Halichondrin B is an
antimitotic drug that inhibits microtubule assembly. To understand the molecular details of its interaction with
tubulin, we investigated the binding of two
halichondrin B analogues,
eribulin (previously,
ER-086526, E7389) and
ER-076349, to
tubulin by quantitative analytical ultracentrifugation.
Eribulin is currently undergoing phase III clinical trials for
cancer;
ER-076349 is a closely related analogue with C.35
hydroxyl instead of C.35 primary
amine [Towle, M. J., et al. (2001)
Cancer Res. 61, 1013]. Below the critical concentration for microtubule assembly and in the presence of
GDP,
tubulin undergoes weak self-association into short curved oligomers.
Eribulin inhibits this oligomer formation 4-6-fold, while
ER-076349 slightly stimulates oligomer formation by 2-fold. This is in contrast to
vinblastine which strongly stimulates large spiral
polymers by 1000-fold under these same conditions.
Vinblastine-induced spiral formation is strongly inhibited by both
eribulin and
ER-076349.
Colchicine binding to the intradimer interface has no significant effect on small oligomer formation or the inhibitory activity of
eribulin on this process. These results suggest that
halichondrin B analogues bind to the interdimer interface or to the beta-subunit alone, disrupt
polymer stability, and compete with
vinblastine-induced spiral formation.
Stathmin is known to form a tight 1:2 complex with
tubulin.
Eribulin strongly inhibits formation of the 1:2
stathmin-
tubulin complex (>3.3 kcal/mol), while
ER-076349 weakens formation of the 1:2 complex by approximately 1.9 kcal/mol. These results suggest that
eribulin is a global inhibitor of
tubulin polymer formation, disrupting
tubulin-
tubulin contacts at the interdimer interface.
ER-076349 also perturbs
tubulin-
tubulin contacts, but in a more
polymer specific manner, reflecting adaptability of the interdimer interface to
drug and
polymer polymorphism. These results suggest
halichondrin B analogues exhibit unique
tubulin-based activities that may underlie the clinical utility of these compounds.