Determination of Drug Binding to Microtubules In Vitro

Abstract Many naturally occurring compounds and their synthetic analogs bind to soluble tubulin or to tubulin in microtubules. These compounds are often important drugs or drug candidates. They can potently alter both the dynamics and the polymer mass of microtubules. The binding affinity of a drug for soluble tubulin heterodimers is a common and relatively readily determined biochemical characteristic of a tubulin-targeted drug. However, it is only one important aspect of the drug–tubulin interaction. In solution and in cells, soluble tubulin is in equilibrium with polymerized microtubules. It is as important to determine drug binding to microtubules as it is to determine binding to soluble tubulin, since drug binding to microtubules frequently alters their function. The affinity of a compound for microtubules often differs vastly from its affinity for soluble tubulin. Here, we present detailed instructions for assessing binding stoichiometry and affinity to assembled unstabilized microtubules using radiolabeled drug. In addition, using examples from binding results with several important drugs including vinca alkaloids, colchicine, eribulin, and taxanes, we discuss aspects of the interactions with microtubules that may alter the experimental design of the drug-binding experiments.