Enzymatic Assay for MCR-1 Inhibition

The enzymatic assay was performed according to a previous reported method with a slight modification^{38 (link)}. Briefly, 0.1 mM full-length MCR-1 was treated with 10 molar equivalents of AuCl or Au(PEt₃)Cl at 25 °C for 18 h and subsequently incubated with 0.1 mM of a fluorescent substrate, nitrobenzodiazole-labeled glycerol-3-phosphoethanolamine (NBD-glycerol-3-PEA, Avanti Lipids, USA) in assay buffer (50 mM HEPES, pH 7.5, 100 mM NaAc, 0.023% DDM) at 25 °C overnight. TLC plate was used to separate NBD-glycerol from the MCR-1 reaction mixture in a mobile phase [ethyl acetate: methanol: water, 7:2:1 (vol/vol)]. The reaction product was analyzed by exposing the TLC plate to UV light (455-485 nm) and visualizing the fluorescent signals with a gel imaging system.