Microtubule regulation by small molecules.

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In part one we have focused on antimitotic peptides because these are among the most potent anti-MT agents known, they have been synthesized and analogs are available, and because they induce the MT subunits to assume unusual and characteristic ring shapes. We are studying the structural and dynamic properties of these ring polymers by analytical ultracentrifugation, cryoelectron microscopy, fluorescence correlation spectroscopy, and protease mapping. The high stability and uniformity of these rings that our studies revealed have led us to attempt crystallization of these polymers to achieve atomic resolution of their structure. We are also examining the effects on microtubule polymerization of synthetic analogs of thalidomide and combretastatin A. We have also demonstrated the role of posttranslational modifications by deacetylases, and the effects of antimicrotubule drugs on the p53 response. In part two, we are seeking to identify small molecules that do not bind well with mammalian tubulin but do bind to parasite tubulin. The tubulin molecule is quite conserved evolutionarily, but differences do exist, and several molecules are known that can target, for example, yeast rather than mammalian tubulin or vice-versa. We are looking for molecules that will target Leishmania, the infectious cause of an important group of human diseases. We have identified several small molecules that show promise as selective agents, binding to Leishmania tubulin preferentially over mammalian tubulin, and preventing parasite multiplication inside human macrophage cells. In addition, we have produced purified tubulin from cultured Leishmania and evaluated it for use in drug screening.