Syntheses of Thailandepsin B Pseudo-Natural Products: Access to New Highly Potent HDAC Inhibitors via Late-Stage Modification

New Thailandepsin B pseudo-natural products have been prepared. Our synthetic strategy offers the possibility to introduce varying warheads via late stage modification. Additionally, it gives access to the asymmetric branched allylic ester moiety of the natural product in a highly diastereoselective manner applying rhodium-catalyzed hydrooxycarbonylation. The newly developed pseudo-natural products are extremely potent and selective HDAC inhibitors. The non-proteinogenic amino acid d -norleucine was obtained enantioselectively by a recently developed method of rhodium-catalyzed hydroamination.     

Crossing of the Cystic Barriers of Toxoplasma gondii by the Fluorescent Coumarin Tetra-Cyclopeptide

{"type":"entrez-nucleotide","attrs":{"text":"FR235222","term_id":"258291874","term_text":"FR235222"}}FR235222 is a natural tetra-cyclopeptide with a strong inhibition effect on histone deacetylases, effective on mammalian cells as well as on intracellular apicomplexan parasites, such as Toxoplasma gondii, in the tachyzoite and bradyzoite stages. This molecule is characterized by two parts: the zinc-binding group, responsible for the binding to the histone deacetylase, and the cyclic tetrapeptide moiety, which plays a crucial role in cell permeability. Recently, we have shown that the cyclic tetrapeptide coupled with a fluorescent diethyl-amino-coumarin was able to maintain properties of cellular penetration on human cells. Here, we show that this property can be extended to the crossing of the Toxoplasma gondii cystic cell wall and the cell membrane of the parasite in its bradyzoite form, while maintaining a high efficacy as a histone deacetylase inhibitor. The investigation by molecular modeling allows a better understanding of the penetration mechanism.     

NMR elucidation of monomer–dimer transition and conformational heterogeneity in histone‐like DNA binding protein of Helicobacter pylori

Helicobacter pylori (H. pylori) colonizes under harsh acidic/oxidative stress conditions of human gastrointestinal tract and can survive there for infinitely longer durations of host life. The bacterium expresses several harbinger proteins to facilitate its persistent colonization under such conditions. One such protein in H. pylori is histone‐like DNA binding protein (Hup), which in its homo‐dimeric form binds to DNA to perform various DNA dependent cellular activities. Further, it also plays an important role in protecting the genomic DNA from oxidative stress and acidic denaturation. Legitimately, if the binding of Hup to DNA is suppressed, it will directly impact on the survival of the bacterium, thus making Hup a potential therapeutic target for developing new anti‐H. pylori agents. However, to inhibit the binding of Hup to DNA, it is necessary to gain detailed insights into the molecular and structural basis of Hup‐dimerization and its binding mechanism to DNA. As a first step in this direction, we report here the nuclear magnetic resonance (NMR) assignments and structural features of Hup at pH 6.0. The study revealed the occurrence of dynamic equilibrium between its monomer and dimer conformations. The dynamic equilibrium was found to shifting towards dimer both at low temperature and low pH; whereas DNA binding studies evidenced that the protein binds to DNA in its dimeric form. These preliminary investigations correlate very well with the diverse functionality of protein and will form the basis for future studies aiming to develop novel anti‐H. pylori agents employing structure‐based‐rational drug discovery approach.     

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.     

Studies of Adriamycin Binding to Histone H1 by Resonant Mirror Biosensor and Fluorescence Spectroscopy

Abstract

Adriamycin is a clinically used antitumor anthracycline antibiotic. Histone H1 is a target for the activity of anthracycline drug at the chromatin level. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of adriamycin with H1, and the binding constant was obtained. By the analysis of fluorescence spectrum and fluorescence intensity, it was shown that adriamycin can quench the intrinsic fluorescence of tyrosine in H1 through a static quenching procedure. The binding constants of adriamycin with H1 were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained, and the binding forces were suggested to be mainly hydrophobic. The interaction of adriamycin and H1 in the presence of denaturant or salt was studied. The effect of Fe³⁺ on the binding constant was also investigated by optical biosensor and fluorescence spectroscopy. Furthermore, the steady‐state Stern–Volmer collisional quenching study of Tyr72 with acrylamide indicated that the association between adriamycin and H1 did not change molecular conformation of H1.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

组蛋白H3赖氨酸27和36位甲基化修饰的鉴定

采用液相色谱-质谱联用技术结合生物信息学分析手段,研究Hela细胞组蛋白H3赖氨酸(Lysine (K))K27和K36位点带有甲基化和二甲基化修饰的多肽鉴定,通过二级质谱碎片解析和二级碎片丰度分析,对组蛋白H3赖氨酸K27和K36上甲基化和二甲基化修饰进行了鉴定和分析。