Koshiki Mino

Identification of Cell-Active Lysine Specific Demethylase 1-Selective Inhibitors

Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

Design, Synthesis, Enzyme-Inhibitory Activity, and Effect on Human Cancer Cells of a Novel Series of Jumonji Domain-Containing Protein 2 Histone Demethylase Inhibitors

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

Identification of SAP155 as the target of GEX1A (Herboxidiene), an antitumor natural product

GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Lysine-Specific Demethylase 1-Selective Inactivators: Protein-Targeted Drug Delivery Mechanism†

Reversible histone methylation, a process controlled by two counteracting enzyme families, the histone methyltransferases and the histone demethylases, plays a pivotal role in the regulation of epigenetic gene expression. Lysine-specific demethylase 1 (LSD1) removes methyl groups from monoand dimethylated Lys4 of histone H3 (H3K4me1/2) through flavin adenine dinucleotide (FAD) dependent enzymatic oxidation. LSD1 also demethylates H3K9me1/2 in prostate cell lines and in cells infected with herpesviruses. Furthermore, histone demethylation by LSD1 is suggested to be associated with certain disease states, including cancer and herpes simplex infection. 3b, 4] trans-2-Phenylcyclopropylamine (PCPA/Tranylcypromine), which was originally found as an inhibitor of monoamine oxidases (MAOs; also FAD-dependent enzymes), is the best-studied LSD1 inhibitor, and biological studies using PCPA have uncovered important roles of LSD1 in several diseases. 4a,c,d] In addition, several groups, including ours, have reported PCPA derivatives with LSD1 inhibitory activity. While many of these LSD1 inhibitors have been suggested to be potential lead compounds for anticancer agents, most of them have various disadvantages, including poor intracellular activity, insufficient inhibitory potency, or inadequate selectivity for LSD1 over MAO A and MAO B. To overcome these issues, we hypothesized that LSD1 could be potently and selectively inactivated by delivering PCPA directly to the LSD1 active site. Herein we describe the design and synthesis of a series of LSD1 inactivators based upon this concept. PCPA inhibits LSD1 by a single-electron-transfer mechanism (Figures 1 and 2 A). In the active site of LSD1, FAD first extracts one electron from the nitrogen atom of PCPA to form a cation radical. Then, opening of the cyclopropyl ring occurs and subsequent covalent bond formation with FAD. In the course of LSD1 inactivation, the nitrogen atom of PCPA is released as an ammonia molecule through hydrolysis of the imine intermediate. Taking this mechanism into account, together with our idea of delivering PCPA directly to the

Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity

Histone Nε-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 μM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

Synthesis and biological activity of optically active NCL-1, a lysine-specific demethylase 1 selective inhibitor.

Optically active (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were synthesized and evaluated for their lysine-specific demethylase 1 inhibitory activity and cell growth inhibitory activity. In enzyme assays, the (1S,2R)-isomer was approximately four times more potent than the (1R,2S)-isomer. In cell growth inhibition assays, the two isomers showed similar activity in HEK293 cells and SH-SY5Y cells, whereas the (1S,2R)-isomer showed approximately four times more potent activity than the (1R,2S)-isomer in HeLa cells.

Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2)

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted. Graphical Abstract Knockdown of LSD1/2 or LSD inhibitor increased a presumed tumor suppressor TFPI-2. This study indicates that LSD2 also contributes to TFPI-2 regulation through histone modification.