Chemical Biology Laboratory
1. Carbohydrate chemistry
1-1. Carbohydrate microarrays
We are preparing and applying carbohydrate microarrays for rapid analysis of glycan-mediated recognition events.
Glycans in cells and organisms play key roles in a wide range of physiological processes through interactions with glycan-binding proteins. Importantly, these biomolecular interactions are also implicated in various pathological processes. As a consequence, elucidation of glycan-mediated binding events and their consequences is highly important and interesting in basic biological research and biomedical applications.
To rapidly assess glycan-associated recognition events, we reported for the first time carbohydrate microarrays containing various glycans in 2002. Since 2002, we have shown that this microarray technology is ideally suited to study glycan-mediated binding events because they enable multiple parallel analyses of glycan-protein interactions using only small amounts of glycan samples. 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.
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So far, we applied this microarray technology for rapid analysis of glycan binding properties of lectins and antibodies, the quantitative measurements of glycan-protein interactions, detection of cells and pathogens, fast assessment of substrate specificities of glycosyltransferases and glycosidases, and rapid identification of functional glycans that elicit cell surface lectin-mediated cellular responses. Nowadays, we are conducting a research to expand this microarray technology.
Representative publication: Angew. Chem. Int. Ed. 2002, 41, 3180; J. Am. Chem. Soc. 2004, 126, 4812; Angew. Chem. Int. Ed. 2005, 44, 2881; Nat. Protoc. 2007, 2, 2747; J. Am. Chem. Soc. 2012, 134, 19287; J. Am. Chem. Soc. 2016, 138, 857; Chem. Sci. 2016, 7, 2084; Org. Lett. 2018, 20, 1240.
Review articles: Acc. Res. Acc. 2017, 50, 1069; Chem. Soc. Rev. 2013, 42, 4310; Chem. Commun. 2008, 7, 4389; Chem. Soc. Rev. 2008, 37, 1579; Chem. Eur. J. 2005, 11, 2894.
1-2. Glycoclusters
We are preparing and applying glycoclusters for detection of pathogens and mammalian cells.
Many pathogens and mammalian cells express glycan-binding proteins (lectins) on their surfaces. It is well-known that binding of monomeric glycans to cell-surface lectins is weak but multivalent interactions between glycans and the lectins are strong.
On this basis, we are preparing glycoclusters that efficiently detect mammalian cells and pathogens expressing lectins on their surfaces. For example, glycan-conjugated, fluorescent and magnetic nanoparticles were prepared and employed to detect Helicobacter pylori that is known to cause chronic gastritis, which may lead to peptic ulcer disease and gastric cancer. These dual-modal glyconanoparticles were also used to enrich the pathogen and to block adhesion of H. pylori to mammalian cells. In addition, peptide-based glycoclusters were prepared and applied to probe mammalian cell-surface lectins.
Representative publication: J. Am. Chem. Soc. 2015, 137, 5961; Mol. BioSyst. 2013, 9, 978; Chem. Asian J. 2011, 6, 2107.
1-3. Synthetic glycoproteins
We are developing methods to prepare homogeneous glycoproteins for biological and biomedical applications.
Owing to 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.
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We are developing a general method for the efficient preparation of homogeneous glycoproteins that utilizes a combination of genetic code expansion and chemoselective ligation techniques. For example, an alkyne tag-containing protein, generated by genetic encoding of an alkynylated unnatural amino acid, is coupled via click chemistry to versatile azide-appended glycans. The glycoproteins produced by this strategy successfully recognize mammalian cell-surface lectins and enter the cells through lectin-mediated internalization. Interestingly, the glycoprotein containing multiple mannose-6-phosphate (M6P) 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. This strategy is the foundation of a highly attractive approach to treat lysosomal storage disorders.
Representative publication: Cell Chem. Biol. 2018, 25, 1255; ACS Chem. Biol. under revision.
2. Organic fluorescent probes
We are synthesizing a variety of organic fluorescent probes that detect and image biologically important species, ions or enzymes in cells.
