Establishment and usage of chemical biology strategies for PTM-related research

Abstract

Inhibitor development is an enormous challenge, despite many technological advances in recent decades. Process of inhibitor development requires still much time, expensive cost, and a lot of effort. Development of effective inhibitor screening platform is important to resolve challenge of inhibitor development and may provide more novel treatment options for patients. Here, novel inhibitor screening platforms were described based on genetic code expansion. Phosphoproteins were biosynthesized as targets by phosphoserine (Sep) incorporation system published in 2011. There are two targets to treat human diseases: Protein-Protein interactions (PPIs) and protein activities of targets. Fluorescence resonance energy transfer (FRET) assay was used for screening inhibitors to inhibit PPIs of targets such as complex of CDC25C and 14-3-3beta involved with human diseases. New hybrid assay based on $TiO_2$ (affinity of phosphate group) decorated GO (fluorescence quenching ability) was developed for screening inhibitors to inhibit protein kinase activity of targets such as pERK2 and PKA (collaboration with prof. Dal-Hee Min). These platforms are suitable for high throughput screening (HTS) assay and can be applied to inhibitor discovery as a general method for targets involved with human diseases. In addition to phosphoprotein, diverse posttranslational modifications (PTMs) occur in proteins. In this study, new strategy was designed for protein ubiquitination by integrating genetic code expansion and TEC reaction. This protein ubiquitination method incorporated thiol and alkene group into ubiquitin (ub) and conjugated between ub and ub by using thiol-ene coupling (TEC) reaction. This strategy facilitates synthesis of diverse ub chains such as homotypic, heterotypic and branched formation of ub chain and site-specific protein ubiquitination without E1-E2-E3 enzymes and ATP. This controllable method is expected to apply inhibitor screening platforms for targeting ubiquitinated proteins and to provide opportunity to many researchers for studying diverse biological processes by protein ubiquitination.