy926 cells line, respectively, and Dr. Marco Sebastiani and Dr. Federica D’Ambrosio for the assistance in some experiments.

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    Authors and Affiliations

    1. Dipartimento di Scienze della Vita, Università degli Studi di Siena, Via A. Moro 2, 53100, Siena, Italy

      F. Fusi, M. Durante, M. Frosini & S. Saponara

    2. Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, via A. Moro 2, 53100, Siena, Italy

      O. Spiga & A. Trezza

    3. Dipartimento NEUROFARBA-Sezione di Farmaceutica e Nutraceutica, Università di Firenze, via Ugo Schiff 6, 50019, Sesto Fiorentino, Italy

      E. Floriddia, E. Teodori & S. Dei

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    1. F. Fusi
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    Correspondence to S. Saponara.

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    F. Fusi and M. Durante contributed equally to this work.

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    Supplementary Fig. 1

    Representative photomicrograph showing morphological changes of A7r5 and EA.hy926 cells treated with 10, 50, and 100 μM FRA77 (panel A), GDE6 (panel B), GDE19 (panel C), and verapamil (panel D) for 24 h. Controls represent DMSO-treated cells. The cytotoxic potential of the compounds were evaluated using the grade scale described in the United States Pharmacopeia 28, edition 2005 (see “Materials and methods”). Control cells maintained their original flattened, spindle-shaped (A7r5), or polygonal (EA.hy926) morphology. Both A7r5 and EA.hy926 cells treated with 10 μM concentration of all compounds still maintain a morphology very close to untreated cells (grade 0). On the contrary, the treatment with either 50 or 100 μM FRA77, GDE6, and GDE19 caused significant changes: cells partially detached and showed the lobulated appearance of apoptotic cells characterized by shrinkage, membrane blebbing, and losing contact with adjacent cells. The density of the adherent cells decreased compared with control conditions, indicating detachment of both A7r5 and EA.hy926 cells exposed to the compounds (grades 2–3). Verapamil did not significantly affect A7r5 cell morphology up to 100 μM concentration, while in EA.hy926 cells it caused apoptotic-like changes, particularly evident at the highest concentration (cells rounding, shrinkage, and severe blebbing, grades 2–3). (OM 5X, scale bar 250 μm) (PPTX 2958 kb)

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    Fusi, F., Durante, M., Spiga, O. et al. In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents. Naunyn-Schmiedeberg's Arch Pharmacol 389, 1033–1043 (2016). https://doi.org/10.1007/s00210-016-1266-y

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