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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 β-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and β-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 β-catenin signaling.

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.

Sickle cell disease (SCD) is caused by a single nucleotide polymorphism on chromosome 11 in the β-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 (α2β2) or HbS (α2βs2) 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.

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 4.2 μM against the Calu-6 human lung adenocarcinoma line, and 4.8 μM 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.

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

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.

Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Affected patients are at risk of multi-organ complications, significant morbidity and early mortality with an average age at death of 42 and 48 years for men and women, respectively. Complications include acute and chronic pain due to vascular occlusion, end organ damage, acute chest syndrome and increased risk of sepsis. SCD is caused by a point mutation on chromosome 11, which codes for the beta chain of adult hemoglobin. Fetal hemoglobin is not affected by the mutation. Persons with SCD who maintain persistent fetal hemoglobin demonstrate a decreased number of pain crises and acute chest episodes along with a decreased risk of mortality compared to other patients with SCD. The only FDA approved fetal hemoglobin inducer, Hydroxyurea, remains highly underutilized due to the risk of complications and unwanted side effects. The discovery of a new agent that promotes re-expression of fetal hemoglobin could revolutionize the care of affected patients by reducing morbidity and mortality. Epigenetic modulation is one possible mechanistic approach to accomplish this goal. Recent studies with lysine specific demethylase-1 (LSD1) inhibitors, including tranylcypromine (TCP) have shown promising results. Unfortunately, TCP also affects monoamine oxidase and thus has significant side effects. Developing a molecule with high specificity, low cross reactivity and negligible toxicity would increase desired effects while reducing unwanted side effects. Objectives: To discover small-molecule inhibitors of LSD1 that exhibit high selectivity for LSD1 and low in-vivo toxicity that can be used to promote re-expression of fetal hemoglobin in SCD patients. Design/Methods: Our laboratory at the Medical University of South Carolina has recently described a library of potent, non-toxic LSD1 inhibitors. Our preliminary results suggest that these inhibitors show promise as fetal hemoglobin inducers. Using K562 cells, an erythroleukemic cell line known to model in-vivo hemoglobin production, we are screening this library to identify the most effective compounds using Western blotting for the gamma globin chain that is unique to fetal hemoglobin. Following identification of the most effective compounds in initial screens, we will confirm results with RT-qPCR. Subsequent studies will involve culturing and treating erythroid progenitor cells from healthy subject samples and from patients with SCD obtained through bone marrow sampling. HPLC will be used for identification and quantification of hemoglobin chains, including fetal hemoglobin, after ex-vivo treatment with selected compounds. Results: Initial results from Western blots for fetal hemoglobin (gamma globin protein) demonstrate that TCP, as well as the experimental compounds C1, 107-3 and 107-15, promote the re-expression of fetal hemoglobin in K562 cells. Experimental compounds were compared to DMSO as negative control, and hemin (10 mM) and hydroxyurea (200 mM) as positive controls. All experimental compounds evaluated are significantly less toxic than hydroxyurea, and do not impact cell viability in trypan blue exclusion studies. Conclusion: Initial studies show promising results for fetal hemoglobin production using these novel LSD-1 inhibitors. Viability data is also encouraging. Continued investigation is warranted to further investigate the efficacy of these compounds. Disclosures No relevant conflicts of interest to declare.