trans-2-Phenylcyclopropylamine Is a Mechanism-Based Inactivator of the Histone Demethylase LSD1

Abstract

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.

Impairment of prostate cancer cell growth by a selective and reversible lysine‐specific demethylase 1 inhibitor

“…17 One milligram peptide of histone H3 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] carrying one or two methyl groups at lysine 4 were incubated with 2 lg of GST-LSD1 in the absence or presence of Namoline. The reaction mixture was incubated in demethylation buffer for 4 hr at 37 C and analyzed by mass spectroscopy.…”

“…7 In this screen, we identified the c-pyrone 3-chloro-6-nitro-2-(trifluoromethyl)-4H-chromen-4-one (1) as a novel LSD1 inhibitor, which we termed Namoline. Namoline inhibits the demethylase activity of LSD1 with a half-maximal inhibitory concentration (IC 50 ) of 51 lM (Fig.…”

“…[7][8][9][10][11][12][13] However, these amine oxidase inhibitors including clorgyline, pargyline, tranylcypromine, polyamines and derivatives thereof, many of them do not selectively target LSD1 and therefore, limits their use as therapeutics owing to potential side effects. In this study, we found a novel and selective LSD1 inhibitor called Namoline by combining protein structure similarity clustering and in vitro screening.…”

Chemical and biochemical approaches in the study of histone methylation and demethylation

“…In the early stage of inhibitor development for LSD1, some mechanism-based or suicide inactivators are identified by targeting FAD, the cofactor of the redox reaction for the demethylation by LSD1. 262 Since LSD1 and monoamine oxidases (MAO) show homology in their catalytic sites, [275][276][277][278] many of MAO inhibitors (e.g., pargyline, chlorgyline, deprenyl, tranylcypromine, phenelzine (PCPA), and nialamide) have been screened for their activity against LSD1. Results demonstrated that these MAO inhibitors showed inhibitory activity against recombinant LSD1 at rather high concentrations.…”

“…Among them, two inhibitors are phenelzine and tranylcypromine with an IC 50 value of 21 μM. 40,277 Recently, based on the crystal structures of FAD-PCPA adduct and the FAD-N-propargyl lysine peptide adduct complex with LSD1 279,280 , Ueda and coworkers designed a series of PCPA-lysine hybrid compounds and evaluated their inhibitory activities targeting at LSD1 and MAO-A and MAO-B. 281 Among the four synthesized compounds, the LSD1 inhibitory activities of compound 1 and 2 ( Figure 17) were over 10-fold more potent than that of PCPA (IC 50 values: PCPA = 32 μM; 1 = 2.5 μM; 2 = 1.9 μM).…”