LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. This drug is a clinically validated antidepressant known to target monoamine oxidases A and B. These two flavoenzymes are structurally related to LSD1 and LSD2. Mechanistic and crystallographic studies of tranylcypromine inhibition reveal a lack of selectivity and differing covalent modifications of the FAD cofactor depending on the enantiomeric form. These findings are pharmacologically relevant, since tranylcypromine is currently administered as a racemic mixture. A large set of tranylcypromine analogues were synthesized and screened for inhibitory activities. We found that the common evolutionary origin of LSD and MAO enzymes, despite their unrelated functions and substrate specificities, is reflected in related ligand-binding properties. A few compounds with partial enzyme selectivity were identified. The biological activity of one of these new inhibitors was evaluated with a cellular model of acute promyelocytic leukemia chosen since its pathogenesis includes aberrant activities of several chromatin modifiers. Marked effects on cell differentiation and an unprecedented synergistic activity with antileukemia drugs were observed. These data demonstrate that these LSD1/2 inhibitors are of potential relevance for the treatment of promyelocytic leukemia and, more generally, as tools to alter chromatin state with promise of a block of tumor progression.

Methylation of Lys residues on histone proteins is a well known and extensively characterized epigenetic mark. The recent discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled. Among the histone demethylases so far identified, LSD1 has the unique feature of functioning through a flavin-dependent amine oxidation reaction. Data base analysis reveals that mammalian genomes contain a gene (AOF1, for amine-oxidase flavin-containing domain 1) that is homologous to the LSD1-coding gene. Here, we demonstrate that the protein encoded by AOF1 represents a second mammalian flavin-dependent histone demethylase, named LSD2. The new demethylase is strictly specific for mono-and dimethylated Lys 4 of histone H3, recognizes a long stretch of the H3 N-terminal tail, senses the presence of additional epigenetic marks on the histone substrate, and is covalently inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a biochemically stable complex with the C-terminal domain of the corepressor protein CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites that are not present in LSD1. We conclude that mammalian LSD2 represents a new flavindependent H3-Lys 4 demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of chromatin-remodeling complexes that are distinct from those involving LSD1.Histones pack the eukaryotic DNA into the nucleosome, the basic unit of chromatin (1). These highly conserved proteins are not merely spools to wind DNA, but they rather regulate gene expression by modulating the activity of the transcriptional machinery. This is achieved through recognition of histone post-translational modifications by specific transcription factors, according to a scheme dictated by the so-called "histone code" (2, 3). Methylation of Lys residues on the histone N-terminal tails is one of the most extensively characterized epigenetic marks, being involved in the regulation of a plethora of fundamental processes such as heterochromatin formation, X-chromosome inactivation, and DNA repair (4, 5). The recent discovery of lysine-specific demethylase 1 (LSD1) 3 demonstrated that lysine methylation can be dynamically controlled and is not a static epigenetic mark as thought in the past (6, 7). LSD1 specifically acts on mono-and dimethylated Lys 4 of histone H3 through an oxidative process that requires FAD as essential redox cofactor (Fig. 1a). More recently, several other histone demethylases have been uncovered; they feature the property of containing a JmjC catalytic domain that carries out the reaction through an iron-dependent mechanism (8). The JmjC enzymes are able to act on trimethylated lysines, which is mechanistically impossible for flavin-catalyzed oxidative demethylation reactions (7). Histone demethylases have been found in association with a number of chromatin-remodeling complexes and are involved in many diverse transcriptional programs (9...

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