[1,2,4]Triazolo[1,5-a]pyrimidine derivatives: Structure-activity relationship study leading to highly selective ENPP1 inhibitors.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.

Photoinduced NO-release from polymer dots doped with an Ir(III) complex and N-methyl-N-nitroso-4-aminophenol.

Nitric oxide (NO) is a signaling molecule that plays a variety of functions in the human body, but it is difficult to use it in biological experiments or for therapeutic purposes because of its high reactivity and instability in the biological milieu. Consequently, photocontrollable NO releasers, which enable spatiotemporal control of NO release, have an important role in elucidating the functions of NO. Our group has developed visible-light-controllable NO-releasing molecules that contain a fluorescent dye structure as a light-harvesting antenna moiety and an N-nitrosoaminophenol structure as an NO-releasing moiety. Here, we aimed to construct an NO-generating system employing an intermolecular photoredox reaction between the two separate components, since this would simplify chemical synthesis and make it easier to examine various dyes as antennae. For this purpose, we constructed polymer nanoparticles doped with both N-methyl-N-nitroso-4-aminophenol (NAP, 1) and an Ir(III) antenna complex (2, 3 or 4) in order to dissolve in aqueous solution without a co-solvent. These polymer nanoparticles released NO upon photoirradiation in vitro in the purple (400-430 nm) or blue (400-460 nm) wavelength region to activate the doped Ir(III) complex.

Substituent Effects at the N-Nitrosoaminophenol Moiety of a Photoinduced-Electron-Transfer-Driven Nitric Oxide Releaser.

Nitric oxide (NO) has multiple physiological activities, including roles in vasorelaxation, neurotransmission, and immune response. Indeed, NO-releasing compounds are utilized as therapeutic agents for cardiovascular diseases based on the potent and rapid vasorelaxation induced by NO. We have developed a series of photoinduced-electron-transfer-driven (PeT-driven) NO releasers composed of a light-harvesting antenna moiety and an NO-releasing N-nitrosoaminophenol moiety, which efficiently release NO upon irradiation with blue (500 nm), green (560 nm), or red (650 nm) light. In this paper, we investigated substituent effects at the 2-position of the N-nitrosoaminophenol moiety by means of spectroscopic, fluorescence, and NO-release measurements. Interestingly, a methyl substituent at this position had no significant effect on the NO-releasing ability, while a nitro group or a methoxy group reduced it. The nitro group may suppress electron transfer to the antenna moiety, while the methoxy group may accelerate electron transfer but suppress deprotonation to afford the phenoxyl radical, which is the key reaction for release of NO. These structure-activity relationships should be helpful for further functionalizing PeT-driven NO releasers.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons.

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium-like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP The timing of neuronal migration termination, controlled by Sema3E-PlexinD1-Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.

Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity.

Lysine methylation is one of the most important modification, which is regulated by histone lysine methyltransferases and histone lysine demethylases. Lysine-specific demethylase 1 (LSD1) specifically demethylates mono- and dimethyl-lysine on histone H3 (H3K4Me/Me2, H3K9Me/Me2) to control chromatin structure, resulting in transcriptional repression or activation of target genes. Furthermore, LSD1 is overexpressed in various cancers. Therefore, LSD1 inhibitors would be not only potential therapeutic agents for cancers but also chemical tools to research biological significance of LSD1 in physiological and pathological events. However, known assay methods to date have some inherent drawbacks. The development of simple method in detecting LSD1 activity has been indispensable to identify useful inhibitors. In this study, we designed and synthesized artificial substrates based on inhibitors of LSD1 to examine LSD1 activity by an absorption increment.

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe.

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

In Cellullo and ex Vivo Availability of a Yellowish-Green-Light-Controllable NO Releaser.

Spatiotemporally controllable nitric oxide (NO) releasers are very attractive chemical tools for investigating the biological activities of NO, which is involved in the regulation of vasodilation, neurotransmission, and immune responses. We previously developed an easily synthesized, yellowish-green-light-controllable NO releaser, NO-Rosa5, and characterized its photoredox reaction mechanism. Here, we aimed to establish the biological applicability of NO-Rosa5 for in cellullo and ex vivo experiments. We successfully demonstrated yellowish-green-light-controlled NO release in HEK293T cells in vitro, as well as photomanipulation of the rat aorta response to NO in an ex vivo system. Furthermore, NO-Rosa5 showed lower toxicity than NOBL-1, a previously reported blue-light-controllable NO releaser, as determined by tetrazolium salt cell viability assay. Overall, our results indicate that NO-Rosa5 is a biocompatible, photocontrollable NO releaser with low toxicity and potentially broad applicability.

