Bispecific Small Molecule Engineering

We will make a bifunctional small molecule to promote proximity induced reactions triggered by endogenous enzymes in vivo. This approach has been used for degrading unwanted proteins by utilizing a ubiquitin-proteasome pathway. We will use this "proteolysis targeting chimera (PROTAC)" strategy by connecting E3 ligase binding small molecule and target protein binding small molecule with a linker. We aim to degrade protein targets of broad range of diseases including a metabolic disease, cancer or degenerative brain disease by this approach.

Modulating Protein-protein Interaction

We will create a protein-protein interaction which occurs conditionally. To induce protein-protein interaction with a small molecule, we will borrow natures architecture as a starting point. We will introduce synthetic non-canonical amino acid (ncAA) at interface of dimeric wildtype protein. Then surrounding residues will be randomized to make a library of protein dimer. A variant that induces optimal fitting of the ncAA will be selected by using a selection system in E. coli. After that, we will screen a set of small molecule derivatives with similar chemical and physical property to the ncAA side chain for the small molecule dependent dimerization event. Finally, we will have small molecule or ncAA dependent protein-protein interaction module which is useful to make a conditional gene expression system of life. This work will directly impact on human health by making live attenuated vaccines.

Adding New Chemistries to the Genetic Code

Over the last two decades, we and others encode a large number of ncAAs with unique chemical and physical properties in prokaryotic and eukaryotic cells, and even in animals. All that is necessary is an orthogonal translation system consisting of an exogenous tRNA and aminoacyl-tRNA synthetase (aaRS) pair that does not cross-react with tRNAs or aaRSs in the host and that recognizes a unique codon, usually a nonsense codon. We will expand current "encodable" ncAA set by directed evolution approach. The novel ncAA will include large or small amino acids, fluorescent amino acids, and other chemically useful amino acids.

Nanobody Engineering

We will ask whether single-domain antibody (nanobody, Nb) can be generally used to selectively binds small molecules. The Nbs (12-15 kDa) are smaller than IgGs (150-160 kDa) while retaining high affinity and stability. Moreover, many Nbs were evaluated as therapeutics as they specifically detect or neutralize disease targets. We will evolve Nb to find fluorescent small molecule specific Nb for bio-imaging applications. At the same time, we will engineer bio-active small molecule specific Nbs to develop novel small molecule target identification protocols.