Thus, the selective inhibition of PA-1 teratocarcinoma cells by CBB3001 indicates that this compound is highly specific for LSD1 inhibition in teratocarcinoma cells that express SOX2 and OCT4. 2.5. express SOX2 and OCT4. encoded gene at 3q26.33 is frequently amplified in squamous cell carcinomas of lung, esophagus and oral cavity, small cell lung carcinomas, and glioblatoma multiforme (GBM).24C26 SOX2 is also overexpressed in many other cancers including breast and ovarian carcinomas.27C31 The expression of SOX2 confers the stem cell properties to cancer cells.12, 27, 28 It was also shown that LSD1 is essential for maintaining the oncogenic potential of MLL-AF9 leukemia stem cells and acute myeloid leukemia.32, 33 Thus, LSD1 serves as a critical epigenetic target for various cancer cells with stem cell properties such as expression of SOX2 or other stem cell proteins.12, 27, 28 Here, we report the development of a new LSD1 inhibitor, which is structurally different from our previous LSD1 inhibitors.11, 12 Our studies revealed that this inhibitor potently inhibits LSD1 activity and in cultured cancer cells. Importantly, this inhibitor selectively impedes the proliferation of teratocarcinoma and embryonic carcinoma cells that express pluripotent stem cell proteins SOX2 and OCT4. However, it has low toxicity towards other cancer cells that do not express these pluripotent stem cell proteins, similar to that of our previously developed LSD1 inhibitors based on the crystal structure of LSD1 protein.11, 12 2. Results and discussion 2.1 Design and organic synthesis of CBB3001 Because the catalytic domain of LSD1 shares significant similarity with other members of the amine oxidase family, most investigation on LSD1 function involves the use of non-selective amine oxidase inhibitors, such as tranylcypromine (trans-2-phenylcycpropylamine, PCPA, Figure 1A), originally developed against two major isoforms of monoamine oxidases, MAO-A and MAO-B, for clinical use as anti-depressants.6, 8, 34C37 Tranylcypromine has been shown to inhibit LSD1 activity with substantially reduced potency as compared to its inhibition of MAOs. It inhibits LSD1 activity through the irreversible modification of the covalently bound FAD at high concentrations (IC50: submillimolar to millimolar), similar RELA to its inhibitory mechanism for MAOs.6, 8, 15, 34, 35, 38, 39 We tried to test a derivative of tranylcypromine, CBB3001, towards LSD1 since the activity of this compound towards LSD1 has never been reported. For the synthesis of CBB3001, we modified the Corey-Chaykovsky chemical synthesis scheme,40C43 as outlined in Figure 1B, to obtain better yield. Open in a separate window Figure 1 The synthesis scheme of CBB3001. A. The structure of tranylcypromine (trans-2-phenylcycpropylamine). B. Scheme for chemical synthesis of CBB3001: i) a) Boc2O, triethylamine, DMAP, DCM; b) Trimethylsulfoxonium iodide, NaH, DMSO. ii) a) Zn, HCl (aq), i-PrOH; b) Boc2O, TEA, dichloromethane (DCM). Compound 3 is CBB3001. C. The structure of CBB3001. 2.1.1 Preparation of tert-butyl 4-(2-nitrocyclopropyl)phenyl carbonate (2) To obtain compound 2 (Figure 1B), the mixture of (E)-4-(2-Nitrovinyl)phenol, Boc2O, DMAP, and triethylamine in dichloromethane was allowed to react at room temperature overnight. Water was then added and the organic layer was separated, washed, dried over Dichlorisone acetate Na2SO4, filtered and evaporated to dryness. The residue was dissolved in DMSO. Trimethylsulfoxonium iodide was added to a suspension of 60% NaH in DMSO and then (E)-demethylation assay. B. CBB3001 or tranylcypromine inhibits LSD1 demethylase activity causes the accumulation of the mono- and di-methylated forms of H3K4 but not trimethylated H3K4.6, 11, 12, 15, 38 To determine whether CBB3001 inhibits LSD1 analysis of CBB3001 on LSD1 demethylation of methylated H3K4 in histone H3. A. effects of CBB3001 on cancer cells. Actively growing HCT116 and PA-1 cells were treated with various concentrations of CBB3001 for 16 hours and examined. Cells were examined and cells images were acquired with 1010 lens of Nikon ECLIPSE Ti-S microscope equipped with NIS-Elements BR 3.1 software. Triplicated cells (technical repeats) were used for examination Dichlorisone acetate and one set of representative treated cells was shown. B. Triplicated treated cells (technical repeats) from Figure 3A were harvested by trypsin digestion, diluted, and Dichlorisone acetate blindly spotted onto a hemacytometer. Cells in four corners of the hemacytometer were counted to obtain average cells per dish. The differences between control and CBB3001 cells in triplicated samples were plotted. Experiments were repeated for three independent times with the same conclusion and one example is shown. Statistically significant differences were determined using a two-tailed equal-variance independent t-test. Different data sets were considered to be statistically significant when the P-value was <0.05 (*), 0.01 (**) or 0.001 (***). C. Total histones were extracted from control and CBB3001 treated cells and the levels of mono-, di-, and trimethylated H3K4 and histone H3 were.