Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. Indeed the ability to cause mitotic arrest in rapidly growing cell cultures in the laboratory is often considered to be an assay of the principal mechanism of these drugs. Despite the fact that we have argued that mitosis is not likely to be a central target for chemotherapy in patient tumors, it is still true that chemotherapy agents can alter mitosis. This is true to some extent for all chemotherapy agents, not only for microtubule-targeting ones. To measure these effects we designed a cell-based assay using a cell that contains an artificial chromosome that codes for a fluorescent protein. Exposure of these cells to various chemotherapy agents results in different kinetics of loss of fluorescence due to differing effects on mitotic fidelity and hence chromosome loss. These results allow quantitation of each chemotherapy agents ability to induce chromosome loss. Our results showed that agents differ remarkably in their potency against mitotic fidelity, and that even microtubule-targeting agents differed significantly from each other. Surprisingly, microtubule-stabilizing drugs cause more chromosome loss than do microtubule-destabilizing drugs. These results may allow insight into the mechanisms of mitotic fidelity as well as providing a rationale to favor one drug clinically over another (if they differ in potential for causing chromosome loss). In pursuit of newer microtubule-targeting agents with more favorable spectra of actions as well as more facile chemistry, we also developed a new method for synthesizing variants of polyketides, a class of compounds containing many clinically important natural compounds. We applied our method to the microtubule-targeting natural product drug dictyostatin, demonstrating that this method allowed synthetic extension of particular sites on precursor molecules to produce new variants of dictyostatin that demonstrated significantly different biological activity. We also pursued the basic structural biology of the tubulin dimer. This knowledge is required to understand how small molecules regulate tubulin and how tubulin binding domains regulate interaction with other proteins. The most basic step in tubulin biology is assembly of the heterodimer, and we used analytical ultracentrifugation as well as fluorescence polarization to show that dimer formation is a reversible, mass-action-driven process. We also examined the role of the tubulin carboxyl terminal tail peptides which mediate interaction of many proteins with tubulin and/or microtubules. We showed that sequence differences between different isotypes of alpha- and of beta-tubuiln, as well as posttranslational modifications of these peptides, are crucial regulators of binding of tubulin to the mitochondrial outer membrane channel protein VDAC, showing that subtle differences between tubulin isotypes expressed in different tissues could alter mitochondrial function. We also showed that these isotype differences are significant for binding of the microtubule-severing enzyme katanin. Since these peptides are displayed on the outer surface of the microtubule and are recognized by katanin, these differences will translate to differing microtubule dynamics in cells expressing different tubulin isotypes. These differences in dynamics are expected to alter cellular morphological plasticity and possibly cell migration.
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