A Covalent and Modulable Inhibitor of the Tubulin-Microtubule System: Insights Into the Mechanism of Cacalol

Inhibitors of the tubulin-microtubule system are part of an effective strategy to treat different kinds of cancer, whose research has allowed scientists to discover and develop new and more selective molecules. Cacalol (1) is a natural product with anti-cancer activity and documented selectivity in breast cells, but with an undescribed molecular mechanism associated with these properties. The main objective of this work is to provide evidence that helps to explain the inhibitory and selective activity reported for cacalol (1) against cancer cell lines and to expand the knowledge about the mechanism of action involved in it. Cacalol derivatives were studied using reactivity approaches, tubulin polymerization assays, mass spectrometry, and molecular modeling techniques to decode the inhibitory binding mechanism. This work demonstrates that an oxidated form of cacalol, the methylenecyclohexadienone 2, is generated in highly oxidant conditions, thus emulating the environment present in cancer cells. This species (2) is responsible for the inhibition of tubulin polymerization by promoting an irreversible binding interaction with the Cys347 in α-tubulin.