Genetically Encoded Epoxide Warhead for Precise and Versatile Covalent Targeting of Proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Genetically encoding reactive warheads in protein binders is an effective strategy for covalent protein targeting.
• Existing methods require expansion to target diverse natural residues under physiological conditions.

Purpose of the Study:

• To genetically encode a novel reactive warhead with broad reactivity and stability.
• To develop covalent protein drugs targeting PD-L1 and KRAS.

Main Methods:

• Genetic encoding of epoxide-containing tyrosine (EPOY).
• Incorporation of EPOY into a nanobody (KN035) for PD-L1 targeting.
• Incorporation of EPOY into a designed ankyrin repeat protein (DarpinK13) for KRAS targeting.

Main Results:

• Achieved the first single genetically encoded reactive warhead targeting 10 nucleophilic residues.
• Demonstrated EPOY-mediated cross-linking with different PD-L1 mutations.
• Developed a covalent KRAS binder with potential for pan-covalent targeting of KRAS mutants.

Conclusions:

• EPOY significantly expands the scope of covalent warheads for both small-molecule and protein drugs.
• Covalent targeting of KRAS, particularly H95, presents a promising strategy for developing covalent pan-KRAS inhibitors.