Structure-activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures

Abstract

Methylation at specific histone lysine residues is a critical post-translational modification that alters chromatin architecture, and dysregulated lysine methylation/demethylation is associated with the silencing of tumor suppressor genes. The enzyme lysine-specific demethylase 1 (LSD1) complexed to specific transcription factors catalyzes the oxidative demethylation of mono- and dimethyllysine 4 of histone H3 (H3K4me and H3K4me2, respectively). We have previously reported potent (bis)urea and (bis)thiourea LSD1 inhibitors that increase cellular levels of H3K4me and H3K4me2, promote the re-expression of silenced tumor suppressor genes and suppress tumor growth in vitro. Here we report the design additional (bis)urea and (bis)thiourea LSD1 inhibitors that feature 3-5-3 or 3-6-3 carbon backbone architectures. Three of these compounds displayed single-digit IC50 values in a recombinant LSD1 assay. In addition, compound 6d exhibited an IC50 of 4.2μM against the Calu-6 human lung adenocarcinoma line, and 4.8μM against the MCF7 breast tumor cell line, in an MTS cell viability assay. Following treatment with 6b-6d, Calu-6 cells exhibited a significant increase in the mRNA expression for the silenced tumor suppressor genes SFRP2, HCAD and p16, and modest increases in GATA4 message. The compounds described in this paper represent the most potent epigenetic modulators in this series, and have potential for use as antitumor agents.

Keywords: Antitumor agent; Epigenetics; Histone demethylase; Lysine-specific demethylase 1; Oligoamine.

Figure 1

Structures of tranylcypromine 1, verlindamycin 2, (bis)thioureas 3–5, 6a–d and 8a–g, and (bis)ureas 7a–e and 9a–d (see Table 1).

Figure 2

Inhibition of recombinant human LSD1 by compounds 1, 2 and 6–9 at a concentration of 10 μM. Percent activity remaining was determined following treatment with each test compound as determined by the luminol-dependent chemilumine scence method. Each data point is the average of three determinations ± standard error of the mean. 1% DMSO was used as the negative control.

Figure 3

Structure/activity correlations for (bis)aralkylthiourea oligoamines 3, 5, 6b, 6c, 6d, 8f and 20. Each data point is the average of 3 determinations ± standard error of the mean. Data for 3, 5 and 20 were previously reported.

Figure 4

Lineweaver-Burk analysis of the inhibition of recombinant LSD1 by compound 6d. Each data point is the average of three determinations that in each case differed by 5% or less. The K_i value of 2.4 μM was calculated by the graphing software (KaleidaGraph).

Figure 5

LSD1 inhibition through the compounds 6b, 6c, and 6d causes the re-expression of aberrantly silenced genes. Calu-6 cells were seeded at a density of 400,000 cells per T25 flask. Upon 60% confluency, the cells were treated with 10 μM of either compound 6b, 6c, or 6d for 24h or 48h. RNA was harvested and qRT-PCR was conducted as described in the Materials and Methods. The data from 4 experiments were compiled (n=12). Data are shown as the fold mean ± S.E. of treated cells when compared to non-treated cells (NT) for each time point. Student’s t tests were conducted comparing the treated to NT groups for each time point. *p<0.1 and **p<0.05.

Figure 6

Panel A. Induction of global H3K4me2 following treatment with a 10 μM concentration of compounds 6b, 6c or 6d. Calu-6 cells were seeded at a density of 400,000 per T25 flask. Upon 60% confluency, the cells were treated with 10 μM of 6b, 6c, or 6d for 24h or 48h. 30 μg of nuclear extract was used for Western blot analysis. Non-treated cells are denoted as NT. Gel bands were quantitated using the Odyssey software, and H3K4me2 bands were normalized to total histone H3 bands. The graphical data are the means ± S.E. from four experiments. Student’s t tests were used to determine statistical significance. *p<0.1 and **p<0.05. Panel B. 5 μg of full-length LSD1 purified protein was incubated with 5 μg of bulk histones in the presence or absence of 5, 10, or 20 μM LSD1 inhibitor. The reaction was incubated at 37°C for 3h. Western blot analysis was performed and H3K4me2 levels were determined using the Odyssey Software program. All of the data were normalized to the histone only control and are shown as a percent of H3K4me2 in relation to the histone only condition. Data points in Panel B represent quantitation of single Western blots from a representative experiment that was repeated 4 times with similar results.

Figure 7

Molecular modeling studies of LSD1 inhibitors. Panel A: Computer-predicted binding mode of compound 6b and 6d in the LSD1 binding site shown as a cartoon. Panel B: Molecular interactions between LSD1 and 6b. Hydrogen bonding interactions of 6b with Ala 539, FAD and Asn 535 are highlighted using arrows. Both the picture were generated using MOE 2012.10

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