Connecting chemical structure to single cell signaling profiles.

A challenge in chemical biology is to study structure-activity relationships (SAR) in vivo in cells. Multiplexed activity profiling (MAP), developed for natural product discovery, is well-suited to address this challenge as it is high throughput, singlecell, and measures multiple hallmark cellular functions. Applying MAP while systematically varying molecular structure (SAR-MAP) could reveal previously unappreciated activity within chemical families and prioritize candidate molecules for further characterization. Here we use SAR-MAP to identify structural features responsible for specific bioactivities of the natural product family, rocaglates. MV411 leukemia cells and healthy human leukocytes are selected for proof-of-concept screening. Testing 600 representative molecules using MAP classifies roughly half of tested rocaglates (9 of 19) as bioactive. SAR-MAP elucidates a methoxy substituent on select rocaglate pyrimidinones as responsible for a desirable anti-leukemia activity. Thus, SAR-MAP can be immediately applied to identify structural variations driving natural product activity in cell lines and primary human cells.