组蛋白赖氨酸特异性去甲基化酶1 抑制剂的研究进展

组蛋白赖氨酸特异性去甲基化酶1（LSD1）能够催化氧化去除组蛋白H3K4 和H3K9 的单、双甲基,该酶在多种恶性肿瘤组织 中高度表达,与肿瘤的发生发展密切相关,是一个新兴的肿瘤治疗靶标。综述LSD1 的结构、催化机制以及近年来LSD1 抑制剂的研究进展。     

Intermolecular insights into allosteric inhibition of histone lysine-specific demethylase 1

BackgroundHistone lysine-specific demethylase 1 (LSD1) has become a potential anticancer target for the novel drug discovery. Recent reports have shown that SP2509 and its derivatives strongly inhibit LSD1 as allosteric inhibitors. However, the binding mechanism of these allosteric inhibitors in the allosteric site of LSD1 is not known yet.MethodsThe stability and binding mechanism of allosteric inhibitors in the binding site of LSD1 were evaluated by molecular docking, ligand-based pharmacophore, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM/GBSA) analysis, quantum mechanics/molecular mechanics (QM/MM) calculation and Hirshfeld surface analysis.ResultsThe conformational geometry and the intermolecular interactions of allosteric inhibitors showed high binding affinity towards allosteric site of LSD1 with the neighboring amino acids (Gly358, Cys360, Leu362, Asp375 and Glu379). Meanwhile, MD simulations and MM/GBSA analysis were performed on selected allosteric inhibitors in complex with LSD1 protein, which confirmed the high stability and binding affinity of these inhibitors in the allosteric site of LSD1.ConclusionThe simulation results revealed the crucial factors accounting for allosteric inhibitors of LSD1, including different protein–ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. Meanwhile, a halogen bond interaction between chlorine atom of ligand and key residues Trp531 and His532 was recurrent in our analysis confirming its importance.General significanceOverall, our research analyzed in depth the binding modes of allosteric inhibitors with LSD1 and could provide useful information for the design of novel allosteric inhibitors.     

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.     

Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes

Introduction

Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE₂, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes.

Methods

Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry.

Results

The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage.

Conclusion

These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions.     

组蛋白去甲基化酶LSD1及其生物学功能

赖氨酸特异性组蛋白去甲基化酶1(Lysine specific demethylase 1, LSD1) 的发现, 表明组蛋白的甲基化修饰是一个动态可调节的过程。结构分析显示, LSD1 是一个黄素腺嘌呤二核苷酸(Flavin adenine dinulcleotide, FAD) 依赖性胺氧化酶, 它能够特异性脱去单甲基化和二甲基化组蛋白H3第4位赖氨酸(H3K4) 和H3K9 位点上的甲基基团。功能研究显示, LSD1 定位于细胞核内, 调控着基因转录的激活和抑制, 被誉为细胞深处的基因“开关”, 在胚胎发育和肿瘤发生过程中起着重要的作用。文章主要综述了LSD1 的结构、作用机制及其调控作用研究的新进展。     

High-throughput TR-FRET assays for identifying inhibitors of LSD1 and JMJD2C histone lysine demethylases

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 μL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.     

Human histone demethylase LSD1 reads the histone code

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys4 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 Lys4 of H3. Substrate recognition is not confined to the residues neighboring Lys4, 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 Km to ionic strength. We have probed LSD1 for its ability to demethylate Lys4 in presence of a second modification on the same peptide substrate. Methylation of Lys9 does not affect enzyme catalysis. Conversely, Lys9 acetylation causes an almost 6-fold increase in the Km value, whereas phosphorylation of Ser10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 microM, suggesting that LSD1 can bind to H3 independently of Lys4 methylation. LSD1 is a chromatin-modifying 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.     

Functional interplay between histone demethylase and deacetylase enzymes

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.     

Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST

Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.     

Investigation of the acetylation mechanism by GCN5 histone acetyltransferase

The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes.     

A SIRT1-LSD1 corepressor complex regulates Notch target gene expression and development

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.