| Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, while others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Utilizing hydrogen-deuterium exchange (HDX) in combination with mass spectrometry (MS) we obtained new information on the conformational effects of Taxol, discodermolide, epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A, on microtubules isolated from chicken erythrocytes (CET). The results of our comparative HDX-MS studies indicate that all of the MSAs have significant conformational effects on the C-terminal H12 helix of alpha-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent alphabeta-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, which is composed of multiple different isotypes, the lateral interactions between the adjacent protofilaments in CET, composed of one alpha- (alpha1) and one beta-tubulin (betaVI) isotype, are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability, but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site-binding drugs are used both in tissue culture and in mouse xenograft models. |
