Epigenetic Reexpression of Hemoglobin F Using Reversible LSD1 Inhibitors: Potential Therapies for Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a single nucleotide polymorphism on chromosome 11 in the ß-globin gene. The resulting mutant hemoglobin S (HbS) is a poor oxygen transporter and causes a variety of vascular symptoms and organ failures. At birth, the DRED epigenetic complex forms and silences the γ-globin gene, and fetal hemoglobin (HbF, 2 α-, and 2 γ-subunits) is replaced by adult HbA (α2ß2) or HbS (α2ßs 2) in SCD patients. HbF is a potent inhibitor of HbS polymerization, thus alleviating the symptoms of SCD. The current therapy, hydroxyurea (HU), increases γ-globin and the HbF content in sickle cells but is highly underutilized due to concern for adverse effects and other complications. The DRED complex contains the epigenetic eraser lysine-specific demethylase 1 (LSD1), which appears to serve as a scaffolding protein. Our recently discovered 1,2,4-triazole derivatives and cyclic peptide LSD1 inhibitors promote the upregulation of γ-globin production in vitro without significant toxicity. Herein, we demonstrate that these LSD1 inhibitors can be used to disrupt the DRED complex and increase the cellular HbF content in vitro and in vivo. This approach could lead to an innovative and effective treatment for SCD.

Spermine oxidase mediates Helicobacter pylori-induced gastric inflammation, DNA damage, and carcinogenic signaling.

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and ß-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and ß-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of ß-catenin signaling.

Dual inhibitors of LSD1 and spermine oxidase.

We have previously described the synthesis and evaluation of 3,5-diamino-1,2,4-triazole analogues as inhibitors of the flavin-dependent histone demethylase LSD1. These compounds are potent inhibitors of LSD1 without activity against monoamine oxidases A and B, and promote the elevation of H3K4me2 levels in tumor cells in vitro. We now report that the cytotoxicity of these analogues in pancreatic tumor cells correlates with the overexpression of LSD1 in each tumor type. In addition, we show that a subset of these 3,5-diamino-1,2,4-triazole analogues inhibit a related flavin-dependent oxidase, the polyamine catabolic enzyme spermine oxidase (SMOX) in vitro.

Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen.

BACKGROUND: The obesity pandemic is associated with multiple major health concerns. In addition to diet and lifestyle, there is increasing evidence that environmental exposures to chemicals known as obesogens also may promote obesity. OBJECTIVES: We investigated the massive environmental contamination resulting from the Deepwater Horizon (DWH) oil spill, including the use of the oil dispersant COREXIT in remediation efforts, to determine whether obesogens were released into the environment during this incident. We also sought to improve the sensitivity of obesogen detection methods in order to guide post-toxicological chemical assessments. METHODS: Peroxisome proliferator-activated receptor gamma (PPARγ) transactivation assays were used to identify putative obesogens. Solid-phase extraction (SPE) was used to sub-fractionate the water-accommodated fraction generated by mixing COREXIT, cell culture media, and DWH oil (CWAF). Liquid chromatography-mass spectrometry (LC-MS) was used to identify components of fractionated CWAF. PPAR response element (PPRE) activity was measured in PPRE-luciferase transgenic mice. Ligand-binding assays were used to quantitate ligand affinity. Murine 3T3-L1 preadipocytes were used to assess adipogenic induction. RESULTS: Serum-free conditions greatly enhanced the sensitivity of PPARγ transactivation assays. CWAF and COREXIT had significant dose-dependent PPARγ transactivation activities. From SPE, the 50:50 water:ethanol volume fraction of CWAF contained this activity, and LC-MS indicated that major components of COREXIT contribute to PPARγ transactivation in the CWAF. Molecular modeling predicted several components of COREXIT might be PPARγ ligands. We classified dioctyl sodium sulfosuccinate (DOSS), a major component of COREXIT, as a probable obesogen by PPARγ transactivation assays, PPAR-driven luciferase induction in vivo, PPARγ binding assays (affinity comparable to pioglitazone and arachidonic acid), and in vitro murine adipocyte differentiation. CONCLUSIONS: We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. CITATION: Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112-119; http://dx.doi.org/10.1289/ehp.1409672.

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

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.

3,5-Diamino-1,2,4-triazoles as a novel scaffold for potent, reversible LSD1 (KDM1A) inhibitors.

The chromatin remodeling amine oxidase lysine-specific demethylase 1 (LSD1) has become an attractive target for the design of specific inhibitors with therapeutic potential. We, and others, have described LSD1 inhibitors that have potential as antitumor agents. Many of the currently known LSD1 inhibitors are poor drug candidates, or are structurally based on the tranylcypromine backbone, thus increasing the potential for off-target effects mediated by other amine oxidases. We now describe a series of potent LSD1 inhibitors based on a novel 1,2,4-triazole scaffold; these inhibitors show a high degree of specificity for LSD1 in vitro, and cause increases in cellular histone 3 dimethyllysine 4 (H3K4me2), a gene transcription activating mark. Importantly, these inhibitors are not toxic to mammalian cells in vitro, and thus they may show utility in the treatment of epigenetically-based diseases where cell death is not a desired endpoint Figure 1. Structures of LSD1 inhibitors 1, verlindamycin 2, (bis)thioureas 3, amidoxime 4, cyclic peptide 5, N3-(2-chloro-6-phenoxybenzyl)-4H-1,2,4-triazole-3,5-diamine 6 and N3,N5-bis(2-methoxybenzyl)-1H-1,2,4-triazole-3,5-diamine 7.