Asako Murata

Biochemical Target Isolation for Novices: Affinity-Based Strategies

Although a number of genomic and biochemical technologies are now used to elucidate the mechanisms of action of bioactive small molecules, affinity-based isolation of molecular targets is a classic, but still powerful, approach. This review highlights recent cases where biochemical isolation of target proteins of bioactive small molecules highlighted general strategies for a successful isolation and identification of molecular targets. This review is intended to be both an update on the most recent findings for those already active in the field of forward chemical genetics and a guide for scientists entering this burgeoning field.

Marine Natural Product Aurilide Activates the OPA1-Mediated Apoptosis by Binding to Prohibitin

Aurilide is a potent cytotoxic marine natural product that induces apoptosis in cultured human cells at the picomolar to nanomolar range; however, its mechanism of action has been unknown. Results of the present study showed that aurilide selectively binds to prohibitin 1 (PHB1) in the mitochondria, activating the proteolytic processing of optic atrophy 1 (OPA1) and resulting in mitochondria-induced apoptosis. The mechanism of aurilide cytotoxicity suggests that PHB1 is an apoptosis-regulating protein amenable to modulation by small molecules. Aurilide may serve as a small-molecule tool for studies of mitochondria-induced apoptosis.

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules.

A Chemical Probe that Labels Human Pluripotent Stem Cells

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Xanthone derivatives as potential inhibitors of miRNA processing by human Dicer: Targeting secondary structures of pre-miRNA by small molecules

In recent years, various biological processes have been found to be regulated by miRNA-mediated gene silencing. A small molecule that modulate the miRNA pathway will provide the biological tool for elucidating mechanisms of miRNA-mediated gene regulation, and can be the drug lead for miRNA related diseases. In this study, we demonstrated that an aminoalkoxy-substituted thioxanthone derivative interferes Dicer-mediated processing of pre-miRNA. Information about the interaction between these xanthone derivatives and pre-miRNAs will enable us to design and develop new small molecule-based inhibitors for miRNA pathway.

Fluorescent indicator displacement assay of ligands targeting 10 microRNA precursors

Fluorescent indicator displacement (FID) assay is a rapid and convenient assay for identifying new ligands that bind to the target molecules. In our previous studies, we have shown that a series of 2,7-diaminoalkoxy xanthone and thioxanthone derivatives can be used as fluorescent indicators for detecting the interaction between RNA and a ligand. The xanthone and thioxanthone fluorochromes showed efficient fluorescence quenching upon binding to target RNA. Subsequent displacement of the bound-fluorochrome with a ligand that binds more strongly to the target RNA led to the recovery of the fluorescence by releasing the fluorochrome from RNA. Here we report a pilot screening of a chemical library that contains 9600 structurally diverse compounds for molecules that bind to a specific miRNA precursor using the FID assay.

Formation of a ligand-assisted complex of two RNA hairpin loops.

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.

Ligand-inducible formation of RNA pseudoknot

Here, we demonstrate that a series of naphthyridine derivatives, naphthyridine carbamate tetramer (NCTn), can bind to the RNA CGG/CGG triad comprised of two single-stranded regions of a hairpin loop and a tail. Complete suppression of the binding by a single mutation indicated simultaneous binding of NCTn between hairpin loop and single stranded tail, leading to the formation of NCTn-induced pseudoknot.

A Ligand That Targets CUG Trinucleotide Repeats

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9-diaminoalkyl-substituted 1,10-phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (Tm ) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time-of-flight (ESI-TOF) mass spectrometry showed that DAP bound to r(CUG)9 but not to r(CAG)9 and r(CGG)9 . The dual luciferase assay clearly indicated DAP bound to the r(CUG)n repeat by affecting the translation in vitro.

Naphthyridine-Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA.

The expansion of CAG repeats in the human genome causes the neurological disorder Huntingtons disease. The small-molecule naphthyridine-azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA-binding to the CAG repeat DNA and RNA, we conducted systematic structure-binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA, modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)9 and r(CAG)9 . NBzA binding to d(CAG)9 was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time-of-flight (ESI-TOF) mass spectrometry. For the binding to r(CAG)9 , both NA and NBzA showed stepwise binding in ESI-TOF MS, and NBzA-binding to r(CAG)9 induced more extensive conformational change than NA-binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.