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.

Histone demethylase LSD1 regulates transcription by demethylating Lys 4 of histone H3. The crystal structure of the enzyme in complex with CoREST and a substrate-like peptide inhibitor highlights an intricate network of interactions and a folded conformation of the bound peptide. The core of the peptide structure is formed by Arg 2 , Gln 5 , and Ser 10 , which are engaged in specific intramolecular H-bonds. Several charged side chains on the surface of the substrate-binding pocket establish electrostatic interactions with the peptide. The three-dimensional structure predicts that methylated Lys 4 binds in a solvent inaccessible position in front of the flavin cofactor. This geometry is fully consistent with the demethylation reaction being catalyzed through a flavin-mediated oxidation of the substrate amino-methyl group. These features dictate the exquisite substrate specificity of LSD1 and provide a structural framework to explain the fine tuning of its catalytic activity and the active role of CoREST in substrate recognition.Lysine methylation is among the most well characterized histone modifications, and its existence has been known since the early days of chromatin research (1, 2). This type of epigenetic mark provides a huge potential for functional responses in that it can occur in different forms (mono-, di-, and tri-methylation) and on different histone sites, each having a specific physiological meaning. Histone methylation has been long thought to be a low turnover epigenetic mark, but the recent discovery of histone demethylases (3, 4) has challenged this view by demonstrating that histone lysine methylation can be actively and dynamically regulated. Two classes of histone demethylases have been uncovered; the enzymes of the JmjC family use iron as cofactor, whereas lysine-specific demethylase 1 (LSD1) 4 employs FAD as the prosthetic group (5).LSD1 catalyzes the oxidative demethylation of mono-and dimethyl Lys 4 of histone H3, generating hydrogen peroxide and formaldehyde (3, 4). The enzyme is implicated as a key component of distinct co-activator and co-repressor complexes in a surprisingly wide range of cellular processes where it participates in the dynamic transition of transcriptional programs (6). Its catalytic activity is finely tuned by the epigenetic marks present on the H3 N-terminal tail (7) and by other protein partners, such as CoREST, that form a stable complex with the enzyme (8, 9). The three-dimensional structure of LSD1 in its native state (10, 11) and in complex with the LSD1-binding domain of CoREST (12) have revealed that the catalytic center is located in the core of the enzyme main body. A protruding tower domain consisting of two remarkably long helices forms the docking site for the co-repressor protein (Fig. 1A).Here, we describe the structural analysis of LSD1-CoREST bound to a 21-amino acid H3 peptide in which pLys 4 ("p" is for peptide) is mutated to Met. The structural analysis illuminates the molecular properties that enable LSD1 to function as a key transcriptional reg...

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono-and dimethylated Lys 4 of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono-or dimethylated Lys 4 of H3. Substrate recognition is not confined to the residues neighboring Lys 4 , but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of K m to ionic strength. We have probed LSD1 for its ability to demethylate Lys 4 in presence of a second modification on the same peptide substrate. Methylation of Lys 9 does not affect enzyme catalysis. Conversely, Lys 9 acetylation causes an almost 6-fold increase in the K m value, whereas phosphorylation of Ser 10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 M, suggesting that LSD1 can bind to H3 independently of Lys 4 methylation. LSD1 is a chromatinmodifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.Histones play a fundamental role in the control of a variety of cellular processes, including gene expression, DNA replication, and repair. Histone function is modulated through covalent modifications by acetylation, methylation, ubiquitination, and sumoylation (1, 2). These modifications have specific effects and act in a combinatorial manner defining the so-called histone code (1, 3). Very recently, Shi et al. (4) and our group (5) have reported on the discovery of the first enzyme able to specifically demethylate Lys 4 of histone H3. The protein was therefore named LSD1 (for lysine-specific demethylase; it is also known as KIAA0601 and BHC110). The existence of histone-demethylating enzymes has always been questioned (6, 7), and with the discovery of LSD1, it was firmly established that histone methylation is a dynamic process under enzymatic control similar to the other known post-translational histone modifications. LSD1 has been typically found in association with CoREST and HDAC1/2 proteins, forming a module found in several multiprotein co-repressor complexes (8 -11). Knock-out of LSD1 by RNA interference was shown to cause increased levels of histone methylation, resulting in the reactivation of a specific target gene (4). It has also been shown that LSD1 is a bona fide co-repressor able to repress a reporter gene and that this function is dependent on the demethylase activity (4). Based on sequence homology analysis, it was predicted that several histone demethylases are likely to exist in mammalian and other eukaryotic organisms (4).From a biochemical and structur...