Strategy for Construction of Homogeneous Glycoproteins in Mammalian Cells.

A general strategy that combines genetic code expansion with bio-orthogonal ligation techniques was developed and utilized to prepare homogeneously glycosylated receptors on the surface of mammalian cells. Using this approach, conjugates of the cell-surface oxytocin receptor (OTR) with oligosaccharides were efficiently generated in the cells. Cell studies revealed that glycans linked to the OTR are not essential for agonist-induced calcium flux and its internalization into cells via an OTR-mediated endocytosis.

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.

Nuclear Chloride Ion-Selective Fluorescent Probe and Its Biological Applications.

Owing to the biological significance of Cl in cells, several chemical fluorescent probes and biosensors have been constructed to monitor this anion in the cytosol and subcellular organelles. However, a fluorescent probe for the selective detection of nuclear Cl has not been described thus far. In the current study, we developed the first nuclear Cl-selective biosensor, Cl-YFP-NLS, whose fluorescence was effectively quenched by this anion, and demonstrated that it is an efficient and powerful tool for determining the levels of nuclear Cl.

A fluorescent probe for selective detection of lysosomal β-hexosaminidase in live cells.

Determining the activity of lysosomal β-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, βGalNAc-Rhod-CM(NEt), which consisted of a βGalNAc-linked rhodol unit serving as a β-hexosaminidase reactive fluorogenic moiety and a N,N'-diethylaminocoumarin (CM(NEt)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of βGalNAc-Rhod-CM(NEt) with β-hexosaminidase promoted generation of Rhod-CM(NEt), thereby leading to an increase in the intensity of fluorescence of Rhod.

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.

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.

-GlcNAcylation of RIPK1 rescues red blood cells from necroptosis.

Necroptosis is a type of cell death with excessive inflammation and organ damage in various human diseases. Although abnormal necroptosis is common in patients with neurodegenerative, cardiovascular, and infectious diseases, the mechanisms by which -GlcNAcylation contributes to the regulation of necroptotic cell death are poorly understood. In this study, we reveal that -GlcNAcylation of RIPK1 (receptor-interacting protein kinase1) was decreased in erythrocytes of the mouse injected with lipopolysaccharide, resulting in the acceleration of erythrocyte necroptosis through increased formation of RIPK1-RIPK3 complex.

A fluorescent probe to simultaneously detect both O-GlcNAcase and phosphatase.

O-GlcNAc modification of proteins often has crosstalk with protein phosphorylation. These posttranslational modifications are highly dynamic events that modulate a wide range of cellular processes. Owing to the physiological and pathological significance of protein O-GlcNAcylation and phosphorylation, we designed the fluorescent probe, βGlcNAc-CM-Rhod-P, to differentially detect activities of O-GlcNAcase (OGA) and phosphatase, enzymes that are responsible for these modifications.

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.

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.

Rhodamine-based cyclic hydrazide derivatives as fluorescent probes for selective and rapid detection of formaldehyde.

We describe fluorescent probes to detect formaldehyde (FA) in aqueous solutions and cells. The probes rapidly respond to FA in aqueous solutions and have great selectivity toward FA over other biologically relevant analytes. The results of cell studies reveal that probe 1 can be utilized to monitor endogenous and exogenous FA in live cells.

A thiol-activated fluorogenic probe for detection of a target protein.

A novel fluorogenic probe for facile and efficient detection of a target protein that binds to a bioactive small molecule was developed. The probe was composed of a thiol-activated fluorogenic moiety bearing an alkyne tag and a protein binding ligand linked to a photoreactive protein labeling group. The new probe in conjunction with affinity chromatography and mass spectrometry was utilized to identify a target protein in cell lysates.

Identification of Pyridinyltriazine Derivatives as Potent panFGFR Inhibitors against Gatekeeper Mutants for Overcoming Drug Resistance.

