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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: Bioorg Med Chem. 2018 Feb 7;26(8):1523–1537. doi: 10.1016/j.bmc.2018.01.031

Figure 2.

Figure 2

Analysis of CBB3001 and tranylcypromine (PCPA) on LSD1-dependent demethylation in vitro.

A. Purified recombinant GST-LSD1 protein used for the in vitro demethylation assay.

B. CBB3001 or tranylcypromine inhibits LSD1 demethylase activity in vitro. Recombinant LSD1 was incubated at 30 °C for 1 hour with the di-methylated H3K4 peptide and various concentrations of CBB3001. The demethylated products, mono-methylated (H3K4Me1) and non-methylated (H3K4me0), were analyzed by mass-spectrometry (MS). From top to bottom panels: 1) 1 μg di-methylated H3K4 peptide without GST-LSD1; 2) 1 μg dimethylated peptide + 2 μg GST-LSD1; 3) 1 μg di-methylated H3K4 peptide + 2 μg GST-LSD1 + 30 μM CBB3001; 4 and 5) 1 μg di-methylated H3K4 peptide + 2 μg GST-LSD1 + 50 μM or 500 μM tranylcypromine in 20 μl reaction as indicated.

C. The MS peak areas of control and various CBB3001 treated samples were integrated as described in Experimental and used to calculate IC50 of CBB3001 at 0, 1, 2, 5, 10, 15, 20, 30, 40, 50, 100 μM, respectively.