Computational design of pH-sensitive binders

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Therapeutic Development

Background:

• Physiological pH gradients are crucial in biological processes, including vesicle transport and the tumor microenvironment.
• Existing methods for creating pH-dependent binders are empirical, labor-intensive, and lack predictability.
• Targeting pH changes offers a promising avenue for developing novel therapeutics.

Purpose of the Study:

• To introduce novel computational principles for designing pH-dependent protein binders.
• To create binders that can be modulated by pH for therapeutic applications.
• To demonstrate the efficacy of these binders in degrading specific protein targets.

Main Methods:

• Designed pH-dependent binders using two computational principles: electrostatic repulsion at interfaces and destabilization via buried histidine networks.
• Introduced histidine residues adjacent to positive charges to weaken binding at low pH.
• Incorporated buried histidine-containing hydrogen-bonding networks to destabilize protein structure under acidic conditions.

Main Results:

• Successfully designed binders that dissociate at acidic pH against multiple targets: ephrin type-A receptor 2, tumor necrosis factor receptor 2, interleukin-6, proprotein convertase subtilisin/kexin type 9, and Neo2.
• Engineered catalytic degraders by fusing designed binders to lysosomal trafficking receptors.
• Achieved target degradation at substoichiometric levels using these catalytic degraders.

Conclusions:

• The described computational methods provide a rational approach for designing pH-sensitive protein therapeutics.
• These pH-dependent binders and catalytic degraders have broad applicability for modulating protein activity in various physiological environments.
• The findings pave the way for developing next-generation protein-based therapies exploiting pH gradients.