CELL-BASED AND BIOCHEMICAL SCREENS FOR SMALL-MOLECULE INHIBITORS OF DYNEIN AND OF HSP70

摘要

Dyneins are protein motor complexes that generate force towards the minus ends of microtubules, and cytoplasmic dynein plays a variety of important roles in cell. A small library of synthetic chemicals based on the nature product purealin was first examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line, but selective activities against a small panel of human carcinoma cell lines. Purealin and some of its analogues showed concentration dependent inhibitory effects against the microtubule-stimulated ATPase activity of both bovine cytoplasmic dynein heavy chain as well as the recombinant motor domain of human cytoplasmic dynein in an uncompetitive pattern, indicating that they do not compete with the binding of ATP. Purealin also weakly inhibited p53 nuclear accumulation after DNA damage. Treatment of cells with small interfering RNAs of cytoplasmic dynein heavy chain were also used as a positive control in this assay, although the lifetime of the protein turned out to be too long for the siRNA approach to be useful in a screening protocol.A strategy was built to screen for dynein inhibitors based on a GFP-GR nuclear translocation assay by using a mouse mammary adenocarcinoma cell line (3617.4) stably expressing the fluorescent protein. A small library of synthetic compounds was screened for inhibition of hormone-stimulated GFP-GR nuclear translocation. Several compounds was found to elicit the desired phenotype, and these compounds inhibited the ATPase activity of cytoplasmic dynein heavy chain motor domain without competing for the hydrolyzable ATP-binding site. Biochemical specificity tests showed that the compounds did not compete for GR binding nor inhibit the ATPase activities of Hsp70, Hsp90 or myosin. Libraries of compounds designed to be Hsp70-perturbing agents were also evaluated. Previous data showed that the Hsp70 chaperone class is induced in certain breast cancer cells and that antisense-mediated knockdown of Hsp70 triggers apoptosis, indicating that Hsp70s represent a new target for breast cancer therapy. A small molecule inhibitor of Hsp70 co-chaperone interaction (MAL3-101), as well as several analogues, showed antiproliferative activity against SK-BR3 breast cancer cells.