A high-throughput selection system for fast-acting covalent protein drugs

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Covalent protein drugs show therapeutic promise but face challenges with slow target engagement.
• Optimizing affinity, stability, and warhead geometry for rapid covalent binding is complex.
• Existing high-throughput selection platforms for fast covalent binders are lacking.

Purpose of the Study:

• To develop a novel yeast display platform for selecting fast-acting covalent proteins.
• To engineer covalent protein therapeutics with rapid kinetics without enhancing intrinsic warhead reactivity.
• To demonstrate the platform's versatility across different protein modalities and therapeutic targets.

Main Methods:

• Yeast display platform coupled with chemoselective modification.
• Selection of covalent nanobodies targeting programmed death-ligand 1 (PD-L1).
• Engineering of covalent interleukin-18 (IL-18) inhibitors and SARS-CoV-2 miniproteins.

Main Results:

• Engineered a covalent PD-L1 nanobody with rapid crosslinking kinetics (k_obs = 0.18 min⁻¹, t₁/₂ = 3.8 min) and enhanced tumor suppression.
• Developed a fast-acting covalent IL-18 inhibitor (k_obs = 0.54 min⁻¹, t₁/₂ = 1.3 min).
• Created a covalent miniprotein targeting the SARS-CoV-2 receptor binding domain (RBD).

Conclusions:

• The developed yeast display platform enables the selection of fast-acting covalent proteins.
• This approach overcomes limitations of slow target engagement in covalent drug development.
• The platform demonstrates broad applicability for engineering diverse covalent protein therapeutics.