Optimizing linker rigidity to improve intracellular behavior of PROTACs targeting hematopoietic prostaglandin D synthase.

Proteolysis-targeting chimeras (PROTACs) are emerging as powerful tools for targeted protein degradation. Among the key factors influencing their efficacy, linker design plays a critical role by affecting membrane permeability, ternary complex formation, and degradation potency. In this study, we conducted a comparative analysis of three novel PROTACs targeting hematopoietic prostaglandin D synthase (H-PGDS), each incorporating linkers with distinct degrees of rigidity-including methylene modifications and spirocyclic structures. Although all compounds exhibited similar binding affinities and degradation activities, the most rigid derivative (PROTAC-3) showed markedly higher intracellular accumulation but formed the least stable ternary complex. These results reveal a trade-off between cell permeability and complex stability, emphasizing the importance of comprehensive linker optimization. Our findings highlight the value of integrating conformational rigidity and spatial design in the rational development of next-generation PROTACs.