Enzyme Induced Solid‐Like Condensates Formation of Engineered Peptide in Living Cells for Prostate Cancer Inhibition

This work describes the rational design and synthesis of hepsin-recognized amphiphilic-branched peptides (DMN-SIPL) that can form solid condensates through liquid-liquid phase separation (LLPS) upon enzymatic reaction. The peptide forms solid-like condensates both in vitro and in living cells, triggered by type-II membrane-associated serine peptidase, hepsin, whose overexpression determines prostate cancer progression. Specifically, integrating self-assembly, hepsin hydrolysis, and hepsin-binding domain generates a branched substrate that acts as a precursor for enzyme-induced LLPS. Upon binding hepsin on the cell membrane, DMN-SIPL forms condensates initiated by hepsin-induced self-assembly. The prostate cancer cells then uptake these condensates via lipid raft-mediated endocytosis. The entrapped hepsin in the condensates further hydrolyzes the DMN-SIPL to stabilize the intracellular condensates. Structure-activity relationship reveals the importance of enzyme-binding motif, enzyme-recognized motif, and the self-assembly motif. Mechanistic studies indicate that the resulting solid-like condensates modulate cancer cell metabolism by inhibiting hepsin upstream protein activation and downstream signal transduction, ultimately inducing cancer cell growth inhibition selectively. As a first example, this work investigates enzymatic LLPS condensate formation in living cells, paving the way to generate functional synthetic biomolecular condensates through LLPS for biomedical applications.