Fig. 7.
N-Alkylisatins are not substrates or inhibitors of P-gp. (A) Relative effect of the isatins on Pgp-mediated extrusion of Calcein AM. Cells (MES-SA/Dx5) were incubated with compounds 1-5 (40 μM), CSA (40 μM) or vehicle control (2% v/v) for 40 min. Data is normalized to CSA treated cells (100% inhibition) and presented as the mean ± SD (n = 12) determined using live cell imaging (IncuCyte ZOOM). ***P < 0.001; ns = not significant. (B) Effect of test compounds on P-gp ATPase activity. P-gp membranes in the presence of compounds 1-5 (in 1% DMSO) or verapamil (positive control) at either 40 μM or 200 μM were treated with MgATP as per the Pgp-GloTM assay protocol. Change in luminescence in relative light units (RLU) was calculated by subtracting the RLU of each test compound from the RLU of sodium orthovanadate (selective inhibitor of P-gp ATPase) to correct for P-gp independent ATP consumption. Change in luminescence is inversely proportional to ATP levels, which are negatively correlated with the activity of P-gp ATPase and therefore P-gp-mediated transport. Red line indicates basal activity. Data are presented as the mean ± SD (n = 3). **P < 0.01; *P < 0.05; ns = not significant.