Directing the oxidative folding of disulfide-rich peptides for enhanced engineering and applications

Disulfide-rich peptides (DRPs) leverage dense disulfide networks to form rigid and stable cores, enabling exceptional proteolytic resistance and precise target complementarity. These attributes drive their utility as high-affinity molecular tools in bioanalytics/chemical biology and clinically validated therapeutics (e.g., ziconotide for chronic pain and insulin for diabetes). However, DRP functionality critically depends on native oxidative folding, where inefficient disulfide pairing causes low production yields, induces functional instability through disulfide isomerizations, and triggers misfolding upon sequence engineering. Recent advances in directed oxidative folding permit precise pathway control, facilitating efficient engineering and discovery of functional DRPs, thereby accelerating diagnostic and therapeutic development. Herein, we summarize novel strategies that actively direct the oxidative folding of DRPs to enhance their engineering and applications. Additionally, we present our perspective on key challenges in DRP design and discovery associated with oxidative folding, and propose future research directions to advance this field.