Abstract
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.