Clorgyline

The androgen responsive prostate cancer cell line, LNCaP and androgen insensitive prostate cancer cell line PC3 (both from the American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 IU/ml penicillin (Invitrogen Corp, Carlsbad, CA). The MAOA specific inhibitor, N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy) propylamine hydrochloride (clorgyline), was purchased from Sigma-Aldrich (St Louis, MO). Docetaxel (provided by Sanofi-Aventis, Bridgewater, NJ) was diluted in 70% ethanol and used at 1 nM, 10 nM, and 100 nM concentrations for LNCaP cells and 50 nM and 200 nM for PC3 cells. For HIF1a expression studies, VCaP cells were grown in DMEM-F12 with 10% FBS. The MAOA specific inhibitor clorgyline (Sigma-Aldrich, St Louis, MO) was diluted in 70% ethanol and used at 1 µM concentration. Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized using the SuperScript II Reverse Transcriptase kit (Invitrogen Corp, Carlsbad, CA). qRT-PCR reactions were done in triplicate, using SYBR Green master mix (Applied Biosystems, Foster City, CA) and analyzed using an Applied Biosystems 7700 sequence detector. Samples were normalized to the expression level of GAPDH.

4-Hydroxyquinoline, kynuramine dihydrobromide, selegiline, and clorgyline were obtained from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade.

Recombinant human monoamine oxidase (rhMAO-A and -B) enzymes were purchased from BD Biosciences (Bedford, MA, USA). Kynuramine, clorgyline, deprenyl, and DMSO were obtained from Sigma Chemical (St Louis, MO, USA).

Checkerboard assays to assess antifungal drug synergy using the fractional inhibitory concentration index (FIC) for combinations of compounds were performed as described (National Committee for Clinical Laboratory Standards 1992 ; Franzot and Casadevall 1997 (link)). In brief, the WT strain H99 was inoculated from plated colonies into PBS at an OD₆₀₀ of 0.25, and subsequently diluted 1:100 into RPMI medium. Nikkomycin Z stock was diluted in PBS (Nikkomycin Z; Sigma [Sigma Chemical], St. Louis, MO). Manumycin A, clorgyline, and 2-bromopalmitate (2BP) were diluted in DMSO (manumycin A, Bioviotica; clorgyline, Sigma; and 2BP, Sigma). Caspofungin was diluted for a final concentration range between 100 and 1.5625 μg/ml, and the test drugs were diluted to the following final concentration ranges: Nikkomycin Z 400 to 0.78125 μg/ml, manumycin A 40 to 0.078 μM, 2BP 400 to 0.78125 µM, and clorgyline 100 to 1.5625 µM. Assays were incubated at 30 and 37°. FIC index values for a combination of compounds A and B were calculated as: $\text{FIC}=\frac{MI{C}_A in combination}{MI{C}_A alone}+\frac{MI{C}_B in combination}{MI{C}_B alone}$ where an FIC index value of < 1.0 is considered synergistic (with < 0.5 considered strongly synergistic), additive if the value was 1.0, autonomous if the value was between 1.0 and 2.0, and antagonistic if the FIC index was > 2.0 (Franzot and Casadevall 1997 (link)).

Dronedarone, N-debutyl-dronedarone, propanoic acid-dronedarone, phenol-dronedarone, and C-dealkyl-dronedarone were synthesized by the Isotope Chemistry and Metabolite Synthesis Entity of the Drug Disposition Domain of Sanofi (Chilly-Mazarin, France). Their chemical structures are shown in Figure 1.
Midazolam (MDZ), 1′-hydroxy-Midazolam (1′-OH-MDZ), tolbutamide, 4-hydroxy-tolbutamide, dextromethorphan, dextrorphan, phenacetin and O-deethyl-phenacetin, dimethyl sulfoxide (DMSO), 1-Aminobenzotriazole (ABT), ketoconazole, clorgyline, alamethicin, ethanolamine, transferrin, linoleic acid, ascorbic acid, insulin, L-arginin, and glucagon were obtained from Sigma (St Louis, MO). Ham F12 and Williams E media, L-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium pyruvate, penicillin, and streptomycin were purchased from Gibco (Paisley, UK). The 1′-hydroxy-Midazolam glucuronide (Glu-O-MDZ) was synthesized by the Isotope Chemistry and Metabolite Synthesis Department of Sanofi (Chilly-Mazarin, France). All other chemicals and solvents used were of analytical grade.

Materials: Monoamine oxidase A and B (MAO-A and MAO-B), acetyl and butyrylcholinesterase (AChE and BChE), kynuramine, benzylamine, acetylthiocholine iodide (ATCI), S-butyrylthiocholine iodide (BTCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), toloxatone, clorgyline, lazabemide, pargyline, donepezil, and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich (St. Louis, MO, USA).

A Bruker model AMX 500 NMR and 400 NMR spectrometers operating on a standard pulse system were used to acquire ¹H and ¹³C NMR and 2D spectra. The instruments ran at 500 and 400 MHz for ¹H while they ran at 125 and 100 MHz for ¹³C. CDCl₃, DMSO-d₆, and acetone-d₆ were used as NMR solvents, and TMS was used as an internal standard. ESI-MS data were recorded on Thermo Orbitrap Fusion (Thermo Scientific). Samples were analyzed in the negative mode of ionization. Samples were directly infused at 3 uL/min. Mass was analyzed in Orbitrap (mass error on the instrument <2 ppm). ESI-MS data were obtained on a Micromass Q-Tof micromass spectrometer. FTMS-ESI was analyzed on Thermo Orbitrap Fusion (Thermo Scientific). The sample was analyzed in the negative mode of ionization. Mass was analyzed in Orbitrap (mass error on the instrument <2 ppm). TLC was performed on precoated silica gel GF254 plates and Column Chromatography was performed on silica gel (200–300 mesh) and Sorbadex-LH20 (Sorbent Technologies, Atlanta, GA, USA). The recombinant human monoamine oxidase-A and monoamine oxidase-B enzymes were obtained from BD Biosciences (Bedford, MA, USA). Kynuramine, clorgyline, phenelzine, deprenyl, and DMSO were procured from Sigma Chemical Company (St. Louis, MO, USA).