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Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site

Significance Natural products provide the inspiration for most drugs, and marine natural products, in particular, are emerging as promising new therapeutics with new targets or mechanisms of action. Pharmacological targeting of tubulin dynamics has been a validated strategy for cancer therapy for decades, yielding structurally diverse natural products and derivatives, including paclitaxel, vincristine, maytansine, and eribulin, targeting six known and different binding sites. We discovered a chemical scaffold from marine cyanobacteria that targets a seventh tubulin binding site. We report the entire spectrum of the discovered chemical and biological novelties, including the isolation, structure determination, and chemical synthesis of the natural product, and the investigation of its mechanism of action, target identification, and binding mode elucidation at the atomic level. Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin−GB1 complex.