Mamoru Hatakeyama

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-κB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-κB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Development of novel magnetic nano-carriers for high-performance affinity purification

We developed novel magnetic nano-carriers around 180 nm in diameter for affinity purification. Prepared magnetic nano-carriers possessed uniform core/shell/shell nano-structure composed of 40 nm magnetite particles/poly(styrene-co-glycidyl methacrylate (GMA))/polyGMA, which was constructed by admicellar polymerization. By utilizing relatively large 40 nm magnetite particles with large magnetization, the magnetic nano-carriers could show good response to permanent magnet. Thanks to uniform polymer shell with high physical/chemical stability, the magnetic nano-carriers could disperse in a wide range of organic solvent without disruption of core/shell structure and could immobilize various kinds of drugs. We examined affinity purification using our prepared magnetic nano-carriers with anti-cancer agent methotrexate (MTX) as ligand. Our magnetic nano-carriers showed higher performance compared to commercially available magnetic beads in terms of purification efficiency of target including extent of non-specific binding protein.

Development and application of high-performance affinity beads: Toward chemical biology and drug discovery

In drug development research, the elucidation and understanding of the interactions between physiologically active substances and proteins that numerous genes produce is important. Currently, most commercially available drugs and physiologically active substances have been brought to market without knowledge of factors interacting with the drugs and the substances. Affinity purification is a useful and powerful technique employed to understand factors that are targeted by drugs and physiologically active substances. However, use of conventional matrices for affinity chromatography often causes a decrease in efficiency of affinity purification and, as a result, more practical matrices for affinity purification have been developed for application in drug discovery research. In this paper, we describe the development of high-performance affinity beads (SG beads and FG beads) that enable one-step affinity purification of drug targets and the elucidation of the mechanism of the action of the drugs. We also describe a chemical screening system using our affinity beads. We hope that utilization of the affinity beads will contribute to the progress of research in chemical biology.

A New Mechanism of Methotrexate Action Revealed by Target Screening with Affinity Beads

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-β-d-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitts lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.

A two-step ligand exchange reaction generates highly water-dispersed magnetic nanoparticles for biomedical applications

The high-temperature thermolysis of fatty acid–iron complexes generates magnetic nanoparticles (MNPs) of a precisely controlled size coated with fatty acids and dispersed in oil. Because they are water-immiscible, MNPs are unsuitable for water-based biomedical applications. Ligand exchange reactions that transform oil- into water-dispersed MNPs have attracted considerable attention, but are difficult to perform. In this paper, we report the successful preparation of size-controlled and highly water-dispersed MNPs, which have 4, 8 and 20 nm diameter by a unique two-step ligand exchange reaction. As temporary ligands, we selected thiomalic acid (TMA), which possesses moderate affinity toward MNPs and is soluble in both oil and water to remove fatty acids by XANES analyses. Next we selected the citric acids as secondary ligands for TMA-exchanged MNPs to be highly dispersed in water to remove TMA from the surface of MNPs. And the resulting highly water-dispersed MNPs are expected to be available as MRI contrast agents and hyperthermia carriers.

Tools and methodologies capable of isolating and identifying a target molecule for a bioactive compound

Elucidating the mechanism of action of bioactive compounds, such as commonly used pharmaceutical drugs and biologically active natural products, in the cells and the living body is important in drug discovery research. To this end, isolation and identification of target protein(s) for the bioactive compound are essential in understanding its function fully. And, development of reliable tools and methodologies capable of addressing efficiently identification and characterization of the target proteins based on the bioactive compounds accelerates drug discovery research. Affinity-based isolation and identification of target molecules for the bioactive compounds is a classic, but still powerful approach. This paper introduces recent progress on affinity chromatography system, focusing on development of practical affinity matrices and useful affinity-based methodologies on target identification. Beneficial affinity chromatography systems with using practical tools and useful methodologies facilitate chemical biology and drug discovery research.

DNA-carrying latex particles for DNA diagnosis 2. Distinction of normal and point mutant DNA using S1 nuclease

Abstract In our previous paper, we reported a DNA diagnosis system in which fully complementary DNA was selectively hybridized with a DNA immobilized on latex particles but one-point mutant DNA was not. The system, however, included a few disadvantages. To overcome them, a single-strand DNA cleaving enzyme (S1 nuclease) was used in this study. S1 nuclease cleaved the unhybridized and loosely hybridized DNAs on the particles and enabled the determination of the fully complementary hybrid. This method saved us a troublesome labeling of the samples.

High-performance affinity beads for identifying anti-NF-kappa B drug receptors.

will not work in cell lines that fail to express either calcineurin or NF-AT. These problems can be circumvented by cotransfection with NF-AT and/or calcineurin expression plasmids. Electroporation has proved to be an easy and effective means of transfecting Jurkat cells with plasmid DNA, whereas other cell lines may require a different means of transfection (liposomes, retrovirus, CaC12, etc.). This assay could be adapted to yield significant information about calcineurin activity in different human cell lines and provide a readout of H202-induced oxidative stress.

High-performance affinity chromatography method for identification of L-arginine interacting factors using magnetic nanobeads.

L-Arginine exhibits a wide range of biological activities through a complex and highly regulated set of pathways that remain incompletely understood at both the whole-body and the cellular levels. The aim of this study is to develop and validate effective purification system for L-arginine interacting factors (AIFs). We have recently developed novel magnetic nanobeads (FG beads) composed of magnetite particles/glycidyl methacrylate (GMA)-styrene copolymer/covered GMA. These nanobeads have shown higher performance compared with commercially available magnetic beads in terms of purification efficiency. In this study, we have newly developed L-arginine methyl ester (L-AME)-immobilized beads by conjugating L-AME to the surface of these nanobeads. Firstly, we showed that inducible nitric oxide synthase, which binds and uses L-arginine as a substrate, specifically bound to L-AME-immobilized beads. Secondly, we newly identified phosphofructokinase, RuvB-like 1 and RuvB-like 2 as AIFs from crude extracts of HeLa cells using this affinity chromatographic system. The data presented here demonstrate that L-AME-immobilized beads are effective tool for purification of AIFs directly from crude cell extracts. We expect that the present method can be used to purify AIFs from various types of cells.

DNA-carrying latex particles for DNA diagnosis. 1. Separation of normal and point mutant DNAs according to the difference in hybridization efficiency

Point mutation of specific genome DNA causes cancer. A system for rapid and sensitive detection of specific genome DNA is required because it enables early detection and treatment of cancer. We developed a new system for DNA diagnosis using DNA-carrying latex particles and succeeded to collect point mutant specifically from the mixture with normal DNA of codon 12 of K-ras gene.