This project focuses on the ability of small molecules to alter the cell's cytoskeleton and, in particular, microtubules (MT). MT are the most rigid of the cytoskeletal polymers, and are central to establishing and maintaining non-spherical cell morphology. In addition, MT have intrinsic polarity, and the cytoplasmic array of MT provides the substrate for directional intracellular movement. Thus, small molecules that alter MT integrity and/or dynamics can scramble intracellular trafficking and alter the physical properties of the cytoplasm. This project has two parts. One is to understand in detail the interaction of MT-active small molecules with MT or the MT subunit protein, tubulin, focusing on anti-mitotic peptide natural products from marine sources, as well as compounds produced from knowledge-directed synthesis. The second part aims to use the knowledge of MT-small molecule interactions to identify drug therapies for parasite diseases.? 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 discovered an anlog of thalidomide that stabilizes microtubules.? 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.
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