[Show abstract][Hide abstract] ABSTRACT: Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3 bearing hydrazine displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.
Full-text · Article · Dec 2015 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.
[Show abstract][Hide abstract] ABSTRACT: The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography-tandem mass spectrometry (MS/MS) or liquid chromatography-MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO-. The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC-MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC-MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA.
Full-text · Article · May 2015 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to be potentially useful. However, most of the conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylnitrobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic visible wavelength range (400–430 nm).
No preview · Article · Oct 2014 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be identified, catalyzes specifically the demethylation of the mono- and dimethyl groups of histone H3 lysine 4, and its dysregulation is thought to contribute to the development of cancer. We have recently reported that NCL1 (4) is the first cell-active LSD1-selective inhibitor. To find LSD1 inhibitors that show higher potency than NCL1 (4), we designed and synthesized an N-alkylated analogue of NCL1 (5), and evaluated its biological activity. In enzyme assays, compound 5 was six times more potent than 4, and compound 5 exhibited cell growth inhibition in cervical cancer HeLa cell line and neuroblastoma SH-SY5Y cell line. Compound 5 should be useful as a lead structure for anticancer drugs.
No preview · Article · Sep 2014 · Medicinal Chemistry Communication
[Show abstract][Hide abstract] ABSTRACT: Hydrogen sulfide (H2S) has been recognized as one of the important gaseous cellular signaling molecules. H2S is considered to be involved in vascular relaxation, neurotransmission, and inflammation. To investigate the physiological and pharmacological actions of H2S, inorganic sulfide salts such as NaSH have been commonly used as H2S donors. However, these approaches suffer from an inability to precisely control the release rate and dosage. Although several H2S releasers have been reported, they are still incontrollable for H2S release except one example. To overcome these shortcomings, we focused on photochemical H2S generation. Controlled H2S release with photoirradiation has the potential to provide a high degree of control over release location, timing, and dosage. We envisaged that modification of H2S with suitable photolabile protecting groups would afford an H2S donor directly controllable with light, which would release H2S upon rapid photodissociation of the two protecting groups. A ketoprofenate photocage offers many advantages including good photochemical properties. In this study, we adopted ketoprofenate photocages for our new H2S photo-releaser, and developed a novel photocontrollable H2S releaser, which releases H2S proportionally to the photo-irradiation time and intensity. Photocontrolled H2S release from this compound was also demonstrated in biological bovine serum systems.
[Show abstract][Hide abstract] ABSTRACT: Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluo-rogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.
No preview · Article · Apr 2014 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: A series of 114 SIRT inhibitor candidates was assembled using 'click chemistry', by reacting two alkynes bearing 2-anilinobenzamide pharmacophore with 57 azide building blocks in the presence of Cu(I) catalyst. Screening identified two SIRT2-selective inhibitors, which were more SIRT2-selective than AGK2, a known SIRT2 inhibitor. These findings will be useful for further development of SIRT2-selective inhibitors.
No preview · Article · Mar 2014 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: We recently discovered N-hydroxy-3-[1-(phenylthio)methyl-1H-1,2,3-triazol-4-yl]benzamide (NCC149) as a potent and selective histone deacetylase 8 (HDAC8) inhibitor from a 151-member triazole compound library using a click chemistry approach. In this work, we present a series of NCC149 derivatives bearing various aromatic linkers that were designed and synthesized as HDAC8-selective inhibitors. A series of in vitro assays were used to evaluate the newly synthesized compounds, four of which showed HDAC8 inhibitory activity similar to that of NCC149, and one of which displayed HDAC8 selectivity superior to that of NCC149. In addition, these top four compounds induced the increase of acetylated cohesin (an HDAC8 substrate) in HeLa cells in a dose-dependent manner, indicating inhibition of HDAC8 in the cells. While none of these compounds enhanced the acetylation of H3K9 (a substrate of HDAC1 and 2), only one compound refrained from increasing α-tubulin acetylation, a substrate of HDAC6, indicating that this compound is more selective for HDAC8 than the other derivatives. Furthermore, this HDAC8-selective inhibitor suppressed the growth of T-cell lymphoma cells more potently than did NCC149. These findings are useful for the further development of HDAC8-selective inhibitors.
[Show abstract][Hide abstract] ABSTRACT: We report the design, synthesis and application of a directly photocontrollable hydrogen sulfide (H2S) donor, which releases H2S proportionally to the intensity and duration of photoirradiation. Photocontrolled H2S release from this donor was also demonstrated in bovine serum. This H2S donor should be suitable for use in various biological systems.
Preview · Article · Nov 2013 · Chemical Communications
[Show abstract][Hide abstract] ABSTRACT: Two-photon-excitation release of nitric oxide (NO) from our recently synthesized photo-labile NO donor, Flu-DNB, was confirmed to allow fine spatial and temporal control of NO release at the subcellular level in vitro. We then evaluated in vivo applications. Femtosecond near-infrared pulse laser irradiation of predefined regions of interest in living mouse brain treated with Flu-DNB induced NO-release-dependent, transient vasodilation specifically at the irradiated site. Photoirradiation in the absence of Flu-DNB had no effect. Further, NO release from Flu-DNB by pulse laser irradiation was shown to cause chemoattraction of microglial processes to the irradiated area in living mouse brain. To our knowledge, this is the first demonstration of induction of biological responses in vitro and in vivo by means of precisely controlled, two-photon-mediated release of NO.
No preview · Article · Aug 2013 · ACS Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Histone Nε-methyl lysine demethylases
KDM2/7 have been identified as potential targets for cancer therapies.
On the basis of the crystal structure of KDM7B, we designed and prepared
a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays
revealed that compound 9 potently inhibits KDM2A, KDM7A,
and KDM7B, with IC50s of 6.8, 0.2, and 1.2 μM, respectively.
While inhibitors of KDM4s did not show any effect on cancer cells
tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative
activity, indicating the potential for KDM2/7 inhibitors as anticancer
Full-text · Article · Aug 2013 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Drug drop off: Given that lysine-specific demethylase 1 (LSD1) could be potently and selectively inactivated by delivering phenylcyclopropylamine (PCPA), a weak and nonselective LSD1 inhibitor, directly to the enzyme's active site, a novel series of LSD1 inactivators (1) were designed. Biological and mechanistic studies indicate that 1 inhibits LSD1 potently and selectively.
No preview · Article · Aug 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: 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 diphenylphosphino-benzoyl 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 showed that P-Rhod can detect not only HNO enzymatically gen-erated in the horseradish peroxidase-hydroxylamine system in vitro, but also intracellular HNO release from Angeli's salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.
No preview · Article · Jul 2013 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: To find histone deacetylase 3 (HDAC3)-selective inhibitors, a series of 504 candidates was assembled using "click chemistry", by reacting nine alkynes bearing a zinc-binding group with 56 azide building blocks in the presence of Cu(I) catalyst. Screening of the 504-member triazole library against HDAC3 and other HDAC isozymes led to the identification of potent and selective HDAC3 inhibitors T247 and T326. These compounds showed potent HDAC3 inhibition with submicromolar IC50s, whereas they did not strongly inhibit other isozymes. Compounds T247 and T326 also induced a dose-dependent selective increase of NF-κB acetylation in human colon cancer HCT116 cells, indicating selective inhibition of HDAC3 in the cells. In addition, these HDAC3-selective inhibitors induced growth inhibition of cancer cells, and activated HIV gene expression in latent HIV-infected cells. These findings indicate that HDAC3-selective inhibitors are promising candidates for anticancer drugs and antiviral agents. This work also suggests the usefulness of the click chemistry approach to find isozyme-selective HDAC inhibitors.