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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Determinants of Ion-Transporter Cancer Cell Death.

Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.

A Glycoengineered Enzyme with Multiple Mannose-6-Phosphates Is Internalized into Diseased Cells to Restore Its Activity in Lysosomes.

In this study we developed an efficient method to prepare glycoengineered ß-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal ß-N-acetylhexosaminidase, more readily than commercialized human ß-hexosaminidase. Moreover, glycoengineered enzymes successfully removed GM2-ganglioside stored in lysosomes of diseased cells, indicating that its activity is restored in diseased cells. We also synthesized and applied a lysosome-targeting fluorogenic substrate to monitor endogenous and supplemental glycoengineered ß-N-acetylhexosaminidase activities in lysosomes. The results of this study indicate that the present strategy, which relies on genetic code expansion and bioorthogonal ligation techniques, is highly attractive to generate multi-M6P-containing lysosomal enzymes that can be used to study lysosomal storage disorders associated with lysosomal enzyme deficiencies.

Carbohydrate Microarrays Containing Glycosylated Fluorescent Probes for Assessment of Glycosidase Activities.

Carbohydrate microarrays, containing glycosylated fluorescent probes, have been constructed using N-hydroxysuccinimide (NHS) ester-conjugated BSA modified surfaces. When the carbohydrate moieties were cleaved from the fluorescent probes in the conjugates by glycosidases, the fluorescence signals of the probes were enhanced. In this study, we have applied these microarrays to profile glycosidase activities and have employed them to determine IC50 values of glycosidase inhibitors.

Analysis of binding properties of pathogens and toxins using multivalent glycan microarrays.

Pathogens infect hosts often through initial binding of their cell surface lectins to glycans expressed on the exterior of host cells. Thus, methods to evaluate the glycan-binding properties of pathogens are of great importance. Because of the multivalent nature of interactions of pathogens with glycans, the ability to assess the glycan density-dependent binding of pathogens is particularly important. In this study, we developed a facile technique to construct multivalent carbohydrate microarrays through immobilization of unmodified glycans on multivalent hydrazide-derivatized glass surfaces. This immobilization strategy does not require the use of multivalent glycoconjugates, which are typically prepared by using multistep sequences. The results of analysis of microarray images, obtained after incubation of multivalent glycan microarrays with cholera toxin B and pathogens such as uropathogenic E. coli and H. pylori, show that the binding affinities of toxins and pathogens for glycans are highly glycan density-dependent. Specifically, toxins and pathogens bind to glycans more strongly as the valency of the glycans on the microarrays is increased from 1 to 4. It is anticipated that the newly developed immobilization method will be applicable to the preparation of multivalent carbohydrate microarrays that are employed to evaluate multivalent glycan binding properties of a variety of pathogens and toxins.

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.