Structure–Activity Relationship of Cycling Molecular Assemblies for Golgi Targeting and Disruption

Targeting the Golgi apparatus with small molecules remains challenging because of limited binding targets and poor selectivity in cells. We recently introduced cycling molecular assemblies (CyMAs), which use a palmitoylation/depalmitoylation enzyme switch to kinetically trap supramolecular nanostructures at the Golgi, but the molecular features governing their activity were unclear. Here, we report a structure-activity relationship (SAR) study of CyMA by systematically varying the peptide backbone, thioester warhead, and N-terminal capping group. Analysis of Golgi localization, trapping kinetics, critical aggregation concentration (CAC), and cytotoxicity revealed that the self-assembling ability is the primary determinant of CyMA activity. The d-Phe-d-Phe backbone exhibits optimal assembly and enables efficient Golgi accumulation at nanomolar concentrations. Warhead and N-terminal modifications further tune trapping kinetics and disrupt potency. Together, these results define design principles for CyMA and establish enzyme-driven self-assembly as a general, modular strategy for developing drugs for Golgi and organelle targeting.