Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies.

Covalent labeling systems that employ protein-tags or chemical probes to convert proteins into fluorescent conjugates are powerful tools for carrying out real time imaging and pulse-chase tracking studies that enable the spatiotemporal role of proteins in complex biological systems to be investigated. In this study, we have covalently modified a specific nucleophilic cysteine residue of the PYP-tag protein with weakly fluorescent α,ß-unsaturated ketone (conjugate addition) and α-halomethyl ketone (SN2 reaction) acceptors to afford highly fluorescent PYP-tag-dimethylaminocoumarin (DMAC) conjugates, whose ligands are covalently bound to the PYP-protein through stable thioether linkers. A chloromethylketone derived DMAC-CMK reagent was found to afford the best kinetic and stability profile for labeling the PYP-tag in cellular systems, with in vitro studies demonstrating that PYP-DMAC-CMK conjugates exhibit excellent photostability and cellular stability profiles which enables them to be used for long-term protein imaging studies in cellular systems. The potential of using this no wash fluorescent labeling PYP-tag-DMAC system to visualise dividing cells undergoing mitosis and for imaging a PYP-tag fused telomere binding protein bound to chromatin in cell nuclei has been demonstrated.

Glucose Tolerance-Improving Activity of Helichrysoside in Mice and Its Structural Requirements for Promoting Glucose and Lipid Metabolism.

An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for 14 days, improved the glucose tolerance in mice without affecting the food intake, visceral fat weight, liver weight, and other plasma parameters. In this study, using hepatoblastoma-derived HepG2 cells, helichrysoside, trans-tiliroside, and kaempferol 3-O-ß-D-glucopyranoside enhanced glucose consumption from the medium, but their aglycones and p-coumaric acid did not show this activity. In addition, several acylated flavonol glycosides were synthesized to clarify the structural requirements for lipid metabolism using HepG2 cells. The results showed that helichrysoside and related analogs significantly inhibited triglyceride (TG) accumulation in these cells. The inhibition by helichrysoside was more potent than that by other acylated flavonol glycosides, related flavonol glycosides, and organic acids. As for the TG metabolism-promoting activity in high glucose-pretreated HepG2 cells, helichrysoside, related analogs, and their aglycones were found to significantly reduce the TG contents in HepG2 cells. However, the desacyl flavonol glycosides and organic acids derived from the acyl groups did not exhibit an inhibitory impact on the TG contents in HepG2 cells. These results suggest that the existence of the acyl moiety at the 6'' position in the D-glucopyranosyl part is essential for glucose and lipid metabolism-promoting activities.

Chemical Tools with Fluorescence Switches for Verifying Epigenetic Modifications.

