Hidehiko Nakagawa

Identification of Cell-Active Lysine Specific Demethylase 1-Selective Inhibitors

Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

Design, Synthesis, Enzyme-Inhibitory Activity, and Effect on Human Cancer Cells of a Novel Series of Jumonji Domain-Containing Protein 2 Histone Demethylase Inhibitors

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Significance Reactions of sulfur-centered nucleophiles with nitrogenous species have been studied independently for more than a century for synthetic/industrial purposes; to understand geochemical, atmospheric, and biological processes; and to explain the origins of life. Various products and reaction mechanisms were proposed. We here identify a singular process comprising a network of cascading chemical reactions that form three main bioactive products at physiological pH: nitrosopersulfide, polysulfides, and dinitrososulfite. These anionic products scavenge, transport, and release NO/HNO or sulfide/sulfane sulfur, each displaying distinct chemistries and bioactivities. Our observations provide a chemical foundation for the cross-talk between the NO and H2S signaling pathways in biology and suggest that the biological actions of these entities can be neither considered nor studied in isolation. Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Synthesis and activity of N-oxalylglycine and its derivatives as Jumonji C-domain-containing histone lysine demethylase inhibitors

N-Oxalylglycine (NOG) derivatives were synthesized, and their inhibitory effect on histone lysine demethylase activity was evaluated. NOG and compound 1 inhibited histone lysine demethylases JMJD2A, 2C and 2D in enzyme assays, and their dimethyl ester prodrugs DMOG and 21 exerted histone lysine methylating activity in cellular assays.

Identification of G protein-coupled receptor 120-selective agonists derived from PPARγ agonists

A weak, nonselective G protein-coupled receptor 120 (GPR120) agonist 10 was found by screening a series of carboxylic acids derived from the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist 3. Modification based on the homology model of GPR120 led to the first GPR120-selective agonist 12. These results provide a basis for constructing new tools for probing the biology of GPR120 and for developing new candidate therapeutic agents.

A Reductant-Resistant and Metal-Free Fluorescent Probe for Nitroxyl Applicable to Living Cells

Nitroxyl (HNO) is a one-electron reduced and protonated derivative of nitric oxide (NO) and has characteristic biological and pharmacological effects distinct from those of NO. However, studies of its biosynthesis and activities are restricted by the lack of versatile HNO detection methods applicable to living cells. Here, we report the first metal-free and reductant-resistant HNO imaging probe available for use in living cells, P-Rhod. It consists of a rhodol derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent rhodol derivative. P-Rhod showed high selectivity for HNO in the presence of various biologically relevant reductants, such as glutathione and ascorbate, in comparison with previous HNO probes. We show that P-Rhod can detect not only HNO enzymatically generated in the horseradish peroxidase-hydroxylamine system in vitro but also intracellular HNO release from Angelis salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.

Design, synthesis, inhibitory activity, and binding mode study of novel DNA methyltransferase 1 inhibitors

To identify novel non-nucleoside DNA methyltransferase (DNMT) inhibitors, we designed and synthesized a series of maleimide derivatives. Among this series, compounds 5-8 were found to be more potent DNMT1 inhibitors than RG108, a DNMT1 inhibitor reported previously by Siedlecki et al. The binding mode analysis of compound 5 is also reported.

Rapid discovery of highly potent and selective inhibitors of histone deacetylase 8 using click chemistry to generate candidate libraries.

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 μM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

An Unexpected Example of Protein‐Templated Click Chemistry

Click chemistry is a popular approach to the synthesis of functionalized molecules, and emphasizes the use of practical and reliable reactions. Copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), which selectively produces anti(1,4)-triazoles in preference to the syn isomer (1,5-triazole), is regarded as a superlative example of click chemistry. The CuAAC reaction can be accelerated by Cu-stabilizing ligands, such as tris[(1-substituted-1H-1,2,3-triazol-4-yl)methyl]amines and tris(2-benzimidazolylmethyl)amines. The catalytic system has received a great deal of use in various fields such as chemical biology and materials science. The 1,3-dipolar cycloaddition of azides with unactivated alkynes occurs much more slowly but is highly chemoselective. This property stimulated the development of “in situ click chemistry” for the field of drug discovery, in which target enzymes are allowed to assemble new inhibitors by linking azides and alkynes that bind to adjacent sites on the protein surface. The linkage reaction does not employ Cu catalysis, but instead relies on acceleration of the otherwise sluggish [3+2] cycloaddition reaction when the reaction partners are held in proximity to each other, often in or near the enzyme active site. In the course of an in situ click chemistry study on histone deacetylase (HDAC), we unexpectedly observed acceleration of the AAC reaction by trace copper associated with the protein in a structurally sensitive manner. Herein we report these findings, which constitute the first example of a Cu-protein complex catalyzing the AAC reaction. HDAC inhibitors are attractive drug candidates for cancer, inflammation, and neurodegenerative disorders. As shown in Figure 1, most HDAC inhibitors consist of a

Photoinduced nitric oxide release from a hindered nitrobenzene derivative by two-photon excitation.

Here, we demonstrated photoinduced NO generation from a 2,6-dimethylnitrobenzene-based compound (Flu-DNB) via a two-photon excitation (TPE) process. After pulse laser irradiation to a solution of Flu-DNB, oxidation products of NO were observed. This is the first account of a non-nitrosyl-chelated metal ion containing NO donor which can be controlled by the TPE technique.