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Mechanistic analysis of a suicide inactivator of histone demethylase LSD1

Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, Maryland, United States
Biochemistry (Impact Factor: 3.19). 07/2007; 46(23):6892-902. DOI: 10.1021/bi700414b
Source: PubMed

ABSTRACT Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.

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    ABSTRACT: Lysine specific demethylase 1 (LSD1) selectively removes methyl groups from mono-and dimethylated histone 3 lysine 4 (H3K4), resulting in gene silencing. LSD1 is overexpressed in many human cancers, resulting in aberrant silencing of tumor suppressor genes. Thus, LSD1 is a validated target for the discovery of antitumor agents. Using a ligand-based approach, we designed and synthesized a series of cyclic and linear peptides that are effective inhibitors of LSD1. Linear peptide 7 and cyclic peptide 9 inhibited LSD1 in vitro by 91 and 94%, respectively, at a concentration of 10 μM. Compound 9 was a potent LSD1 inhibitor (IC 50 2.1 μM; K i 385 nM) and had moderate antitumor activity in the MCF-7 and Calu-6 cell lines in vitro. Importantly, 9 is significantly more stable to hydrolysis in rat plasma than the linear analogue 7. The cyclic peptides described herein represent important lead structures in the search for inhibitors of flavin-dependent histone demethylases. KEYWORDS: Chromatin architecture, chromatin remodeling, lysine specific demethylase, cyclic peptide, KDM inhibitors, histone, histone 3 lysine 4 H istone proteins occur as octamers that consist of one H3−H4 tetramer and two H2A−H2B dimers. 1 These proteins interact with double-stranded DNA in such a way that approximately 146 base pairs are wrapped around the histone octamer to form a nucleosome. Lysine-rich histone tails, consisting of up to 40 amino acid residues, protrude through the nucleosomal DNA strand, and act as a site for one of several post-translational modifications (PTMs) of chromatin (acety-lation, methylation, phosphorylation, ubiquitylation, sumoyla-tion, ADP ribosylation, deamination and proline isomer-ization), allowing alteration of higher order nucleosome structure. 2,3 There are numerous lysine methylation sites on histone tails, and PTMs at specific lysine marks can promote transcriptional activation or silencing. The flavin-dependent histone demethylase LSD1, also known as BHC110 and KDM1A, 4,5 catalyzes the oxidative demethylation of histone 3 methyllysine 4 (H3K4me1) and histone 3 dimethyllysine 4 (H3K4me2). Methylated histone 3 lysine 4 (H3K4) is a transcription-activating chromatin mark at gene promoters, and aberrant demethylation of this mark by LSD1 is known to silence expression of tumor suppressor genes important in human cancer. 6 By contrast, H3K9 methylation results transcriptional repression. 7 More broadly, LSD1 is known to modulate activation or repression of a number of important genes. 8 Because it is overexpressed in a number of human cancers (neuroblastoma, retinoblastoma, prostate cancer, breast cancer, lung cancer, and bladder cancer), 9−12 LSD1 has emerged as an important target for the development of specific inhibitors as a new class of antitumor drugs. 13 To date, a handful of small molecule inhibitors of LSD1 have been described, as shown in Figure 1. Effective LSD1 inhibitors include tranylcypromine-based analogues such as 1 and 2, 14,15 oligoamines such as verlindamycin 3 6 and related isosteric ureas and thioureas, 16,17 and peptide based LSD1 inhibitors 4 and 5. 18−21 Forneris et al. described a 21-mer peptide analogous to the histone 3 lysine 4 substrate region of LSD1, wherein Lys4 was replaced by a methionine (compound 6, Figure 1). 22 This linear peptide was a potent inhibitor of recombinant LSD1 with a K i value of 0.04 μM, and inhibited LSD1 bound to CoREST with a K i value of 0.05 μM. 22 Recently, a tranylcypromine-K4H3(1-21) peptide with a K i of 120 nM was reported. 23 Cyclic peptides are generally considered to be more stable against proteolytic enzymes than their linear counterparts 24 and can facilitate elucidation of bioactive conformations that are important for biological activity. To date, a cyclic peptide that acts as an inhibitor of LSD1 has not been described. Peptides having less than 16 amino acid residues bind poorly to LSD1, and optimal binding appears to require 21 amino acid residues. 19 Thus, we used ligand-based techniques to design and synthesize a series of linear and cyclic peptides based on the 21 amino acid H3K4 binding region. Because it is a potent

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