Validation TeamThe Chemical Library and Bioprobe Research Group collects many natural compounds and stores them in the RIKEN Natural Product Depository (NPDepo) that was constructed recently.
A good chemical library should contain many compounds with various useful biological activities, therefore our team will validate the activity of the compounds in NPDepo by actual screening of the library through various assay systems.
For this purpose, we will develop high throughput screening systems using chemical array and liquid handling robots to screen the large library.
Besides developing new screening systems to identify compounds with novel biological activities, our team also conducts basic research to study the mechanisms behind the biological activities of the compounds.
【Researchers' theme】
Small molecule inhibitors of cell cycle regulatory proteins
 N. WATANABE, Ph.D. |  We are studying the regulation of cell cycle regulatory proteins, especially proteins that regulate mitosis. A complex of mitotic cyclin and cyclin dependent kinase (CDK) is the main regulator of mitosis and there are several proteins that regulate the activity of the complex. One such protein we have focused on is Wee1, a protein kinase that inactivates cyclin/CDK until cells become ready for mitosis. Upon the onset of mitosis, Wee1 is inactivated both by protein phosphorylation and degradation. As shown in the figure, we have recently identified a protein kinase cascade that induces the degradation of human somatic type Wee1 (Wee1A) through protein ubiquitination and proteasome dependent manner. We also study an HIV-1 encoded protein, Vpr, that also inhibits the entry of mitosis. Vpr is important in AIDS pathogenesis, therefore we would like to know its mode of action. Using the knowledge about the action of these cell cycle regulatory proteins, we are currently focusing on the isolation of small molecule inhibitors of them. We recently established a screening system for the inhibitors of phosphorylation dependent protein-protein interaction such as polo box domain (PBD) dependent binding that is important for the Wee1 degradation upon the onset of mitosis. We also made a screening system for Vpr inhibitors using budding yeast, Saccharomyces cerevisiae, and isolated inhibitors of Vpr. |
Stress meets development in p38 MAP kinase
 T. SUDO, Ph.D. | We are exposed to various stresses, ranging from drastic environmental changes to long-lasting unseen moderate stimuli, resulting in acute or chronic diseases and more seriously leading to be fatal. p38 MAP kinase has been shown to play important roles in stress responses including inflammatory response, apoptosis and differentiation. We have been studying the physiological roles of p38, through the establishment and the analysis of p38 KO mouse, and also by elucidation of the function of the specific interaction of p38 with p62/SQSTM1 under cytokine induced signaling, to regulate stress signaling. In the course of the study, we found molecular mechanisms that would be possible targets for drug discovery and we developed a bio microplate reader "HiTS" in collaboration with Scinics Corp. to facilitate a new screening method. We will continue our challenges to develop the ways to prevent, to diagnose and to cure the diseases caused by p38 mal-functioning. |
Studies for tumor metastasis for antitumor drug development
 S. SIMIZU, Ph.D. |  Recently, a novel photo-cross-linked chemical microarray has been developed in our laboratory. We are currently using transient transfected-HEK293T cell lysates, expressing a fusion of red fluorescence protein (RFP) and the protein of interest for the chemical array. This protocol simplifies the traditionally complicated process of1) purifying the protein of interest, 2) preparing antibodies for the purified protein, 3) labeling the purified protein with the antibodies and 4) refolding. The method also prevents the non-specific binding (compared to the RFP protein alone). With the newly developed method, the size of the disease-related gene library has been steadily increasing. |
Application of chemical biology to higher plants.
 T. ITO, Ph.D. |  Discovery of novel genes involved in plant productivity is one of the objectives for plant science. To this end, we have been studying pollen maturation process and environmental stress response in higher plants by use of a model organism, Arabidopsis thaliana. Male sterile trait is applicable to agriculture such as hybrid seed production. To produce engineered male sterile plants, elucidation of pollen maturation process is essential. We showed that Arabidopsis MALE STERILITY1 (MS1) functions as a key transcription factor and regulates many downstream genes necessary for pollen maturation. We are (1) investigating these downstream gene functions, and (2) searching for bioprobes interacting with MS1 and the downstream proteins. There is a steady increase in farmland inappropriate for cultivation because of drought and high salinity. To elucidate the response mechanism against environmental stresses such as drought and salinity, we focus on abscisic acid (ABA), so called “a stress phytohormone”. To isolate bioprobes affecting ABA biosynthetic/signal transduction pathway, we are (3) searching for bioprobes which inhibit or activate ABA functions. |
Proteomics-based analysis of the effect of small molecules.
