Identification of Jumonji AT-Rich Interactive Domain 1A Inhibitors and Their Effect on Cancer Cells

Jumonji AT-rich interactive domain 1A (JARID1A), one of the jumonji C domain-containing histone demethylase (JHDM) family members, plays key roles in cancer cell proliferation and development of drug tolerance. Therefore, selective JARID1A inhibitors are potential anticancer agents. In this study, we searched for cell-active JARID1A inhibitors by screening hydroxamate compounds in our in-house library and the structural optimization based on docking study of the hit-compound to a homology model of JARID1A. As a result, we identified compound <b>6j</b>, which selectively inhibits JARID1A over three other JHDM family members. Compound <b>7j</b>, a prodrug form of compound <b>6j</b>, induced a selective increase in the level of trimethylation of histone H3 lysine 4, a substrate of JARID1A. Furthermore, compound <b>7j</b> synergistically enhanced A549 human lung cancer cell growth inhibition induced by vorinostat, a histone deacetylase inhibitor. These findings support the idea that JARID1A inhibitors have potential as anticancer agents

Rapid Discovery of Highly Potent and Selective Inhibitors of Histone Deacetylase 8 Using Click Chemistry to Generate Candidate Libraries

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including <b>C149</b> (IC₅₀ = 0.070 μM), which was more potent than PCI-34058 (<b>6</b>) (IC₅₀ = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of <b>C149</b> binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI₅₀ = 3–80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents

Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction

<div><p>Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.</p></div

Molecular mechanisms of dynAP-induced transformation of NIH3T3 cells.

<p>(A) Immunoblots of key signaling molecules. Numbers on the right sides indicate relative band intensities of NIH3T3dynAP cells when those of NIH3T3LacZ cells were defined as 1. *non-specific band because its mobility differed from the FOXO3a band. (B) DynAP-induced increases of rictor protein and mRNA expression. β-Actin was used as a control. (C) Rictor knockdown in NIH3T3dynAP cells decreases AktSer473 phosphorylation. Apparent molecular masses of proteins on immunoblots were as follows. dynAP, 42 kDa; Akt, 56 kDa; p53, 53 kDa; S6K, 70 kDa; 4E-BP1, 18 kDa; FOXO3a, 97 kDa; rictor, 200 kDa, β-actin, 45 kDa.</p

Analyses of tumor tissues.

<p>Tumor tissues were obtained from mice in the first experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135836#pone.0135836.g003" target="_blank">Fig 3A</a>. (A) Hematoxylin and eosin staining of tumors derived from NIH3T3dynAP, NIH3T3H-Ras, and NIH3T3H-RasdynAP cells. Arrows indicate capillaries filled with erythrocytes. Cell-cell adhesion appeared to be weak in NIH3T3dynAP cell-derived tumor tissue. (B) Sections stained with an antibody against CD34 (endothelial cell marker) and control sections stained without the antibody. The control sections were sections consecutive to those stained with the antibody. (C) Quantification of the CD34-positive area (left panel) and CD34-positive number (right panel). Data are the means ± SD (n = 9). Tumors obtained from 27 mice (nine mice for NIH3T3dynAP cells, nine for NIH3T3H-RasdynAP cells, and nine for NIH3T3H-Ras cells) were processed to prepare sections. One section of each tumor was randomly selected and two fields of each section were analysed.</p

Tumor formation.

<p>(A) Tumor weights in nude mice that received NIH3T3 cells expressing dynAP, H-Ras, or H-RasdynAP. Tumor weights were measured at the indicated days after injection of the cells. Data were analyzed by the Mann-Whitney test. (B) Gross characteristics of tumors. Tumor tissues derived from NIH3T3dynAP and NIH3T3H-RasdynAP cells were highly vascularized compared with those derived from NIH3T3H-Ras cells.</p

Expression of dynAP in NIH3T3 reduces E-cadherin expression and promotes cell motility.

<p>Cell motility was quantified by wound healing assays. Data are the means ± SD (n = 6).</p

Rictor knockdown in NIH3T3dynAP cells represses dynAP-induced transformation.

<p>(A) Focus formation in 2D culture. (B) Colony formation on soft agar. (C) Spheroid formation in 3D culture. Left panel shows microscopic images of spheroids, and right panel shows quantification of spheroids. All data are the means ± SD (n = 3).</p

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1‑Selective Inactivators

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide <b>3</b>, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide <b>3</b> is a mechanism-based LSD1 inhibitor. Furthermore, peptides <b>37</b> and <b>38</b>, which consist of cell-membrane-permeable oligoarginine conjugated with peptide <b>3</b>, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide <b>37</b> decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1

Identification of the KDM2/7 Histone Lysine Demethylase Subfamily Inhibitor and its Antiproliferative Activity

Histone <i>N</i>^ε-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 <b>9</b> 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 <b>9</b> exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents