1H NMR (500 MHz, CDCl3 + (PhNH)2): = 5

1H NMR (500 MHz, CDCl3 + (PhNH)2): = 5.78 (s,1H), 5.25 (t, 1H, = 6.3 Hz), 4.24 (q, 2H, = 7.1 Hz), 2.30C2.24 (m, 5H), 2.21 (s, 2H), 2.14 (s, 2H), 1.36 (t, 3H= 7.1 Hz), 1.21 (s, 6H), 1.19 (s, 6H). cells and increased it is toxicity toward a standard cell range marginally. In conclusion, adjustment from the geranyl sidechain of BM can lead to brand-new CYP3A4 enzyme inhibitors with solid antitumor results. 0.05, ** 0.01). Data factors reveal the means extracted from triplicate incubations SEM. Desk 1 Inhibition from the CYP2C9, CYP2C19, and CYP3A4 enzymes by BM, SL-BM, and positive handles. connections, and A370 shaped a hydrophobic relationship using the methyl band of KET. The favorably billed sidechain of R372 interacted using the incomplete negative charge from the oxo band of KET. Open up in another window Body 3 The docked (reddish colored) binding setting of KET overlaps using its crystallographic binding setting (blue), which is situated above the heme band (not proven). Desk 2 Binding properties from the ligands towards the CYP3A4 focus on. X represents the amino acid-ligand connections. 0.05, ** 0.01). The info factors represent the means SD (= 6). 3. Dialogue A fresh nitroxide moiety formulated with bergamottin analog (10) continues to be synthesized and examined for make use of as an inhibitor of CYP (2C9, 2C19, and 3A4) enzymes and ROCK inhibitor-2 in comparison to bergamottin (1) and known inhibitors of the enzymes. The cytotoxicity toward cancer and noncancer cell lines was investigated also. BM induced a 50% inhibition from the metabolite development at 0.2- and 0.4-fold concentrations vs. the substrates in the CYP3A4 and CYP2C19 assays, respectively (Desk 1). The IC50 beliefs of BM toward these enzymes had been in the reduced micromolar range, which agrees well using the reported data [14 previously,34,35,36]. Furthermore, BM demonstrated to also end up being an inhibitor of CYP2C9, showing 50% inhibition of metabolite formation at approximately a three-fold concentration vs. the substrate. Previous studies also reported the significant inhibitory effect of BM on CYP2C9 enzymes [11,14,35,36,37]. As our results demonstrated, SL-BM only slightly inhibits CYP2C9 and is almost a 15-fold weaker inhibitor of CYP2C19 than BM (Table 1). However, SL-BM was a five-fold stronger inhibitor of CYP3A4 compared to BM, showing a strong inhibitory efficacy similar to that of the positive control ketoconazole. The enhanced inhibitory activity of SL-BM compared to that of BM was also supported by docking experiments, where the binding of SL-BM was more favorable than that of BM (?Gbind(?10.4 vs. ?9.2 kcal/mol)). The difference in the inhibitory activities of SL-BM and BM may be attributed to the H-acceptor property of the nitroxide, as it was suggested by Row et al. [11]. BM and SL-BM seemed to be nontoxic to normal cells since they did not significantly decrease the viability of NIH3T3 fibroblasts in our toxicity assay. As far as we know, this is the first report about the anticancer activity of bergamottin toward HeLa cells. As shown in previous reports, although BM showed an inhibition effect on many cancer cell lines, such as HT-1080 fibrosarcoma [17], U266 multiple myeloma [18], HepG2 liver cancer, BGC-823 gastric cancer, HL-60 promyelotic leukemia [38], and A549 lung cancer cells [16], we did not observe BM to be significantly cytotoxic toward the HeLa cell line. Nevertheless, the insertion of a nitroxide moiety (10, IC50. = 17.32 M) resulted in the cancer-specific cytotoxic activity of the parent compound (1, IC50 50 M). Therefore, compound 10 may be a good starting point for the development of new CYP3A4 enzyme inhibitors with elevated anti-proliferative effects. 4..The antitumor activity of the new SL-bergamottin was also investigated. was also supported by docking studies, suggesting that the binding positions of BM and SL-BM to the active site of CYP3A4 are very similar, but that SL-BM had a better ?Gbind value than that of BM. The nitroxide moiety markedly increased the antitumor activity of BM toward HeLa cells and marginally increased its toxicity toward a normal cell line. In conclusion, modification of the geranyl sidechain of BM can result in new CYP3A4 enzyme inhibitors with strong antitumor effects. 