Protein-protein interactions are involved in a broad range of cell function and signaling. Various protein binders have been developed to detect or modulate such interactions for research, diagnostic, and therapeutic applications. However, existing protein binders usually bind to their targets in noncovalent mode only, imposing limitations on affinity, stability, and completeness. In addition, covalent bonding between a drug and its target offers multiple desirable therapeutic properties over noncovalent interactions of conventional drugs. Such covalent mode has been implemented in small molecule drugs with great success in recent years, yet the therapeutic potential of covalent protein drugs remains largely untapped. To change this paradigm, this project will develop covalent protein binders and generate covalent protein drugs. New latent bioreactive amino acids will be designed and genetically incorporated into protein binders, which will react with a natural residue of the target only upon protein binding, selectively creating a stable covalent linkage between the two proteins. Covalent protein binders specific for immune-checkpoints and membrane receptors associated with cancer will be generated. The distinct effects of these covalent protein binders on cancer cell signaling and function will be assessed in vitro, and their cytotoxic activity and anti-tumor efficacy as covalent protein drugs will be evaluated in xenografted mouse models. The success of this project will instigate a new dimension for researching cell signaling and function through highly selective, stable, and covalent modulation of proteins. A general platform technology will be established for the development of covalent protein drugs, leading to a new generation of biotherapeutics with a fundamentally different binding mechanism for cancer treatment.

Relevance Statement We will develop an innovative method to generate covalent protein binders for cell receptors. The success of this project will allow researchers to more effectively target proteins involved in various diseases including cancer for mechanistic studies, and may lead to a new generation of protein therapeutics.