Advanced forward chemical genetic approaches to facilitate target-identification.

摘要

Forward chemical genetics has emerged as a new field providing powerful tools for the discovery of noble proteins and their roles in biological pathways. It is a three-step process---synthesis of small-molecule library, screening of the library compounds in search for novel phenotypes, and identification of the small-molecule target(s). Though recent technical advances have greatly increased the number of small molecules and also dramatically increased screening capacity, the identification of target proteins remains challenging. This work is focused on improving target-identification strategies to facilitate forward chemical genetic studies.;First, we developed a tagged library approach for chemical genetic target identification. The most challenging part of target identification is conjugating the active lead compound to an affinity matrix without reducing activity of the lead compound. To overcome this well-known problem, tagged library contains a built in linker tag from the beginning. By applying this tagged library to forward chemical genetic screening, we discovered an insulin sensitizer and successfully identified its target protein without the need of lead optimization, demonstrating the power of the tagged library approach.;Secondly, we have developed diversity-oriented fluorophore libraries as tools for chemical genetics. Aided by their intrinsic fluorescence property, target protein(s) can be labeled in living cells without the need of genetic modification or bulky tagging. When applied to forward chemical genetic study, this live-cell target labeling technique greatly improved target identification process in terms of speed and credibility.