UCSF

Targeted covalent inhibitors have had a major impact on human health and disease. In the past decade, several drugs with a covalent mechanism of action have been approved for the treatment of various cancers. Unfortunately, due to the lack of chemical diversity found naturally in protein biologics, we have been limited to designing covalent inhibitors that are small molecules or peptides. In recent years, however, through the use of genetic code expansion, we have been able to genetically encode new functionalities into proteins. These new functionalities include incorporating bioreactive unnatural amino acids (Uaas) into proteins, which allow for the formation of covalent bonds between binding proteins and their targets. Developing covalent biologics has great implications for the development of novel therapeutics and diagnostic tools. Chapter 1 introduces the reader to the recent chemical advancement of encoding bioreactive Uaas into proteins as well as successful development of anticancer and antiviral covalent protein drugs. Here, we describe the development of a new electrophilic bioreactive Uaa as well as two applications of protein binders with covalent mechanisms. In chapter 2, we describe the development of genetically encoding a SuFEx reactive meta-fluorosulfate tyrosine (mFSY) into proteins to expand the toolbox of bioreactive Uaas. mFSY has the ability to covalently target different side chain orientations as compared to known fluorosulfate Uaas fluorosulfate-L-tyrosine (FSY) and fluorosulfonyloxy-benzoyl-L-lysine (FSK). Chapter 3 describes the application of covalent biologics for targeted radionuclide therapies. We develop a covalent nanobody that binds to its target irreversibly, achieving exceptional tumor uptake, fast blood clearance, and low background. This radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining systematic clearance. Finally, in chapter 4 we demonstrate the selectivity a covalent mechanism of action contributes to protein binding by developing a covalent neuregulin capable of specifically crosslinking with HER4; this covalent binding alters the signaling axis of HER4 in a novel and unexpected manner.