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.

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions.nucleosome | histone tails | molecular recognition | chromatin modification | small-angle X-ray scattering T he mechanisms underlying the recruitment of histone-modifying complexes to specific chromatin regions, nucleosome components, or DNA sequences represent a challenge in the field of chromatin biology (1-3). Nucleosome recognition by histone-modifying enzymes is the result of two opposite requirements: binding to the target locus to locally shape and regulate chromatin accessibility and function, and nucleosome release to avoid the formation of a sticky complex bound too tightly to a chromatin region. A key feature is that it is essential to adopt a productive recognition mode so that the histone tails can be captured to undergo the necessary modifications (4, 5). To investigate these issues in nucleosome recognition, we have studied LSD1-CoREST, a flavin-dependent lysine 4 of histone protein H3 (H3-Lys4) demethylase (known also as KDM1A). This heterodimeric enzyme features several properties that make it an insightful model system for our purposes. First, the unique binding mode of the H3 tail to LSD1 has been largely studied by several biochemical and structural works (6-9). These data demonstrated that recognition by the enzyme active site requires a long stretch of 20 N-terminal amino acids and a specific pattern of posttranslational modifications. In addition, LSD1 alone is unable to recognize the nucleosomal substrate and needs the CoREST partner to effectively demethylate the nucleosomal H3 tail (10-12). Indeed, LSD1 has a characteristic "tower" domain, which protrudes from the catalytic core and represents the site for binding the C-terminal SANT2 (Swi3, Ada2, N-Cor, and TFIIIB) domain of CoREST corepressor (13-15). Furthermore, LSD1 typically acts in concert with histone deacetylases (HDAC 1/2) (9, 12). The stable and conserved LSD1-CoREST1-HDAC recognizes the nucleosomal substrate and exerts repressive activity through synergistic...

The binding mode of newly discovered histone demethylase inhibitors could have applications in the design and repurposing of drugs.