Selective fluorescent labeling of cellular proteins and its biological applications.

Proteins, which are ubiquitous in cells and critical to almost all cellular functions, are indispensable for life. Fluorescence imaging of proteins is key to understanding their functions within their native milieu, as it provides insights into protein localization, dynamics, and trafficking in living systems. Consequently, the selective labeling of target proteins with fluorophores has emerged as a highly active research area, encompassing bioorganic chemistry, chemical biology, and cell biology. Various methods for selectively labeling proteins with fluorophores in cells and tissues have been established and are continually being developed to visualize and characterize proteins. This review highlights research findings reported since 2018, with a focus on the selective labeling of cellular proteins with small organic fluorophores and their biological applications in studying protein-associated biological events. We also discuss the strengths and weaknesses of each labeling approach for their utility in living systems.

Glycosidase-targeting small molecules for biological and therapeutic applications.

Glycosidases are ubiquitous enzymes that catalyze the hydrolysis of glycosidic linkages in oligosaccharides and glycoconjugates. These enzymes play a vital role in a wide variety of biological events, such as digestion of nutritional carbohydrates, lysosomal catabolism of glycoconjugates, and posttranslational modifications of glycoproteins. Abnormal glycosidase activities are associated with a variety of diseases, particularly cancer and lysosomal storage disorders. Owing to the physiological and pathological significance of glycosidases, the development of small molecules that target these enzymes is an active area in glycoscience and medicinal chemistry. Research efforts carried out thus far have led to the discovery of numerous glycosidase-targeting small molecules that have been utilized to elucidate biological processes as well as to develop effective chemotherapeutic agents. In this review, we describe the results of research studies reported since 2018, giving particular emphasis to the use of fluorescent probes for detection and imaging of glycosidases, activity-based probes for covalent labelling of these enzymes, glycosidase inhibitors, and glycosidase-activatable prodrugs.

Glycan microarrays from construction to applications.

Through their specific interactions with proteins, cellular glycans play key roles in a wide range of physiological and pathological processes. One of the main goals of research in the areas of glycobiology and glycomedicine is to understand glycan-protein interactions at the molecular level. Over the past two decades, glycan microarrays have become powerful tools for the rapid evaluation of interactions between glycans and proteins. In this review, we briefly describe methods used for the preparation of glycan probes and the construction of glycan microarrays. Next, we highlight applications of glycan microarrays to rapid profiling of glycan-binding patterns of plant, animal and pathogenic lectins, as well as other proteins. Finally, we discuss other important uses of glycan microarrays, including the rapid analysis of substrate specificities of carbohydrate-active enzymes, the quantitative determination of glycan-protein interactions, discovering high-affinity or selective ligands for lectins, and identifying functional glycans within cells. We anticipate that this review will encourage researchers to employ glycan microarrays in diverse glycan-related studies.

Near-infrared (NIR) fluorescence-emitting small organic molecules for cancer imaging and therapy.

Near-infrared (NIR) fluorophores have unique features that endow them with several advantages over conventional shorter wavelength emitting dyes. As a result, they have been widely utilized as fluorescence and photoacoustic imaging agents, as well as photodynamic and photothermal therapeutic agents. However, non-targeting NIR fluorescence-emitting organic molecules have the drawback of low selectivity toward tumors, which potentially results in severe side effects caused by damage to normal tissues. Thus, the development of NIR fluorophore-based substances that target tumors is a highly active area in medicinal chemistry research. Research efforts carried out thus far have led to the development of a number of NIR fluorophore-based, tumor imaging and therapeutic agents. The discussion in this review focuses on the results of research reported in the 2012-2021 period, giving particular emphasis to studies of NIR small organic dye-based imaging and therapeutic agents that are designed utilizing cancer-selective strategies.

Multivalent glycans for biological and biomedical applications.

Recognition of glycans by proteins plays a crucial role in a variety of physiological processes in cells and living organisms. In addition, interactions of glycans with proteins are involved in the development of diverse diseases, such as pathogen infection, inflammation and tumor metastasis. It is well-known that multivalent glycans bind to proteins much more strongly than do their monomeric counterparts. Owing to this property, numerous multivalent glycans have been utilized to elucidate glycan-mediated biological processes and to discover glycan-based biomedical agents. In this review, we discuss recent advances (2014-2020) made in the development and biological and biomedical applications of synthetic multivalent glycans, including neoglycopeptides, neoglycoproteins, glycodendrimers, glycopolymers, glyconanoparticles and glycoliposomes. We hope this review assists researchers in the design and development of novel multivalent glycans with predictable activities.

The Glycan Microarray Story from Construction to Applications.

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands.

Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species.

Reactive oxygen (ROS) and nitrogen (RNS) species cause oxidative and nitrosative stresses, respectively. These stresses are implicated not only in diverse physiological processes but also in various pathological processes, including cancer and neurodegenerative disorders. In addition, some ROS and RNS in the environment are pollutants that threaten human health. As a consequence of these effects, sensitive methods, which can be employed to selectively monitor ROS and RNS in live cells, tissues and organisms as well as in environmental samples, are needed so that their biological roles can be understood and their concentrations in environmental samples can be determined. In this review, fluorescent, luminescent and colorimetric ROS and RNS probes, which have been developed since 2011, are comprehensively discussed.

Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.

Engineering of cell-surface receptors for analysis of receptor internalization and detection of receptor-specific glycosylation.

The epidermal growth factor receptor (EGFR) is a cell-surface glycoprotein that is involved mainly in cell proliferation. Overexpression of this receptor is intimately related to the development of a broad spectrum of tumors. In addition, glycans linked to the EGFR are known to affect its EGF-induced activation. Because of the pathophysiological significance of the EGFR, we prepared a fluorescently labeled EGFR (EGFR128-AZDye 488) on the cell surface by employing the genetic code expansion technique and bioorthogonal chemistry. EGFR128-AZDye 488 was initially utilized to investigate time-dependent endocytosis of the EGFR in live cells. The results showed that an EGFR inhibitor and antibody suppress endocytosis of the EGFR promoted by the EGF, and that lectins recognizing glycans of the EGFR do not enhance EGFR internalization into cells. Observations made in studies of the effects of appended glycans on the entry of the EGFR into cells indicate that a de-sialylated or de-fucosylated EGFR is internalized into cells more efficiently than a wild-type EGFR. Furthermore, by using the FRET-based imaging method of cells which contain an EGFR linked to AZDye 488 (a FRET donor) and cellular glycans labeled with rhodamine (a FRET acceptor), sialic acid residues attached to the EGFR were specifically detected on the live cell surface. Taken together, the results suggest that a fluorescently labeled EGFR will be a valuable tool in studies aimed at gaining an understanding of cellular functions of the EGFR.

NUAK2 Inhibitors, KHKI-01128 and KHKI-01215, Exhibit Potent Anticancer Activity Against SW480 Colorectal Cancer Cells.

BACKGROUND/AIM: NUAK family kinase 2 (NUAK2) is a promising target for cancer therapeutics due to its reported role in protein phosphorylation, a critical process in cancer cell survival, proliferation, invasion, and senescence. This study aimed to identify novel inhibitors that disrupt NUAK2 activity. We have already identified two KRICT Hippo kinase inhibitor (KHKI) compounds, such as KHKI-01128 and KHKI-01215. Our aim was to evaluate the impact of KHKI-01128 and KHKI-01215 on NUAK2 activity and elucidate its mechanism in colorectal cancer cells. MATERIALS AND METHODS: To evaluate anticancer properties of these inhibitors, four in vitro assays in the SW480 cell line (time-resolved fluorescence resonance energy transfer assay, KINOMEscan kinase profiling, viability, and apoptosis assays) and two pharmacological mechanism analyses (Gene Set Enrichment Analysis and western blotting) were performed. RESULTS: KHKI-01128 and KHKI-01215 exhibited potent inhibitory activity against NUAK2 (half-maximal inhibitory concentration=0.024±0.015 µM and 0.052±0.011 µM, respectively). These inhibitors suppressed cell proliferation, with half-maximal inhibitory concentrations of 1.26±0.17 µM and 3.16±0.30 µM, respectively, and induced apoptosis of SW480 cells. Gene Set Enrichment Analysis revealed negative enrichment scores of -0.84 for KHKI-01128 (false-discovery rate=0.70) and 1.37 for KHKI-01215 (false-discovery rate=0.18), indicating that both effectively suppressed the expression of YES1-associated transcriptional regulator (YAP) target genes. CONCLUSION: These results suggest that KHKI-01128 and KHKI-01215 are potent NUAK2 inhibitors with promising potential for pharmaceutical applications.

Intramolecular cyclization of N-cyano sulfoximines by N-CN bond activation.

Metal-free halogenated anhydrides promote the intramolecular cyclization of N-cyano sulfoximines. Trifluoro- or trichloroacetic anhydride (TFAA or TCAA, respectively) activate the N-cyano groups of N-cyano sulfoximines, leading to the intramolecular cyclization of 2-benzamide-N-cyano sulfoximines 1. This method results in excellent yields of thiadiazinone 1-oxides 2. A full intramolecular cyclization pattern was suggested by (i) labeling experiments with 13C, (ii) isolating of N-trifluoroacetyl sulfoximine 1ac, and (iii) confirming the generation of the intermediate 1ad by LC/MS analysis.