 M. MUROI, Ph.D. |  New biologically active small molecules have been isolated from microbial metabolites by the cell-based assay system in our laboratory. However, identification of molecular targets of the new compounds is usually difficult and a time-consuming process. Proteomics was therefore applied to predict the targets of such active small molecules. Depending on the targets of the small molecules, profiles of expression level and modification of proteins within the cells will be altered. Using 2-Dimensional Fluorescence Differential Gel Electrophoresis (2D-DIGE), proteome expression profiles have been obtained from the cells treated with the authentic small molecules and based on the expression profiles we analyze the newly isolated compounds. |
Chemical array-based screening of bioprobes
 Y. KONDOH, Ph.D. |
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In chemical genetics, the technology for high-throughput screening of a bioprobe regulating a protein function is very important. The identification of small-molecule bioprobes for a protein of interest can facilitate not only the functional analysis of the protein but also the development of clinical drugs. We have developed chemical array "NPDepoArray" for ultra-high throughput screening of bioprobes. The chemical array contains 2-3 thousands of small molecules immobilized with a unique photo-cross-linking approach in a functional-group-independent manner. Chemical array-based screens have enabled the discovery of small molecules that bind target proteins of interest.
By the present, 15,000 of small molecules; natural products, its derivatives, drugs, etc, were microarrayed as chemical array series “NPDepoArray” and new stored small molecules are also microarrayed. We are now searching bioprobes for a variety of proteins associated with human diseases by using chemical array.Screening for new bioprobes and the analysis of its mechanism of action
 M. KAWATANI, Ph.D. | We are studying the development of new bioactive small-molecule compounds that regulate growth and differentiation of mammalian cells. We obtain unique bioactive compounds from the chemical library of RIKEN NPDepo by cell- or enzyme-based screening, elucidate their molecular targets and mechanisms of action, and verify the effectiveness as a therapeutic agent using a disease-model animal. Our current work is focused on the following subjects;  1) Screening for small molecules that disrupt osteoclast function and the analysis of its mechanism of action.2) Analysis of the mechanism of action of antiproliferative compounds GUT-70 and BNS-22. 3) Screening for differentiation-inducing small molecules of human leukemia HL-60 cells and the analysis of its mechanism of action. 4) High-content screening based on cellular phenotype. 5) Screening of aminoacyl-tRNA synthetase inhibitors. |
Investigation of the molecular mechanism in autophagy
 A. NAKAI, Ph.D. | Autophagy, an evolutionarily conserved process for bulk degradation of cytoplasmic components, serves as a cell survival mechanism in starving cells. In autophagy, cytoplasmic proteins or dysfunctional organelles are sequestered in a double-membrane-vesicle, termed autophagosome, delivered to the lysosome by fusion and then degraded. Although altered autophagy has been observed in various diseases, it remains unclear whether autophagy plays a beneficial or detrimental role in the cell. Especially, key modulaters, direct interaction for autophagy, are not still found out. As a result, to determine the molecular mechanism of autophagy in cells, we are trying to perform screening of small molecules that modulate autophagy. The aim of this study is to find small molecules that can control autophagy and develop of therapeutic agents.  |
Analysis of interactions between the bioprobe and their target protein by x-ray crystallography.
 H. OKUMURA, Ph.D. |  In the products metabolized by the microorganisms, there are a lot of compounds to show the bioactivity in the cell cycle inhibition, anti-cancer, etc. These are also useful to investigate the biological phenomenon as bioprobes. Structural information about the binding mode of the bioprobe to the target protein is important and indispensable for the investigation of the interactions between the bioprobe and the target protein and for the medicine development. Protein x-ray crystallography is a powerful tool to clarify the interaction of these molecules. In order to investigate the details of the interactions, we perform the co-crystallization of bioprobe/protein complexes and the structure analysis using SPring-8 RIKEN Beamline. |
Discovery of bioactive compound through morphology-based chemical-genetic screens
 Y. FUTAMURA, Ph.D. | Chemical-genetics is the study of gene-production in a cellular context using exogenous ligands. In this approach, various small molecules that can bind directly to specific proteins and modulate their protein functions are required. Since phenotype-based chemical-genetic screens have been used to help discover small, cell-permeable bioactive molecules, we thus have searched colorful chemical tools on the basis of their ability to interconvert cellular states from pathologic to wild type, or alter cellular morphology of interest from secondary metabolites of microorganisms and the library of natural products, NPDepo. The compound identified in this manner would be a powerful tools and potential therapeutic agents because they would be capable of inducing or suppressing formation of a disease state in a rapid and conditional manner. Furthermore, by studying the molecular targets of such active compounds it might be possible to learn about the molecular basis of biological processes and to get more fruitful drug development outcomes. This study serves as a potent driving force for advancing chemical biology and thus drug discovery research. |
Optimization of Chemical Array Using Various Kinds of Linkers
T.SAITO, Ph.D. | The chemical array can mount several thousands of small molecules on a slide glass, and it reveals the interactions between proteins and small molecules. In order to fix the small molecules on the slide glass, the chemical structure for bonding which is called linker is necessary. The linker, called photo affinity linker, has trifluoromethyldiazirine group, and it can combine with various kinds of small molecules by UV irradiation. The reaction is independent from the functional group of the small molecules. In this work, the length of the linker and trifluoromethyldiazirine linker containing the dummy linker is focused in order to optimize the interaction of small molecules. |
