[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.

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.

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.

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.

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.

Development of Nucleoside Diphosphate-Bearing Fragile Histidine Triad-Imaging Fluorescence Probes with Well-Tuned Hydrophobicity for Intracellular Delivery.

Fluorescence-guided cancer surgery can dramatically improve recurrence rates and postoperative quality of life of patients by accurately distinguishing the boundary between normal and cancer tissues during surgery, thereby minimizing excision of normal tissue. One promising target in early stage cancer is fragile histidine triad (FHIT), a cancer suppressor protein with dinucleoside triphosphate hydrolase activity. In this study, we have developed fluorescence probes containing a nucleoside diphosphate moiety, which dramatically improves the reactivity and specificity for FHIT, and a moderately lipophilic ester moiety to increase the membrane permeability. The ester moiety is cleaved by ubiquitous intracellular esterases, and then, FHIT in the cells specifically cleaves nucleoside monophosphate. The remaining phosphate moiety is rapidly cleaved by ubiquitous intracellular phosphatases to release the fluorescent dye. We confirmed that this probe can detect FHIT activity in living cells. A comprehensive evaluation of the effects of various ester moieties revealed that probes with CLogP = 5-7 showed good membrane permeability and were good substrates of the target enzyme; these findings may be helpful in the rational design of other multiple phosphate-containing probes targeting intracellular enzymes.

Ascorbate-assisted nitric oxide release from photocontrollable nitrosonium ion releasers for potent ex vivo photovasodilation.

We found that N-nitrosoaminoanisole derivatives tethered to dyes work as photocontrollable nitrosonium cation releasers and are converted to potent nitric oxide releasers in the presence of sodium ascorbate. The N-nitrosoaminoanisole derivative 2 worked as a more potent photovasodilating reagent ex vivo than previously reported nitric oxide releasers.

FKBP51 and FKBP52 regulate androgen receptor dimerization and proliferation in prostate cancer cells.

The growth of prostate cancer is dependent on the androgen receptor (AR), which serves as a ligand-specific transcription factor. Although two immunophilins, FKBP51 and FKBP52, are known to regulate AR activity, the precise mechanism remains unclear. We found that depletion of either FKBP51 or FKBP52 reduced AR dimer formation, chromatin binding, and phosphorylation, suggesting defective AR signaling. Furthermore, the peptidyl-prolyl cis/trans isomerase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52-AR signaling, also inhibited AR dimer formation. Finally, elevated expression of FKBP52 was associated with a higher rate of prostate-specific antigen recurrence in patients with prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation.

A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole.

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase whose inhibitors are useful for the regulation of the actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole scaffold. The reversible trans-cis isomerization by visible light stimuli enabled us to manipulate ROCK activities in vitro and in cells.

Synthesis of Fluorescent Probes Targeting Tumor-Suppressor Protein FHIT and Identification of Apoptosis-Inducing FHIT Inhibitors.

For the early diagnosis of cancer, leading to a better chance of full recovery, marker genes whose expression is already altered in precancerous lesions are desirable, and the tumor-suppressor gene FHIT is one candidate. The gene product, FHIT protein, has a unique dinucleoside triphosphate hydrolase (AP3Aase) activity, and in this study, we designed and synthesized a series of FHIT fluorescent probes utilizing this activity. We optimized the probe structure for high and specific reactivity with FHIT and applied the optimized probe in a screening assay for FHIT inhibitors. Screening of a compound library with this assay identified several hits. Structural development of a hit compound afforded potent FHIT inhibitors. These inhibitors induce apoptosis in FHIT-expressing cancers via caspase activation. Our results support the idea that FHIT binders, no matter whether inhibitors or agonists of AP3Aase activity, might be promising anticancer agents.

Live-Cell Imaging of Sirtuin Activity Using a One-Step Fluorescence Probe.

Sirtuins (SIRTs) are a family of NAD+-dependent histone deacetylases (HDACs). In mammals, dysfunction of SIRTs is associated with age-related metabolic diseases, cancers, and even aging. Therefore, the detection of SIRT activity in living cells or tissues would be helpful for diagnosis of a wide range of SIRT-associated diseases. Here, we present methodology to measure SIRT activity in living cells by using our newly developed SIRT fluorescence probe, KST-F-DA.

A Set of Highly Sensitive Sirtuin Fluorescence Probes for Screening Small-Molecular Sirtuin Defatty-Acylase Inhibitors.

