Screening for new bioprobes and the analysis of its mechanism of action

Senior Research Scientist
M. Kawatani

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.

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.

Terpendole E biosynthetic gene cluster: analysis and application to drug development

Senior Research Scientist
T. Motoyama

Mitotic kinesin Eg5 is a protein essential for cell cycle progression and attracting attention as a target for anticancer drugs with low side effects. Terpendole E is the first kinesin Eg5 inhibitor of natural product origin. However, yield of terpendole E production is low and biological activity of terpendole E is relatively low. Aim of this research is to solve these problems by analyzing the biosynthetic gene cluster for terpendole E of the producing filamentous fungus Chaunopycnis alba.
This project consists of the three research subjects as follows.

  1. Isolation of the gene cluster for terpendole E and functional analysis of each genes.
  2. Isolation of new terpendole E analogs by modifying the biosynthetic gene cluster.
  3. Isolation of activating compounds for terpendole E biosynthetic gene cluster from RIKEN natural products depository NPDepo and application of the compounds to overproduction of terpendoles.
Goal of this research is to get large amounts of new terpendoles that can be a lead compound for medicine development.

Proteomics-based analysis of the effect of small molecules.

Makoto Myroi
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.

Discovery of bioactive compound through morphology-based chemical-genetic screens

Yushi Futamura
Research Scientist
Y. Futamura

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.

Systematic fractionation and isolation of microbial metabolites

Toshihiko Nogawa
Research Scientist
T. Nogawa

There is a lot of microbial species with an amazing capability to produce various secondary metabolites. Such metabolites contain unique structures and various biological activities. Due to their wide variety of physicochemical properties and a small abundance, we have not yet isolated all of the secondary metabolites and have not yet investigated their potentially useful activities. We therefore have been constructing the chemical and fraction libraries of the secondary metabolites.
In order to construct the libraries, we have developed a novel method to systematically and exhaustively isolate the metabolites. The method is also useful in effectively finding the novel and valuable compounds. Each fraction is analyzed on LC-MS to reveal the UV and mass spectra of each metabolite within the fraction. The results are stored into the database, which provides useful information to identify known compounds, and to discover novel metabolites. The metabolites and the fractions are tested for their biological activities using various high throughput screenings to investigate their properties.。

Discovery of bioprobes by the chemical array-based approach

Special Postdoctoral
T. Kawamura

Obtainment of unique bioprobes, small molecules which can modulate the functions of biomolecules, is required for chemical biology. To obtain bioprobes, we constructed a new type of chemical array which enabled us to perform high-throughput screening for candidate bioprobes.
However, additional validation studies, to investigate whether the candidates obtained through chemical array screening can modulate the functions of the target proteins in vitro and in intact cells, are required to obtain bioprobes. This study aims to discover new bioprobes especially modulating the functions of cancer-related proteins, and investigate the functions of the proteins. In addition, we attempt to establish a new method to discover bioprobes modulating cancer-related proteins using the chemical array-based approach.