0.05, ** 0.01). Data points indicate the means obtained from triplicate incubations SEM. Table 1 Inhibition of the CYP2C9, CYP2C19, and CYP3A4 enzymes by BM, SL-BM, and positive controls. interactions, and A370 formed a hydrophobic interaction with the methyl group of KET. The positively charged sidechain of R372 interacted with the partial negative charge of the oxo group of KET. Open in a separate window Figure 3 The docked (red) binding mode of KET overlaps with its crystallographic binding mode (blue), which is located above the heme ring (not shown). Table 2 Binding properties of the ligands to the CYP3A4 target. X represents the amino acid-ligand interactions. 0.05, ** 0.01). The data points represent the means SD (= 6). 3. Discussion A new ROCK inhibitor-2 nitroxide moiety containing bergamottin analog (10) has been synthesized and evaluated for use as an inhibitor of CYP (2C9, 2C19, and 3A4) enzymes and compared to bergamottin (1) and known inhibitors of these enzymes. The cytotoxicity toward cancer and noncancer cell lines was also investigated. BM induced a 50% inhibition of the ROCK inhibitor-2 metabolite formation at 0.2- and 0.4-fold concentrations vs. the substrates in the CYP2C19 and CYP3A4 assays, respectively (Table 1). The IC50 values of BM toward these enzymes were in the low micromolar range, which agrees well with the previously reported data [14,34,35,36]. Furthermore, BM proved to also be an inhibitor of CYP2C9, showing 50% inhibition of metabolite formation at approximately a three-fold concentration vs. the substrate. Previous studies also reported the significant inhibitory effect of BM on CYP2C9 enzymes [11,14,35,36,37]. As our results demonstrated, SL-BM only slightly inhibits CYP2C9 and is almost a 15-fold weaker inhibitor of CYP2C19 than BM (Table 1). However, SL-BM was a five-fold stronger inhibitor of CYP3A4 compared to BM, showing a strong inhibitory efficacy similar to that of the positive control ketoconazole. The enhanced inhibitory activity of SL-BM compared to that of BM was also supported by docking experiments, where the binding of SL-BM was more favorable than that of BM (?Gbind(?10.4 vs. ?9.2 kcal/mol)). The difference Rabbit Polyclonal to KITH_HHV1 in the inhibitory activities of SL-BM and BM may be attributed to the H-acceptor property of the nitroxide, as ROCK inhibitor-2 it was suggested by Row et al. [11]. BM and SL-BM seemed to be nontoxic to normal cells since they did not significantly decrease the viability of NIH3T3 fibroblasts in our toxicity assay. As far as we know, this is the first report about the anticancer activity of bergamottin toward HeLa cells. As shown in previous reports, although BM showed an inhibition effect on many cancer cell lines, such as HT-1080 fibrosarcoma [17], U266 multiple myeloma ROCK inhibitor-2 [18], HepG2 liver cancer, BGC-823 gastric cancer, HL-60 promyelotic leukemia [38], and A549 lung cancer cells [16], we did not observe BM to be significantly cytotoxic toward the HeLa cell line. Nevertheless, the insertion of a nitroxide moiety (10, IC50. = 17.32 M) resulted in the cancer-specific cytotoxic activity of the parent compound (1, IC50 50 M). Therefore, compound 10 may be a good starting point for the development of new CYP3A4 enzyme inhibitors with elevated anti-proliferative effects. 4. Materials and Methods 4.1. Chemistry 4.1.1. GeneralThe mass spectra were recorded with a Thermoquest Automass Multi system (ThermoQuest, CE, Instruments, Milan, Italy) operated in EI mode (70 eV). Elemental analyses were carried out with a Fisons EA 1110 CHNS elemental analyzer (Fisons Instruments, Milan, Italy) The melting points were determined with a Boetius micro-melting point apparatus (Franz Kstner Nachf. K. G., Dresden, Germany). The 1H NMR spectra were recorded with a Bruker Avance 3 Ascend 500 system (Bruker BioSpin Corp., Karsluhe, Germany) operated at 500 MHz, and the 13C NMR spectra were obtained at 125 MHz in CDCl3 or DMSO-d6 at 298 K. The in situ reduction of the nitroxides was achieved by the addition of five equivalents of hydrazobenzene (DPPH/radical). The IR spectra were obtained with a Bruker.