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 in this study. Some, such as the new MT-stabilizing compound peloruside, are natural products, as is the clinically established MT-stabilizer taxol. Others, such as analogs of the MT-stabilizing epothilones, are semisynthetic derivatives based on known natural compounds. Others still are totally synthetic compounds. We have investigated two new binding sites on tubulin for anti-MT drugs, as well as the results of drug binding at these, or the longer-known sites, on the properties of MT and the effects on cells. The new binding sites are for the synthetic MT destabilizer, oryzalin, and the natural product MT stabilizer, peloruside. The effects on cells involve these drugs as well as more established drugs, especially clinical agents. Oryzalin and other dinitroanilines are effective herbicides due to a high selectivity for plant tubulin over mammalian tubulin. We have shown that these compounds, which destabilize MT, also show selectivity for protozoal parasite tubulin compared to mammalian tubulin. We have continued our effort to understand this selectivity by mapping the binding site for oryzalin on tubulin using detailed analysis of mutations in parasite tubulin that confer resistance to this compound. We hope to use this detailed knowledge to design compounds that bind better and with improved selectivity to parasite tubulin, thereby affording clinically useful antiparasite drugs. We have used a similar approach to define the binding site and mode of action of peloruside. We have already shown by mass spectrometric studies and molecular modeling that this compound binds to a site on beta tubulin quite distinct from that of taxol, a clinically important MT-stabilizing drug. Selecting and mapping mutations in human tubulin that confer resistance to peloruside have confirmed our mass spectrometry studies, and allowed an improved understanding of the binding site, how occupancy alters MT stability, and how this differs from taxol action. We hope to use this knowledge to understand the differing mechanisms of peloruside and taxol, and provide a basis for combination of these drugs clinically. It is already clear from the binding site mapping and from preclinical studies that taxol and peloruside stabilize MT by different mechanisms. Structural study of the two binding sites suggests a differing balance of longitudinal and lateral stabilization in the MT polymer, suggesting that the mechanical properties of the MT may differ with the two drugs. Unperturbed MT are the most rigid intracellular protein polymers known, and taxol increases their flexibility 10-fold. We are measuring the rigidity of individual fluorescent MT after binding of taxol or peloruside in order to relate differences in binding site structures to differences in MT properties. This understanding could provide an explanation for the synergistic effect observed for combinations of these drugs in preclinical cellular models. Inside the cell, MT occur in arrays. The intrinsic polarity of individual MT is shared throughout the arrays, and this asymmetry underlies nearly all intracellular transport and signaling. We previously showed the role of MT array polarity in intracellular signaling by p53, and we have extended this to demonstrate the MT basis of the directional transport of G-protein-coupled receptors from the ER to the cell surface. On a larger scale, we have investigated the role of MT in overall cell polarity and directed movement. Cells respond to many environmental signals by moving up or down gradients of molecules or other signals such as light and substrate rigidity. Using the methods developed in our lab to study durotaxis, the preferential movement of cells from softer to more rigid surfaces, we will use MT-targeting drugs to define the role of MT polarity in this directed cell movement. The roles of MT extend throughout the life of the cell, not only in mitosis, but also in the 98+% of the cell cycle that is not mitosis. These vital roles include those from above establishing cellular polarity, supporting intracellular transport and signaling, and allowing directionality in cell movements. MT-targeting drugs are active in all cells, not only in mitotic ones, and indeed some targets of clinical use of anti-MT drugs are post-mitotic cells. We have argued that even in clinical settings where intuition says that mitosis is the target, such as in patient tumors, data indicate that MT-targeting drugs are effective due to interference with non-mitotic processes, such as those mentioned above. We plan to combine the experimental approaches described to obtain a better understanding of the non-mitotic processes that are targeted by the action of anti-MT drugs in order to improve the clinical usefulness of these agents.

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