(Bis)urea and (bis)thiourea inhibitors of lysine-specific demethylase 1 as epigenetic modulators

Abstract

The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 overexpression is thought to contribute to the development of cancer. We recently reported a series of (bis)guanidines and (bis)biguanides that are potent inhibitors of LSD1 and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of isosteric ureas and thioureas that are also potent inhibitors of LSD1. These compounds induce increases in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2 and transcription factor GATA4. These compounds represent an important new series of epigenetic modulators with the potential for use as antitumor agents.

Figure 1

(Bis)guanidine and (bis)biguanides with potent antitrypanosomal activity in vitro.

Figure 2

Effect of compounds 2d and 3-33 on LSD1 activity in vitro. Percent of LSD1 activity remaining was determined following 24-hour treatment with 10 μM of each test compound as determined by the luminol-dependent chemiluminescence method.

Figure 3

Effect of compounds 25-27 on the expression of global H3K4me1 and H3K4me2. Calu-6 human anaplastic non-small cell lung carcinoma cells were treated with a 10 μM concentration of 25, 26 or 27 for 24 h (panel A and B) or 48 h (panel C and D) as described in the methods section. Panel A and C shows global H3K4me1 expression and panel B and D shows global H3K4me2 expression. Proliferating cell nuclear antigen (PCNA) was used as a loading control. Shown are Western blot images from a single representative experiment performed in triplicate. Relative protein expression levels were determined by quantitative Western analysis using the Odyssey infrared detection system shown as bar graphs. The results represent the mean of three treatments ± SD. The protein expression level for control samples was set to a value of 1.

Figure 4

Effect of compounds 25-27 on the re-expression of secreted frizzle-related protein 2 (SFRP2, Panel A) and the transcription factor GATA4 (Panel B) mRNA. Calu-6 human anaplastic non-small cell lung carcinoma cells were treated with either a 5 or 10 μM concentration of 25, 26 or 27 for 24 hours as described in the methods section. cDNA was then synthesized from mRNA, amplified and measured by qPCR. Each data point is the average of 3 determinations that differed in all cases by 5% or less.

Figure 5

Effect of compounds 25-27 on Calu-6 human anaplastic non-small cell lung carcinoma cell viability as measured by standard MTS assay. Cells were treated with increasing concentrations of each test compound for 96 hours prior to measurement of cell viability. %NT refers to the percent of viable cells remaining at time T (96 hours) as compared to the number of cells seeded, N₀. Each data point is the average of 3 determinations that differed in all cases by 5% or less.

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