Owing to their high sensitivity, simplicity and fast response times, fluorescent probes have attracted considerable attention for applications in both optical imaging and analytical sensing. Fluorescent probes are typically small molecules that can be employed for quantitative determination of analytes in a highly selective and sensitive manner.
We are developing sensitive and selective fluorescent probes for monitoring and imaging of ions, species or enzymes in cells. For example, Lyso-MQAE and Mito-MQAE were prepared for studies of lysosomal and mitochondrial chloride ions in cells. By using these probes, we have found that chloride ions exit from lysosomes into the cytosol when lysosomal membrane permeabilization takes place by substances.
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In addition, we have shown that mitochondrial concentrations are lower than cytosolic concentrations and that when mitochondrial outer membrane permeabilization occurs by substances, cytosolic chloride ions enter mitochondria of the cells. Moreover, new activity-based probes for glycosidases were created to detect, capture and identify intracellular glycosidases.
Representative publication: Anal. Chem. 2020, 92, 12116; Chem. Sci. 2019, 10, 56 (cover paper); J. Am. Chem. Soc. 2010, 132, 601; Biomaterials 2012, 33, 7818; Nat. Protoc. 2014, 9, 1245; J. Am. Chem. Soc. 2006, 128, 14150; Nat. Protoc. 2007, 2, 1740; Angew. Chem. Int. Ed. 2012, 51, 2878.
Review articles: Chem. Soc. Rev. 2020, 49, 143; Chem. Soc. Rev. 2016, 45, 2976; Chem. Soc. Rev. 2014, 43, 16 (cover paper); Chem. Soc. Rev. 2011, 40, 4783; Chem. Soc. Rev. 2011, 40, 2120.
3. Target-oriented drug delivery systems
We are preparing novel drug delivery systems that specifically target disease cells, particularly cancer.
Every year, a number of new anticancer agents are developed over the world. However, the discovery of safe and efficacious anticancer drugs is a highly difficult task mainly owing to their lack of selectivity, which causes unwanted normal cell death (i.e., toxic side effects). To minimize toxic side effects of anticancer agents, elegant methods that more specifically target cancer cells are required in order to improve chemotherapy efficacy.
In this regard, we are developing novel drug delivery systems that specifically target disease cells. For example, we have devised multiple-targeting delivery systems that specifically kill and detect cancer by targeting receptors and intracellular enzymes that are overexpressed in cancers. We found that this delivery system more selectively kills cancer than does the single-targeting delivery system.
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Representative publication: Org. Lett. 2019, 21, 4628; Chem. Sci. 2013, 4, 947 (back cover paper); Angew. Chem. Int. Ed. 2004, 43, 1675.
4. Elucidation of cellular functions of proteins
We are conducting a study to understand cellular functions of proteins by incorporating unnatural amino acids into proteins in cells.
The genetic code expansion technique allows site-specific incorporation of various unnatural amino acids (UAAs) into proteins in cells. This technology has become a powerful tool in protein engineering owing to its technical simplicity and applicability to any protein.
We have developed the efficient fluorescence resonance energy transfer (FRET) system which consisted of Hsp70-YFP and fluorescent amino acid (ANAP)-incorporated Bax, which was generated by using genetic code expansion technology, and applied the FRET system to elucidate mechanisms on how apoptosis-inducing substances dissociate Bax from Hsp70. We are further applying the FRET system to understand effects of various substances on translocation of other types of proteins in cells.
Representative publication: J. Am. Chem. Soc. 2019, 141, 4273 (cover paper); ChemBioChem 2020, 21, 59.
5. Discovery of bioactive molecules
5-1. Small molecules regulating apoptosis & autophagy
We are discovering potent apoptosis and/or autophagy regulating small molecules.
Apoptosis, or programmed cell death, is an important biological process to maintain tissue homeostasis and to remove unwanted or damaged cells. Tumor cells are known to be resistant to apoptotic cell death via multiple anti-apoptotic processes. Thus, it is of great importance to discover potent apoptosis inducers that can be utilized as anticancer agents.
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Autophagy is a self-eating process that contributes to cell survival under starvation conditions. It is known that levels of autophagy in cancer cells are generally higher than those in normal cells, leading to suggestions that autophagy-disrupting agents may be potentially useful in treatment of cancers.
