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  • Accordingly we prepared three compounds and as

    2020-05-25

    Accordingly, we prepared three compounds (, , and ) as shown in . Condensation of commercially available SB-334867 free base australia with glycine methyl ester gave amide . A Friedel–Crafts reaction between compound and 4-methyl valeryl chloride in the presence of aluminum chloride mainly produced undesired compound rather than desired ketone . Therefore, we reacted compound with iodomethane under basic conditions to obtain compound . Reduction of ketone by EtSiH yielded compound . Treatment of compound with boron tribromide produced phenol . An S2 reaction between compound and -butyl-(3-bromopropyl)carbamate gave ether . Compound was allowed to react in the presence of trifluoroacetic acid to produce amine , and subsequent reaction with the corresponding acid chlorides yielded the desired compounds and . Subsequently, we evaluated the ability of synthetic compounds , , and to inhibit E1 activity using recombinant human E1 and biotin-tagged ubiquitin. The ubiquitin was added to preincubated solutions of E1 and each inhibitor, and the resulting mixtures were incubated for 30 min at 37 °C. After the reactions had been quenched by surfactants, the ubiquitin–E1 conjugate was detected by western blotting analysis using a streptavidin–horseradish peroxidase (HRP) conjugate. As expected, compounds and inhibited the formation of the ubiquitin–E1 conjugate, but compound did not (A). We found that was more potent than when we investigated the compounds at concentrations of 100 and 1000 μM. This suggests that the inhibitory activity of depends on an interaction with Tyr 579 (E). We also conducted an in-depth evaluation of the dose dependency of (B). Although the inhibitory activity of was weaker than that of PYR-41 (, ), which is a representative E1 inhibitor, exhibited dose-dependent inhibition in the range 10–1000 μM. We then determined whether compounds and inhibited the ubiquitin–E2 conjugate in the presence of E1. E2 was co-incubated with ubiquitin and E1 in the presence or absence of the inhibitors (C). Compound inhibited the formation of both ubiquitin–E2 and ubiquitin–E1. Compound did not inhibit ubiquitin–E1 formation (A), and had no effect on ubiquitin–E2 formation. Furthermore, compound (10–1000 μM) exhibited dose-dependent inhibition of ubiquitin–E2 formation (D). Taken together, these results indicate that compound inhibits the formation of both ubiquitin–E1 and ubiquitin–E2. We then attempted to determine the mechanism by which compound acts as an inhibitor. PYR-41 () is an irreversible E1 inhibitor. However, it is assumed that E1 inhibition by compound is reversible. Therefore, we tested the reversibility of E1 inhibition by compound . After pre-incubation of E1 with the inhibitor, the mixture was reacted with biotin-tagged ubiquitin (2.5 μM) for 30 min. Additional biotin-tagged ubiquitin (final concentration, 5.0 μM) was then added to the reaction mixture, which was then either incubated for a further 30 min (, lanes 2, 4, and 6) or not incubated (, lanes 1, 3, and 5), before quenching. Western blotting analysis revealed that additional incubation with ubiquitin had no effect on E1 inhibition by irreversible inhibitor PYR-41 () (, lanes 3 and 4), whereas E1 activity, which was inhibited by , was recovered by the addition of ubiquitin (, lanes 5 and 6). These results suggest that inhibition by compound is reversible. Finally, we investigated the effect of compound on cancer cells to explore the potential clinical usefulness of E1 inhibition. It has been reported that E1 is closely associated with the proliferation of breast cancer cells. Inhibition of E1 induces an accumulation of p53 protein, which is controlled by the ubiquitin–proteasome system (UPS), and suppresses the growth of breast cancer cell lines. Therefore, we evaluated the effect of compound on MCF-7 human breast cancer cells. As reported, treatment of the MCF-7 cells with PYR-41 () increased the p53 levels (, lane 2). We observed a similar phenomenon when the MCF-7 cells were treated with compound (100 μM) (, lane 3). These results suggest that compound inhibits E1 in cancer cells. We then evaluated the inhibition of the growth of MCF-7 cells using an alamarBlue assay. As shown in , compound inhibited the growth of MCF-7 cells, and had a half-maximal growth inhibition (GI) value of 63.4 μM. The GI value of the reversible inhibitor was not as high as that of PYR-41 (), which was consistent with the evaluation of compound and PYR-41 () (B and D). To summarize the cellular assay, these results suggest that reversible E1 inhibitors are active in MCF-7 cells.