Human sirtuins (SIRT1-7) regulate not only deacetylation but also deacylation of fatty acid-derived acyl moieties (defatty-acylation) at the ε-amino group of lysine residues. SIRT-subtype-specific defatty-acylase activity modulators are needed for detailed investigation of the biological roles of these enzymes, and to find suitable small molecules, we require appropriate screening systems. Here, we designed and synthesized a set of SIRT defatty-acylase activity probes with various quencher moieties and peptide sequences based on our previously developed one-step FRET-based SIRT probe SFP3, using improved methodology. Scanning of this set of probes with SIRT isozymes revealed that certain probe/isozyme combinations showed especially high responses. To illustrate the utility of the combinations thus identified, we applied compound 18/SIRT2 for inhibitor screening of a large chemical library. This enabled us to discover a new small molecule SIRT2-specific defatty-acylase inhibitor.

Development of a Red-Light-Controllable Nitric Oxide Releaser to Control Smooth Muscle Relaxation in Vivo.

We designed and synthesized a novel Si-rhodamine derivative, NORD-1, as a red-light-controllable nitric oxide (NO) releaser, on the basis of photoredox parameter analysis. Red-light-responsive NO release from NORD-1 was confirmed by ESR spin trapping and quantified with an NO electrode and by means of Griess assay. The NO release cross section (ε656 nm·ΦNO) of NORD-1 was calculated to be 3.65 × 102, which is larger than that of a previously reported yellowish-green-light-controllable NO releaser, NO-Rosa5. The photoresponsiveness of NO release from NORD-1 was precise and efficient enough to induce vasodilation ex vivo under Magnus test conditions. Finally, we showed that intracavernous pressure (ICP) could be controlled in rats in vivo with the combination of NORD-1 and a red-light source without increasing systemic blood pressure, which is a serious side effect of usual NO releasers, such as nitroglycerin and isopentyl nitrite. NORD-1 is expected to be a useful chemical tool for NO research, as well as a candidate agent to control the circulatory system.

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.

Identification of Potent In Vivo Autotaxin Inhibitors that Bind to Both Hydrophobic Pockets and Channels in the Catalytic Domain.

Autotaxin (ATX, also known as ENPP2) is a predominant lysophosphatidic acid (LPA)-producing enzyme in the body, and LPA regulates various physiological functions, such as angiogenesis and wound healing, as well as pathological functions, including proliferation, metastasis, and fibrosis, via specific LPA receptors. Therefore, the ATX-LPA axis is a promising therapeutic target for dozens of diseases, including cancers, pulmonary and liver fibroses, and neuropathic pain. Previous structural studies revealed that the catalytic domain of ATX has a hydrophobic pocket and a hydrophobic channel; these serve to recognize the substrate, lysophosphatidylcholine (LPC), and deliver generated LPA to LPA receptors on the plasma membrane. Most reported ATX inhibitors bind to either the hydrophobic pocket or the hydrophobic channel. Herein, we present a unique ATX inhibitor that binds mainly to the hydrophobic pocket and also partly to the hydrophobic channel, inhibiting ATX activity with high potency and selectivity in vitro and in vivo. Notably, our inhibitor can rescue the cardia bifida (two hearts) phenotype in ATX-overexpressing zebrafish embryos.

Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples.

We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we "count" the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity-based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester-hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.

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.

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 an ENPP1 Fluorescence Probe for Inhibitor Screening, Cellular Imaging, and Prognostic Assessment of Malignant Breast Cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein that is involved in bone metabolism and insulin resistance, hydrolyzes 2',3'-cGAMP (a STING ligand that promotes innate immunity), and is associated with cancer stemness in breast cancers and glioblastoma. Therefore, ENPP1 is considered a candidate therapeutic target and/or biomarker for early diagnosis of malignant tumors. In this study, we designed and synthesized a sensitive ENPP1 fluorescence probe, Tokyo Green (TG) mAMP. We used it to screen a chemical library for non-phosphate ENPP1 inhibitors. Structural optimization of a selected hit afforded a potent and specific ENPP1 inhibitor. We further found that ENPP1 mRNA expression in tissue samples from patients with triple-negative breast cancer was significantly inversely related to recurrence-free survival (RFS) and overall survival (OS), and TG-mAMP assay revealed a significant difference in ENPP1 activity between ENPP1 high-expressing and ENPP1 low-expressing samples. Our results suggest that TG-mAMP assay might be a rapid and inexpensive tool for predicting the prognosis of patients with malignant breast cancers.

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.