Acid-Catalyzed Hydrolysis and Intramolecular Cyclization of N-Cyano Sulfoximines for the Synthesis of Thiadiazine 1-Oxides.

Herein, we describe a novel approach for the practical synthesis of thiadiazine 1-oxides 10. The first example of an intramolecular cyclization with 2-N-cyano-sulfonimidoyl amides 9 to form the desired thiadiazine 1-oxides 10 was developed. One-pot acid-induced hydrolysis of the cyano group and the intramolecular cyclocondensation protocol readily provided various heterocyclic frameworks in good to moderate yields. Notably, the crystal structures of N-urea sulfoximine 11 and thiadiazine 1-oxide 10i have been determined using X-ray crystallography.

Efficient Preparation and Bioactivity Evaluation of Glycan-Defined Glycoproteins.

Owing to the generation of heterogeneous glycoproteins in cells, it is highly difficult to study glycoprotein-mediated biological events and to develop biomedical agents. Thus, general and efficient methods to prepare homogeneous glycoproteins are in high demand. Herein, we report a general method for the efficient preparation of homogeneous glycoproteins that utilizes a combination of genetic code expansion and chemoselective ligation techniques. In the protocol to produce glycan-defined glycoproteins, an alkyne tag-containing protein, generated by genetic encoding of an alkynylated unnatural amino acid, was quantitatively coupled via click chemistry to versatile azide-appended glycans. The glycoproteins produced by the present strategy were found to recognize mammalian cell-surface lectins and enter the cells through lectin-mediated internalization. Also, cell studies exhibited that the glycoprotein containing multiple mannose-6-phosphate residues enters diseased cells lacking specific lysosomal glycosidases by binding to the cell-surface M6P receptor, and subsequently migrates to lysosomes for efficient degradation of stored glycosphingolipids.

Bacterial Lectin-Targeting Glycoconjugates for Selective Elimination of Pathogenic Bacteria.

Herein we report a strategy to eradicate pathogenic bacteria selectively, which utilizes bacterial lectin-targeting glycoconjugates that contain an epitope or a photosensitizer to promote antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT), respectively. Our results show that death promoted by using the designed synthetic glycoconjugates coupled with ADCC or PDT takes place selectively in pathogenic bacteria expressing lectins on their surfaces.

Cancer cell death using metabolic glycan labelling techniques.

Herein we describe a method for inducing cancer cell death, which relies on the use of a H2O2-responsive glycan metabolic precursor in conjunction with antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT).

A lysosomal chloride ion-selective fluorescent probe for biological applications.

Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions, measurements of their levels are of great importance to understand lysosome-associated biological events. However, appropriate probes to selectively detect Cl- ions within acidic lysosomes have not been developed to date. In this study, we prepared MQAE-MP, a lysosomal Cl--selective fluorescent probe, and applied it to gain information about biological processes associated with lysosomes. The fluorescence of MQAE-MP is pH-insensitive over physiological pH ranges and is quenched by Cl- with a Stern-Volmer constant of 204 M-1. Because MQAE-MP detects lysosomal Cl- selectively, it was employed to assess the effects of eleven substances on lysosomal Cl- concentrations. The results show that lysosomal Cl- concentrations decrease in cells treated with substances that inhibit proteins responsible for lysosomal membrane stabilization, induce lysosomal membrane permeabilization, and transport lysosomal Cl- to the cytosol. In addition, we investigated the effect of lysosomal chloride ions on the fusion of autophagosomes with lysosomes to generate autolysosomes during autophagy inhibition promoted by substances. It was found that changes in lysosomal Cl- concentrations did not affect the fusion of autophagosomes with lysosomes but an increase in the cytosolic Ca2+ concentration blocked the fusion process. We demonstrate from the current study that MQAE-MP has great potential as a lysosomal Cl--selective fluorescent probe for studies of biological events associated with lysosomes.

Trifunctional Fluorogenic Probes for Fluorescence Imaging and Isolation of Glycosidases in Cells.

For both fluorescence imaging and isolation of glycosidases in cells, we prepared novel activity-based, trifunctional fluorogenic probes that consist of (1) a sugar moiety as a glycosidase substrate, (2) a fluoromethylated coumarin for fluorescent labeling, and (3) an alkyne tag for click reaction to enable isolation of the labeled enzyme. One probe, ß-GlcNAc-CM-F, was employed to fluorescently detect endogenous O-GlcNAcase in cells and to isolate the labeled enzyme by affinity chromatography.