Although FGFR inhibitors hold promise in treating various cancers, resistance to the FGFR inhibitors caused by acquired secondary mutations has emerged. To discover novel FGFR inhibitors capable of inhibiting FGFR mutations, including gatekeeper mutations, we designed and synthesized several new pyridinyltriazine derivatives. A structure-activity relationship (SAR) study led to the identification of as a highly potent panFGFR inhibitor against wild-type and mutant FGFRs.

A fluorogenic probe targeting two spatially separated enzymes for selective imaging of cancer cells.

We describe a fluorogenic probe BocLys(Ac)-AB-FC targeting both histone deacetylases (HDACs) and cathepsin L, which are overexpressed in spatially separated subcellular organelles of cancer cells. The results show that this fluorogenic probe can be used for selective cancer cell imaging without interference arising from normal cells.

Novel Small Molecules Capable of Blocking mtRAS-Signaling Pathway.

RAS mutants are involved in approximately 30% of all human cancers and have been regarded as undruggable targets owing to relatively smooth protein surface and obscure binding pockets. In our previous study, we have demonstrated that GNF-7, a multi-targeted kinase inhibitor, possesses potent anti-proliferative activity against Ba/F3 cells transformed with NRAS-G12D. Based on our further analysis using Ba/F3 cells transformed with mtRAS, we discovered a series of pyrimido[4,5-]pyrimidin-2-one analogues as mtRAS-signaling pathway blockers.

Synthetic Na/K exchangers promote apoptosis by disturbing cellular cation homeostasis.

A number of artificial cation ionophores (or transporters) have been developed for basic research and biomedical applications. However, their mechanisms of action and the putative correlations between changes in intracellular cation concentrations and induced cell death remain poorly understood. Here, we show that three hemispherand-strapped calix[4]pyrrole-based ion-pair receptors act as efficient Na/K exchangers in the presence of Cl in liposomal models and promote Na influx and K efflux (Na/K exchange) in cancer cells to induce apoptosis.

Anti-cancer effects of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one derivatives on hepatocellular carcinoma harboring FGFR4 activation.

Hepatocellular carcinoma (HCC) is disease with a high mortality rate and limited treatment options. Alterations of fibroblast growth factor receptor 4 (FGFR4) has been regarded as an oncogenic driver for HCC and a promising target for HCC therapeutics. Herein, we report that GNF-7, a multi-targeted kinase inhibitor, and its derivatives including SIJ1263 (IC < 1 nM against FGFR4) are highly potent FGFR4 inhibitors and are capable of strongly suppressing proliferation of HCC cells and Ba/F3 cells transformed with wtFGFR4 or mtFGFR4.

Real-Time Spatial and Temporal Analysis of the Translocation of the Apoptosis-Inducing Factor in Cells.

Translocation of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus is crucial for AIF-mediated apoptosis. However, the lack of methods for real-time spatial and temporal analysis of translocation of functional AIF is a large hurdle to gain a detailed understanding of this process. In this study, a genetic code expansion technique was developed to overcome this hurdle.

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.

Identification of Thieno[3,2-]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of as a lead.

An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation.

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP).

A triple-targeting delivery system carrying two anticancer agents.

To improve tumor selectivity, a triple-targeting delivery system (Oct-FK(PBA-Az)-Dox) carrying two anticancer agents (apoptozole (Az) and doxorubicin (Dox)) was designed and synthesized. The results showed that both anticancer agents in Oct-FK(PBA-Az)-Dox are liberated in the presence of both HO and cathepsin B, which are normally present at high levels in tumors.

Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance.

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling.

Maintenance of the Neuroprotective Function of the Amino Group Blocked Fluorescence-Agmatine.

Agmatine, an endogenous derivative of arginine, has been found to be effective in treating idiopathic pain, convulsion, stress-mediated behavior, and attenuate the withdrawal symptoms of drugs like morphine. In the early stages of ischemic brain injury in animals, exogenous agmatine treatment was found to be neuroprotective. Agmatine is also considered as a putative neurotransmitter and is still an experimental drug.