Self-Assembling Ability Determines the Activity of Enzyme-Instructed Self-Assembly for Inhibiting Cancer Cells

[Image: see text] Enzyme-instructed self-assembly (EISA) represents a dynamic continuum of supramolecular nanostructures that selectively inhibits cancer cells via simultaneously targeting multiple hallmark capabilities of cancer, but how to design the small molecules for EISA from the vast molecular space remains an unanswered question. Here we show that the self-assembling ability of small molecules controls the anticancer activity of EISA. Examining the EISA precursor analogues consisting of an N-capped d-tetrapeptide, a phosphotyrosine residue, and a diester or a diamide group, we find that, regardless of the stereochemistry and the regiochemistry of their tetrapeptidic backbones, the anticancer activities of these precursors largely match their self-assembling abilities. Additional mechanistic studies confirm that the assemblies of the small peptide derivatives result in cell death, accompanying significant rearrangement of cytoskeletal proteins and plasma membranes. These results imply that the diester or diamide derivatives of the d-tetrapeptides self-assemble pericellularly, as well as intracellularly, to result in cell death. As the first case to correlate thermodynamic properties (e.g., self-assembling ability) of small molecules with the efficacy of a molecule process against cancer cells, this work provides an important insight for developing a molecular dynamic continuum for potential cancer therapy, as well as understanding the cytotoxicity of pathogenic assemblies.