PROJECT SUMMARY Cyanobacteria are among the most ancient organisms on Earth and have evolved chemical weapons for defensive purposes, which we are exploiting for anticancer drug discovery. Our past research has exemplified that marine cyanobacteria produce compounds with exceptionally potent activity and/or possess unusual or first- in-class inhibitors with novel mechanisms of action. We apply a broad yet focused screening platform to maximize discovery rate of new anticancer agents. We also integrate a synthetic chemistry component into our program in order to perform rigorous biological studies to rapidly add value to our discoveries and to help solve the supply problem. Specifically, we will carry out field collections of marine cyanobacteria and prepare fraction libraries for screening in a set of innovative pathway-focused cell-based assays, utilizing various isogenic colorectal cancer cell lines and reporter gene assays, as well as newly developed unbiased phenotypic assays. We will perform dereplication, identify cyanobacteria through molecular analysis and establish phylogenetic relationships among benthic cyanobacteria. Samples will be prioritized based on bioactivity profiles and genetic uniqueness for further processing. Prioritized samples will be subjected to bioassay-guided isolation of the active compounds. Structures will be determined using NMR and mass spectrometry. Basic validation of bioactivity and secondary assays will be performed before prioritizing targets for further investigation. Selected compounds with intriguing structure and promising validated bioactivity will be targeted for total synthesis so that a rigorous biological characterization can be performed to pinpoint the molecular changes induced in the cancer cell and to determine potential direct targets. For novel noncytotoxic compounds that show potential antimetastatic activity, we will apply an orthogonal target-based screening platform.