CHEMICAL BIOLOGY TOOLS FOR PROTEIN HISTIDINE PHOSPHORYLATION

Abstract

Protein histidine (His) phosphorylation plays essential roles in cell signaling, metabolism and epigenetics in a cell, dynamically regulating the target protein functions. It is regulated by protein histidine kinases and phosphatases, and its mis-regulation has been implicated in human diseases including cancer, which make them promising drug targets. However, further studies of phosphohistidine (pHis) have been hampered because of the lack of research tools stemming from innate chemical instability of pHis. Traditional tools rely on laborious and non-continuous methods such as radiolabeling, so they are not suitable for enzyme kinetics and screening for drugs discovery. With this regard, this thesis describes the development of chemical biology tools for protein histidine kinases and phosphatases. Chapter I discusses the progress on the development of high-throughput assay for protein histidine kinases (HKs) activity, particularly those in two-component signaling system (TCS) which are master regulators of antibiotic resistance in pathogenic bacteria. Accordingly, these HKs are attractive therapeutic targets for drug-resistant bacteria. To our delight, our assay conveniently measured HK activity and has been applied in high-throughput screening (HTS) for the discovery of HK inhibitors. Chapter II describes the development of fluorescent probes for protein histidine phosphatase 1 (PHPT1) activity, which implicates critical roles in cancer metastasis. Design, synthesis, validation and optimization of the probes were successfully achieved. Our probes showed excellent specificity towards PHPT1 over other known pHis phosphatases and provided physiologically relevant kinetic parameters of PHPT1. In addition, the activity of nanomolar concentrations of PHPT1 was sensitively measured in human cell lysates, presenting ways of monitoring PHPT1 activity in complex biological mixtures. Overall, our studies presented herein offer significant contributions towards advancing the development of chemical biology tools for protein histidine phosphorylation. These studies will provide new directions for protein phosphorylation research and lay the foundations for novel antibiotic and anticancer drugs discovery by targeting kinases and phosphatases.