Development of a fluorescent probe for detection of citrulline based on photo-induced electron transfer.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.

A yellowish-green-light-controllable nitric oxide donor based on N-nitrosoaminophenol applicable for photocontrolled vasodilation.

Nitric oxide (NO) has been known as a gaseous chemical mediator, which modulates several physiological functions. Spatial and temporal control of NO release facilitates further study and medical application of NO. Herein, we report design and synthesis of a novel NO donor, NO-Rosa. NO-Rosa has a rosamine moiety, which absorbs yellowish green light. Upon irradiation with yellowish green light (530-590 nm), NO is released from NO-Rosa, presumably via photoinduced electron transfer from the N-nitrosoaminophenol moiety to the rosamine moiety. NO release from NO-Rosa was detected by ESR spin trapping and a NO fluorescent probe. Cellular NO release control was achieved in HEK293 cells using a NO fluorescent probe, DAF-FM DA. Furthermore, temporally controlled NO-induced vasodilation was demonstrated by treatment of a rat aortic strip with NO-Rosaex vivo and irradiation by yellowish green light. NO-Rosa is expected to be utilized for further study of NO-related physiological functions, utilizing its ability of spatiotemporal release of NO as a photocontrollable compound with harmless yellowish-green light.

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One-Step Cleavage of the Dabcyl Quencher.

Sirtuins (SIRTs) are a family of NAD+ -dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age-related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT-mediated hydrolytic release of a 4-(4-dimethylaminophenylazo)benzoyl (Dabcyl)-based FRET quencher moiety from the ϵ-amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C-terminal fluorophore. The probe SFP3 detected activities of SIRT1, -2, -3, and -6, which exhibit deacylase activities towards long-chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST-F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST-F can visualize endogenous SIRT1 activity in living cells.

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity.

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.

Identification of tissue-restricted bioreaction suitable for in vivo targeting by fluorescent substrate library-based enzyme discovery.

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

Development of photo-controllable hydrogen sulfide donor applicable in live cells.

Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

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.

A double bond-conjugated dimethylnitrobenzene-type photolabile nitric oxide donor with improved two-photon cross section.

Photocontrollable NO donors enable precise spatiotemporal release of NO under physiological conditions. We designed and synthesized a novel dimethylnitrobenzene-type NO donor, Flu-DNB-DB, which contains a carbon-carbon double bond in place of the amide bond of previously reported Flu-DNB. Flu-DNB-DB releases NO in response to one-photon activation in the blue wavelength region, and shows a greatly increased two-photon cross-section (δu) at 720 nm (Flu-DNB: 0.12 GM, Flu-DNB-DB: 0.98 GM). We show that Flu-DNB-DB enables precisely controlled intracellular release of NO in response to 950 nm pulse laser irradiation for as little as 1s. This near-infrared-light-controllable NO source should be a valuable tool for studies on the biological roles of NO.

Diced electrophoresis gel assay for screening enzymes with specified activities.

We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.

A BODIPY-picolinium-cation conjugate as a blue-light-responsive caged group.

Caged compounds protected with photolabile protecting groups (PPGs) are useful for controlling various biological events with high spatiotemporal resolution. Most of the commonly used PPGs are controlled by ultraviolet light irradiation, but it is desirable to have PPGs controlled by visible light irradiation in order to minimize tissue damage. Here, we describe a boron-dipyrromethene (BODIPY)-picolinium conjugate (BPc group) that functions as a blue-light-controllable PPG. ESR experiments indicate that the photolysis mechanism is based on intramolecular photoinduced electron transfer. We illustrate the applicability of the BPc group to biologically active compounds by employing it firstly to photocontrol release of histamine, and secondly to photocontrol release of a soluble guanylyl cyclase (sGC) activator, GSK2181236A, which induces photovasodilation. The BPc group is expected to be a useful PPG for controlling various biological events with blue light irradiation.