Research

Protein medicinal and material chemistry

A general methodology for the site-specific incorporation of unnatural amino acids with new physicochemical properties into proteins in vivo has recently been developed, providing powerful tools for studying protein structure and function. Taking advantage of this methodology, we express proteins containing unnatural amino acids with chemically orthogonal functional groups, which thereby can be further modified with various molecules such as fluorophores, lipids and polyethylene glycols (Figure 1a). The chemoselective reactions are also applied for producing protein-protein conjugates as potential protein-based therapeutics and materials (Figure 1b).

Due to structural diversity of unnatural amino acids as protein building blocks, it is now possible to examine the structure-activity relationship of the protein-based drugs (Figure 1c). As such, we are trying to engineer many of previously reported binding proteins and protease inhibitors with unnatural amino acids, aiming at improved biological activity and selectivity.

Figure 1. Protein medicinal and material chemistry via unnatural protein engineering

 

Identification of new biological pathways by photocrosslinking

Site-specific incorporation of a photo-crosslinking amino acid, p-benzoyl-L-phenylalanine (Bpa), into proteins provides a powerful tool to capture transient protein-protein interactions by covalent bond formation between interacting protein molecules upon UV irradiation (Figure 2). Based on this proven methodology, we are trying to identify the proteins that are involved in secretion and post-translational modification of certain classes of proteins in E. coli.

   Figure 2. Photocrosslinking of interacting proteins

 

Novel RNA-binding molecules as potential therapeutics

A variety of RNA-mediated biological processes are major targets of therapeutic intervention. In order to identify highly effective RNA-binding molecules from small molecule libraries, we are developing a fluorescence resonance energy transfer (FRET)-based high-throughput RNA binding assay system (Figure 3). The parallel setup of HTS system for many different RNAs will be valuable for observing a selectivity profile of the RNA-binding molecules.

Figure3. FRET-based RNA binding assay

 

We are creating small molecule libraries with many different molecular scaffolds by diversity-oriented synthesis. Covalent conjugates of two or more known RNA-binding structures are also prepared, aiming at enhanced binding affinities by cooperative binding.

In parallel, we evolve sequence-specific RNA-binding peptide scaffolds by in vitro phage or bacterial display techniques. A microhelix derived from the acceptor stem of tRNAs is used as a model RNA system. Because the sequence of this tRNA region is specifically recognized by its cognate aminoacyl-tRNA synthetase (aaRS), the aaRS’s peptide scaffold is used as a feasible starting point for evolution of the sequence-specific RNA-binding peptide.

 

Peptidomimetic buiding blocks

A novel isosteric structure of an a-amino acid derived from isopropylidene malonic acid (Figure 4a) can potentially serve as a general building block of ketomethylene peptidomimetics. The ketomethylene is an isostere of a peptide, in which the amide nitrogen is replaced with methylene carbon in a peptide bond (Figure 4b). Due to the unique chemical reactivity of the ketone functional group, the structure can be further diversified by many different reactions (Figure 4c).

We are developing synthetic methods that efficiently afford the isosteric structures of natural amino acids and various unnatural amino acids as peptidomimetic building blocks. Subsequently, peptide substrate analogs, in which the building block replaces the amino acid residue located in the scissile peptide bond of the given protease substrate as well as the corresponding residue in the known inhibitors are prepared to target many therapeutically important proteases.

Figure 4. Ketomethylene isosteres of peptides