Supplementary Materialsijms-21-02389-s001. with = 3). placement presented the highest affinity towards OCTs in MCF-7 and MDA-MB-231 cells (IC50 values 1057 7.5 mol/L and 1383 Lamivudine 14.0 mol/L, respectively). Of the tested sulfonamides, compounds Lamivudine 7 and 8 showed the lowest affinity towards OCTs in MCF-7 cells. 2.3. Cellular Uptake of Metformin Derivatives 2.3.1. General CharacterizationThe first step of the studies included an establishment of a relationship between the concentration of the tested compound and its cellular uptake. These studies enable us to assess whether metformin derivatives are transported into MCF-7 and MDA-MB-231 cells or only bound to them on the cell surface area. Body 2 presents the uptake of sulfonamides 1C9 at a focus of 800 mol/L after 10-minute incubation. As observed in Body 2, all chloro-substituted benzenesulfonamides (substances 1C3) had been uptaken effectively in MCF-7 cells. For example, the uptake of substance 2 was 2.669 0.040 nmol/min/mg of protein, which value was 25-fold greater than that of the mother or father medication approximately, metformin. Substance 2 was seen as a a moderate affinity (Desk 1) towards OCT transporters; as a result, we presume that substance could be carried using a transporter apart from OCT, which exists in MCF-7 however, not in MDA-MB-231 cells generally, such as for example PMAT (Supplementary Body S1). This declaration could possibly be verified by low uptake of substance 2 in MDA-MB-231 cells fairly, which confirmed over three-fold lower PMAT appearance. In turn, substance 3 was carried into MCF-7 and MDA-MB-231 cells Adipoq at a equivalent price (0.84 0.06 nmol/min/mg proteins and 0.42 0.15 nmol/min/mg protein), and it had been characterized by a minimal affinity towards OCTs. Hence, the compound uses another transporter system. Open in another window Body 2 The uptake of sulfonamide derivatives of metformin into MCF-7 and MDA-MB-231 cells at an 800 mol/L focus after 10 min incubation at 37 C. Metformin uptake was 0.107 0.006 nmol/min/mg of proteins in MCF-7 cells and 0.117 0.010 nmol/min/mg of proteins in MDA-MB-231 cells . In the entire case of sulfonamides with bromide substituent in the aromatic band, a similar design of uptake to chloride sulfonamides was reported for substances 5 and 6. Alternatively, substance 4 was carried into MDA-MB-231 cells around 130-flip better than in MCF-7. This phenomenon might be caused by a relatively high affinity towards OCTs in MDA-MB-231 cells (IC50 = 919.60 13.0 mol/L) and much higher OCT3 expression in these cells in comparison to MCF-7 . However, it should be stated that this measured expressions were only at the RNA level. Thus, further proteomic studies are needed. Derivatives with fluorine substituent in the aromatic ring were characterized by a greater uptake in MCF-7 cells than in MDA-MB-231 cells. For instance, the uptake rate of compound 7 was 1.592 0.943 nmol/min/mg protein in MCF-7 cells, while in MDA-MB-231, it was 0.110 0.01 nmol/min/mg protein. Compound 7 possesses a low affinity towards OCTs in both cell lines; therefore, we presume it might utilize another transporter mechanism, including PMAT. The most curious results were obtained for compound 9, which was characterized by a quite high affinity towards OCTs (Table 1) in both cell lines. However, its uptake was moderate (0.357 0.112 nmol/min/mg protein) in MCF-7 and very low in MDA-MB-231 cells (0.021 0.002 nmol/min/mg proteins). We presume that this phenomenon might stem from the higher affinity for MATE transporters, which might work in combination with OCTs and mediate the elimination of this compound outside the cells, since they also serve as efflux transporters . 2.3.2. Kinetic Analysis of Sulfonamide Uptake in MCF-7 and MDA-MB-231 CellsThe first stage of analysis consisted of determination of the relationship between the concentration of the test compound and its uptake in cells and the analysis of obtained Michaelis-Menten curves. The results of the Lamivudine kinetic parameters of the received curves are presented in Supplementary Table S1. In several cases, the Km Lamivudine and Vmax parameters could not be calculated, since the uptake was linear, and no transporter saturation was observed over Lamivudine the entire concentration range. The cases in which an analysis of the kinetic parameters was possible enable us to summarize that intracellular transportation in the MCF-7 cell range was far better than in MDA-MB-231, because the Vmax/Kilometres ratios, matching to uptake efficiency, had been higher in MCF-7 cells. The in-depth evaluation included the change.