We are developing potent apoptosis and/or autophagy regulating small molecules. For instance, we discovered a small molecule named apoptozole that induces apoptosis in cancer cells by using high-throughput screening of small molecule libraries. We found that this molecule has apoptosis inducing activity and autophagy suppressing activity by inhibiting lysosomal Hsp70. In addition, we developed a small molecule, PZ-6-QN, that blocks the survivin-Smac interaction in mitochondria and induce apoptosis in cancer cells. We are also interested in uncovering novel autophagy regulating small molecules by using cell- and protein-based high-throughput methods.
Representative publication: Chem. Asian J. 2019, 14, 4035; Cell Chem. Biol. 2018, 25, 1242; Chem. Biol. 2015, 22, 391; J. Am. Chem. Soc. 2011, 133, 20267; Angew. Chem. Int. Ed. 2008, 47, 7466.
Review articles: Chem. Soc. Rev. 2012, 41, 3245 (cover paper).
5-2. Supramolecular chemical biology
We are studying bioactivity and cellular functions of supramolecules that recognize ions and biomolecules.
Maintenance of ion homeostasis of cells is essential for sustaining life processes, including proliferation, differentiation and apoptosis. Various cancer cells exhibit different patterns of ion channels, resulting in the dysregulation of ion concentrations and fluxes. Synthetic or semi-synthetic ion carriers or transporters that are able to alter intracellular ion concentrations are considered as attractive anticancer agents. In particular, small molecule-based anion transporters have received great attention due to their potential as therapeutic agents.
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We have for the first time shown that synthetic chloride ion transporters, calix[4]pyrroles, promote apoptosis by inducing sodium chloride influx, an increased level of reactive oxygen species, the release of cytochrome c from the mitochondria and caspase activation. Later, we have also found that a squaramide-based chloride ion transporter disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations.
Recently, we have elucidated the mode of actions of supramolecules on cancer cell death. We have also shown that synthetic glycan receptors promote apoptosis through caspase activation by binding to cell surface mannosides. Based on such findings, we are discovering bioactive supramolecules that can be used as therapeutic agents with international research collaborations.
Representative publication: Chem, 2019, 5, 2079; Nat. Chem. 2017, 9, 667; Nat. Chem. 2014, 6, 885-892; Chem. Sci. 2015, 6, 7284.
5-3. Small molecule-based cellular alchemy
We identified the first small molecule named neurodazine that differentiates human/mouse skeletal muscle cells into neurogenic cells.
Damaged tissues are normally regenerated by differentiation of cells. However, certain types of cells, such as neurons and cardiomyocytes, are not well regenerated under diseased conditions. In this case, cell therapy (or cell transplantation) is needed to regain function. Over a last decade, various cells have been generated from multipotent and pluripotent stem cells by introducing specific genes into cells. However, insertion of exogenous genetic materials into the host genome may be associated with unpredictable side effects.
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To avoid this issue, we have made an attempt to identify small molecules that convert somatic cells into specific cells (small molecule-based cellular alchemy). For example, we identified the first small molecule named neurodazine that differentiates human/mouse skeletal muscle cells into neurogenic cells by using high-throughput screening approach. This substance together with neurodazole was also found to have neurogenesis inducing activity in mouse embryonic carcinoma P19 cells, fibroblasts and neuroblastoma cells. Furthermore, we have shown that a sirtuin-1 selective inhibitor, EX-527, differentiates P19 cells into neurons with electrophysiological properties. Our goal of this research field is to uncover various small molecules that promote differentiation of somatic cells into a certain type of cells.
Representative publication: Angew. Chem. Int. Ed. 2014, 53, 9271; J. Am. Chem. Soc. 2007, 129, 9258; Nat. Protoc. 2008, 3, 835; Chem. Biol. 2015, 22, 1512; Mol. BioSyst. 2015, 11, 2727; Sci. Rep. 2016, 6, 34324.
Review articles: Chem. Soc. Rev. 2017, 46, 6241.