Systematic search and isolation of microbial secondary metabolites

Toshihiko Nogawa
Research Scientist
T. Nogawa

Microbes such as Streptomyces and fungi produce various structurally unique secondary metabolites with interesting biological activities. Such metabolites have been used for medicinal drugs, agrochemicals, and bioprobes to investigate biological functions in a chemical biology study. To search and isolate such important metabolites efficiently, we have constructed a microbial metabolite fraction library, which contains semi-purified extracts generated by basic chromatographic techniques. The library is analyzed by DAD-LC/MS to afford physicochemical information including retention time, UV and mass spectra of each metabolite within the fraction. Based on the information, we have constructed an original database named NPPlot (Natural Products Plot). It is a distribution map in which metabolites are appeared as dots in 2D area by retention time and molecular weight. Combination of the fraction library and NPPlot is a powerful tool for screening of structurally novel and interesting metabolites, and we have isolated various interesting metabolites by the system.

Exploring the chemical inducer for differentiation of mushroom focused on “chemical ecology”


Special Postdoctoral
Researcher
J. Otaka

Microorganisms produce diverse and structurally unique secondary metabolites such as terpenoid, polyketide, alkaloid. What is the role of these compounds in nature? One appropriate interpretation of significance for producing natural products is a “chemical signals” involving biological interaction such as hormone. Because of our interests in chemical signal into morphological differentiation, we selected mushrooms showing dramatic phenomenon from spore stage to fruiting body formation. To isolation and identification of inducer for mushroom differentiation, we adopted chemical ecology-oriented exploring method. An underlying concept in this method is that secondary metabolites emitted by other microorganism affect mushroom differentiation.

Chemical synthesis of bioactive compounds


Senior Scientist
T. Shimizu

Many bioactive compounds have been found by the screening of compounds from microbial source and RIKEN NPDepo in the chemical biology research group. They are expected to be utilized for medicinal drugs, agrochemicals, and also bioprobes as biochemical tools for investigating cell function. Derivatives of these compounds have been chemically synthesized for the identification of the target protein, structure-activity relationship study, and optimization of the active structure. Structure determination and synthesis of compounds for the elucidation of biosynthetic pathway are also carried out.

Chemical array-based screening of bioprobes

Yasumitsu Kondo
Senior Research Scientist
Y. Kondo

arrayer 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.array
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.

Utilization of MorphoBase, an encyclopedia of cell morphology database, for bioprobe- and drug-discovery

Yushi Futamura
Research Scientist
Y. Futamura

Tumor cells often dynamically and specifically change shape depending on the mode of action of a treated drug, and experienced cell biologists can judge the presumed molecular target of a test compound by simple observation of typical morphological changes. This prompted us to accumulate information on morphological changes induced by various compounds with known mechanisms, and construct a database linking morphology to drug function, named “MorphoBase.” Recently, we have developed a high-content imaging method and a phenotype profiling system based on statistical analyses of multiparametric phenotype responses, to identify the molecular targets of several bioprobes. Now we are going to determine the mode of action of compounds of interest, and explore small drug-like compounds with unknown mechanism of action from microbial sources and a chemical library NPDepo with the aid of MorphoBase.

Development of bioprobes targeting cancer metabolism

kawatani
Senior Research Scientist
M. Kawatani

Cancer cells must rewire cellular metabolism to satisfy the demands of growth and survival. Although altered tumor metabolism is now a generally regarded hallmark of cancer, the detailed mechanisms remain to be fully elucidated, due to their characteristic features of complexity, diversity and adaptability. To better understand and regulate cancer metabolism, chemical biology approach would be a powerful strategy. We develop new bioprobes targeting cancer metabolism using our original resources and basic technologies for chemical biology research. By using these bioprobes, we challenge the elucidation of cancer metabolism and the development of new therapeutic strategies in cancer.

Proteomics-based analysis of the effect of small molecules.

Makoto Muroi
Senior Research Scientist
M. Muroi

proteomics 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.

Fungal secondary metabolites: biosynthesis, roles in symbiosis, and applications

motoyama
Senior Research Scientist
T. Motoyama

Filamentous fungi (molds) are versatile organisms. Filamentous fungi have ability to make medicines like penicillin and statins, and fermentation foods like sake. Furthermore, some filamentous fungi are known as plant and animal pathogens and mycotoxin producers. Filamentous fungi have been a major source of secondary metabolites. Recently, it has become apparent that huge numbers of secondary metabolism genes of unknown functions are sleeping in the genomes of filamentous fungi. We predict that some secondary metabolites are involved in biological interactions including symbiosis. We aim to elucidate the roles of fungal secondary metabolites in biological interactions, and to develop agrochemicals and medicines from fungal secondary metabolites involved in the interactions.
This project consists of the three research subjects as follows.

  1. Activation of fungal secondary metabolite biosynthetic gene clusters and analysis of the biosynthetic mechanisms
  2. Elucidation of the roles of fungal secondary metabolites in biological interactions including symbiosis
  3. Development of agrochemicals and medicines from fungal secondary metabolites

Analysis of fungal secondary metabolites biosynthesis and regulatory mechanism

yun
Research Scientist
C.S. Yun

Fungi produce variety kind of small molecules known as secondary metabolites and attracted intense research interest due to their bioactivities. The recent sequencing of fungal genomes has shown that fungi are richer in secondary metabolism-related genes than previously believed. However, the majority of these genes are silent or quiescent under laboratory culture conditions. Our research theme is activate these silent secondary metabolite biosynthetic genes to find out useful chemical compounds in fungi. Recently, we found biosynthetic gene of mycotoxin, tenuazonic acid in fungi by activation of tenuazonic acid biosynthetic gene. Now we are investigating the regulatory mechanism of tenuazonic acid production in fungi. We also trying to find out novel fungal secondary metabolites and elucidate their regulatory mechanism by using chemical biology methods.

Chemical biology approach for modulating plant defense system

yoshida
Postdoctoral Researcher
K. Yoshida

Discovery of small molecules that regulate plant stress responses is of great importance not only to the elucidation of plant defense system but to the agricultural aspects such as protection of crops in a changing climate often observed recently. The bioprobes that modulate phytohormone abscisic acid (ABA) signaling, which is involved in multiple processes in plants such as water stress control, seed dormancy, immune and growth would contribute to unveil signaling mechanisms whereas ABA receptors are encoded by multiple genes (e.g. 14 genes in the model plant Arabidopsis thaliana), making it difficult to genetically clarify their physiological roles in plants. So far chemical array screening identified ABA antagonists (RK438 and RK460) that physically bind to one of A. thaliana ABA receptors PYR1 from approximately 20,000 compounds. Further screening of agonists and antagonists that interact to other ABA receptors would present the potential of chemical biology approach to strengthen studies on plant defense.