Tranylcypromine

HeLa and MDA-MB231 cells were cultured in DMEM supplemented with 10% FCS, 10U/ml penicillin and 10μg/ml streptomycin. For siRNA transient transfection, 3 10⁵ cells per ml were seeded in 6-well plate and 25pmol/ml of siRNAs against LSD1 (Invitrogen), ERRα (Dharmacon and Invitrogen) or control (medium GC Stealth RNA interference negative control duplexes, Invitrogen) (Table 1) were transfected with INTERFERin (Polyplus Transfection) according to the manufacturer’s protocol. Plasmid transfections were performed with Exgen500 (Euromedex) for HeLa cells and JetPRIME (Polyplus Transfection) for MDA-MB231 cells. LSD1-K661A mutant was generated by recombinant PCR and verified by sequencing. pSG5-flagERRαΔA/B and pSG5-flagERRαΔA/BΔAF2 have been described elsewhere [35 (link)]. Cells were harvested 48 hours after transfection. Cycloheximide (Sigma-Aldrich) was used at 50–100μg/ml, MG132 (Sigma-Aldrich) at 50μM, pargyline (Sigma) at 3mM and tranylcypromine (Sigma) at 200μM.

The pooled human liver microsomes (HLMs) and human recombinant enzyme used in the incubation studies were purchased from BD Gentest Co. (Woburn, MA, USA). Coumarin, 7-hydroxyCoumarin, tolbutamide, 4-hydroxytolbutamide, (S)-mephenytoin, 4′-hydroxymephenytoin, metoprolol, α-hydroxymetoprolol, midazolam maleate, 1-hydroxymidazolam, propranolol, quinidine hydrochloride, tranylcypromine, fluconazole, ticlopidine, quinidine, ketoconazole, furafylline, and diethyldithiocarbamate were purchased from Sigma Chemicals (St. Louis, MO, USA). Formic acid, ethylic acid, ammonium formate, MgCl₂, NADP⁺, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and potassium phosphate (monobic and dibaic) were chromatographic-grade chemicals purchased from Sigma Chemicals (St. Louis, MO, USA). Acetonitrile and methanol were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO, USA). PrimeScript^® RT reagent Kit With gDNA Eraser and SYBR^® Premix DimerEraser™ were purchased from Takara Biotechnology (Dalian, China). Glycyrrhetinic acid (GA) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

Cornin (≥ 98%, Fig. 1) and testosterone (≥ 98%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, 4′-hydroxydiclofenac (≥ 98%), 4-hydroxymephenytoin (≥ 98%),NADP+, phenacetin (≥ 98%), acetaminophen (≥ 98%), 6β-hydroxytestosterone (≥ 98%), corticosterone (≥ 98%), 7-hydroxyCoumarin (≥ 98%), sulfaphenazole (≥ 98%), tranylcypromine (≥ 98%), chlorzoxazone (≥ 98%), quinidine (≥ 98%), 6-hydroxychlorzoxazone (≥ 98%), paclitaxel (≥ 98%), clomethiazole (≥ 98%), and furafylline (≥ 98%) were obtained from Sigma Chemical Co. Montelukast (≥ 98%) was obtained from Beijing Aleznova Pharmaceutical (Beijing, China). Coumarin (≥ 98%), dextromethorphan (≥ 98%), diclofenac (≥ 98%), and ketoconazole (≥ 98%) were purchased from ICN Biomedicals. Pooled HLMs were purchased from BD Biosciences Discovery Labware. All other reagents and solvents were of analytical reagent grade.
Fig. 1

The chemical structure of cornin

Tranylcypromine, GSKLSD1, and GSK2789552 are commercially available irreversible LSD1 inhibitors and were purchased from Sigma-Aldrich (Tranylcypromine and GSKLSD1) and Chemitek (GSK2789552). MTT assays were carried out according to standard protocol. Cells were seeded at a density of 3500 cells/well and incubated overnight. The following day, media was removed and fresh media with a range of drug doses were added to cells. At the end of 24, 48, and 72 h following drug addition, MTT reagent was added and spectrophotometer readings were taken at 595 nm and 650 nm.

Clorgyline, pargyline, selegiline (l-deprenyl), phenelzine and tranylcypromine were purchased from Sigma-Aldrich, Dorset, UK. Compounds ASS234 [50 (link)], Contilisant [36 (link)], and F2MPA [19 (link)] were synthesized by Dr. J. Marco-Contelles (Madrid, Spain). Commercial human MAO-A and MAO-B expressed in insect cell membranes (Sigma-Aldrich, Dorset, UK) were used for steady-state kinetic experiments. Human MAO-A expressed in yeast cells (Saccharomyces cerevisiae) was induced, solubilized and purified as before [51 (link),52 ].