Epigenetic DNA and histone modifications alter chromatin conformation and regulate gene expression. A major DNA modification is methylation, which is catalyzed by DNA methyltransferase (Dnmt) and results in gene suppression. Compared to DNA, histones undergo a greater variety of modification types, one of which is the acetylation of lysine. While histone acetyltransferase (HAT) catalyzes acetylation and activates gene expression, histone deacetylase (HDAC) removes the modification and causes gene suppression. As precise regulation of these epigenetic marks on DNA and histones is critical for cellular functions, their dysregulation causes various diseases including cancer, metabolic syndromes, immune diseases, and psychiatric diseases. Therefore, elucidation of the epigenetic phenomena is important not only in the field of biology but also in medical and pharmaceutical sciences. Furthermore, this field is also attracting industrial interest, because small-molecule inhibitors modulate enzymatic activity for epigenetic modification and are used for cancer treatment. Under these circumstances, various methods for detecting epigenetic modifications have been developed. However, most methods require cell lysis, which is not suitable for real-time detection of enzymatic activity. Since fluorescent probes are attractive chemical tools to solve this issue, chemists made considerable efforts to create fluorescent probes for epigenetics. To date, we have particularly focused on HDAC activity and DNA methylation and have developed fluorescent probes for their detection. The first part of this review describes our recent efforts to develop fluorescent probes for detecting HDAC activity. Since the discovery of HDAC activity in the late 1960s, no fluorescent probe has been developed that can detect enzymatic reactions in a simple, one-step procedure despite its biological and medical importance. We designed fluorescent probes to overcome this limitation by devising two different types of fluorescence switching mechanisms, which are based on aggregation-induced emission (AIE) and intramolecular transesterification. Using these probes, we detected HDAC activity simply by mixing the probes and HDAC for the first time. In the second part, a hybrid approach using a protein-labeling system was employed to detect DNA methylation in living cells. So far, live-cell detection of DNA methylation was conducted by imaging the localization of Fluorescent Proteins (FPs) fused to a methylated DNA-binding domain. However, FP lacks a fluorescence switch and emits fluorescence without binding to methylated DNA. We created a hybrid probe that comprises a fluorogen and a protein and enhances fluorescence intensity upon binding to methylated DNA. To create the hybrid probe, we applied our protein labeling system using the PYP-tag that we previously developed. This method successfully visualized methylated DNA in living cells and verified its dynamics during cell division. Both of the above-mentioned fluorescent probes have great potential for use not only in HDAC and DNA methylation but also in other epigenetics-associated modifications. For example, the mechanism of the HDAC probes can be used to detect histone demethylation. The hybrid probe can be converted to a sensor for imaging acetylated or methylated histones. In this review, we mainly describe how we designed the probes using chemical principles and solved the current obstacles with the probe design and discuss the future prospects of these probes.

SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In†Vivo Imaging of Cell Metabolism.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA.

Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity.

Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity.

Histone deacetylases (HDACs) are key enzymatic regulators of many cellular processes such as gene expression, cell cycle, and tumorigenesis. These enzymes are attractive targets for drug development. However, very few simple methods for monitoring HDAC activity have been reported. Here, we have developed a fluorogenic probe, K4(Ac)-CCB, which consists of the histone H3 peptide containing acetyl-Lys and a coumarin fluorophore with a carbonate ester. By the simple addition of the probe to a HDAC solution, enzyme activity was clearly detected through spontaneous intramolecular transesterification, which renders the probe fluorescent. In addition, K4(Ac)-CCB can be applied to the evaluation of HDAC inhibitor activity. This is the first report to demonstrate the monitoring of HDAC activity by using a one-step procedure. Thus, our novel fluorogenic probe will provide a powerful tool for epigenetic research and the discovery of HDAC-targeted drugs.

Photocontrolled compound release system using caged antimicrobial peptide.

A novel photocontrolled compound release system using liposomes and a caged antimicrobial peptide was developed. The caged antimicrobial peptide was activated by UV irradiation, resulting in the formation of pores on the liposome surface to release the contained fluorophores. The compound release could be observed using fluorescence measurements and time-lapse fluorescence microscopy. UV irradiation resulted in a quick release of the inclusion compounds (within 1 min in most cases) under simulated physiological conditions. The proposed system is expected to be applicable in a wide range of fields from cell biology to clinical sciences.

Photoactive yellow protein-based protein labeling system with turn-on fluorescence intensity.

Protein labeling provides significant information about protein function. In this research, we developed a novel protein labeling technique by utilizing photoactive yellow protein (PYP). PYP is a small protein (14 kDa) derived from purple bacteria and binds to 7-hydroxycoumarin-3-carboxylic acid as well as to a natural ligand, 4-hydroxycinnamic acid, through a thioester bond with Cys69. Based on the structure and fluorescence property of this coumarin derivative, we designed two fluorescent probes that bind to PYP. One has an azido moiety, which allows stepwise labeling by click chemistry, and the other is a fluorogenic probe. The live-cell imaging and specific labeling of PYP were achieved by using both probes. The flexibility of the probe design and the small size of the tag protein are great advantages of this system against the existing methods. This novel labeling technique can be used in a wide variety of applications for biological research.