Cytoplasmic dynein is the major retrograde molecular motor responsible for transport of membranous organelles, viruses, mRNA and proteins with nuclear localization signals. It is also involved in chromosome movement, mitotic checkpoint inactivation and spindle orientation, and many other basic cellular processes. Cytoplasmic dynein is a large (ca. 2 MDa) complex of proteins. Its movement along microtubules is powered by ATP hydrolysis by its 380 kDA motor domains, which are associated with the >500 kDa heavy chain subunits. Other than weak and nonspecific redox perturbers and mimics of ATP or phosphate anion, only one small molecule inhibitor of dynein, the natural product purealin, which we have recently synthesized and examined, is known. It is, however, only weakly active in cells. New inhibitors with potency and specificity for dynein and subsets of dynein function would be invaluable cell biology tools. The goal is to develop a refined suite of high throughput cell and biochemical assays for chemical library screening find inhibitors of cellular cytoplasmic dynein.
Aim 1 is to develop a cell-based phenotypic multiparameter fluorescence screen to detect dynein inhibition in interphase cells, as well as to detect possible mitotic block due to inhibition of dynein. Preliminary data provides support of three cellular events, transport from the cytoplasm to the nucleus of: (i) p53 after mild DNA damage; (ii) stably expressed green fluorescent protein-labeled glucocorticoid receptor after binding with an agonist; and (iii) a fluorescent adenovirus. These will be examined and the most useful selected. As some phenotypes could be due to interaction of the small molecules with off-target proteins, Aim 2 is to implement high throughput biochemical screens to confirm that a small molecule's molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin. The investigators, a team of experts in these areas, will design and perform the assays as well as interpret results, and have previous direct or related experience with most of the assays. The product of this work will be a refined, streamlined set of rapid cell and biochemical screens for dynein inhibitors that can be implemented in molecular library screening centers.
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| Daghestani, Hikmat N; Zhu, Guangyu; Johnston, Paul A et al. (2012) Characterization of inhibitors of glucocorticoid receptor nuclear translocation: a model of cytoplasmic dynein-mediated cargo transport. Assay Drug Dev Technol 10:46-60 |
| Brummond, Kay M; Mao, Shuli; Shinde, Sunita N et al. (2009) Design and synthesis of a library of tetracyclic hydroazulenoisoindoles. J Comb Chem 11:486-94 |
