Allele-specific pair of methyltransferases / acetyltransferases and their cofactors (Chemical Biology):

Enlarge Image Figure 1

The activities of protein lysine/arginine methyltransferases (PKMT/PRMT) and histone acetyltansferases (HAT) determine the patterns of chromatin modifications and orchestrate numerous DNA-based processes, such as epigenetic silencing, chromatin remodeling and transcription activation. The errors in the processes have been linked to many human diseases, such as neurologic disorders, congenital malformation, immune disorders and in particular cancers. To elucidate the transient but essential functions of PKMT/PRMT/HAT, traditional biological and genetic tools are often limited due to the lethal or slow responses of these enzymes at gene level. In contrast, the chemical genetic approach using target-specific small molecules can rapidly and selectively affect a particular enzymatic activity in a whole organism.

The common feature of PKMT/PRMT/HAT-involved protein modification is to exploit small, organic cofactors S-adenosyl-L-methionine (SAM) or acetyl-CoA as methyl or acetyl donor, respectively. By taking this advantage, we plan to develop orthogonal pairs of engineered PKMT/PRMT/HAT and their allele-specific cofactor derivatives to target a single enzymatic activity. This goal will be approached by mutually altering cofactor structures and their binding pockets in the targeted enzymes. The consequent allele-specific systems will be adapted for protein substrate identification, temporal regulation of cellular activities as well as downstream target characterization [Figure 1].

Novel cofactor derivatives and target-specific, high-affinity inhibitors (Chemical Synthesis):

Enlarge Image Figure 2

Another project in this laboratory is to synthesize a variety of molecular scaffolds that mimic the structures of several enzyme cofactors such as S-adenosyl-L-methionine (SAM), acetyl-CoA and ATP. These molecules are expected to interact with native or engineered (see the first project in this lab) enzyme targets as either cofactor analogue inhibitors or cofactor surrogates. The consequent disruption of the targeted enzymes is achieved by either blocking the enzymatic activities or exploiting toxic cofactor derivatives. These target-driving inhibitors will also be tested as potential drug candidates for their pharmaceutic activities. The cofactor derivatives containing fluorescent and reactive functional groups can serve as in vivo chemical markers of particular enzymatic activities. Our laboratory emphasizes on both rational design on the basis of enzymatic reactions (substrate-enzyme interactions, enzymatic transition state) and compound library screening to diversify cofactor mimic scaffords and increase target-selectivity as well as biological potency (Figure 2).

Assays and enzymatic synthesis of structurally/functionally-diverse cofactor derivatives in vivo and in vitro (Synthetic Biology):

Enlarge Image Figure 3

Besides chemical synthesis, this laboratory is also interested in in vivo and in vitro enzymatic synthesis of structurally-diverse cofactor derivatives (Synthetic Biology). The enzymatic synthesis in contrast to chemical synthesis is featured as fewer steps, better yields and ready adaptation for in vivo synthesis (in situ synthesis to avoid poor membrane permeability of some molecules). We are exploiting several main pathways of the synthesis of S-adenosyl-L-methionine (SAM) and acetyl-CoA, and engineering native enzymes to promiscuous enzymes. The newly engineered enzymes are expected to take non-native substrates to generate diverse cofactor derivatives (Figure 3). The cell lines containing these promiscuous enzymatic activities will be used as the platform to elucidate downstream effects and new targets of protein lysine/arginine methylation and histone acetylation. We are also developing ultra-sensitive assays to visualize and detect the lysine/arginine methylation and histone acetylation activities in vivo and in vitro.