Received for publication February 7, 2007.
Revised March 29, 2007.
Accepted for publication April 17, 2007.

Identification and Characterization of a New Tubulin-Binding Tetrasubstituted Brominated Pyrrole

Abstract

We studied the mechanism of action of 3,5-dibromo-4-(3,4-dimethoxyphenyl)- 1H-pyrrole-2-carboxylic acid ethyl ester (JG-03-14) and found that it is a potent microtubule depolymerizer. JG-03-14 caused a dose-dependent loss of cellular microtubules, formation of aberrant mitotic spindles, accumulation of cells in the G₂/M phase of the cell cycle, and Bcl-2 phosphorylation. These events culminated in the initiation of apoptosis, as evidenced by the caspase 3 dependent cleavage of PARP. JG-03-14 has antiproliferative activity against a wide range of cancer cell lines, with an average IC₅₀ of 62 nM, and it is a poor substrate for transport by P-glycoprotein. JG-03-14 inhibits the polymerization of purified tubulin in vitro, consistent with a direct interaction between the compound and tubulin. JG-03-14 potently inhibited the binding of [³H]colchicine to tubulin, suggesting that it binds to tubulin at a site overlapping the colchicine site. JG-03-14 had antitumor effects in the PC3 xenograft model, in which it caused greater than 50% reduction in tumor burden after 14 days of treatment. Molecular modeling studies indicate that the dimethoxyphenyl group of JG-03-14 occupies a space similar to that of the trimethoxyphenyl group of colchicine. However, the 2,3,5-trisubstituted pyrrole group, which is connected to the dimethoxyphenyl moiety, interacts with both and tubulin in space not shared with colchicine, suggesting significant differences compared with colchicine in the mechanism of binding to tubulin. Our results suggest that this tetrasubstituted pyrrole represents a new, biologically active chemotype for the colchicine site on tubulin.

Key words: MDR/p-Glycoprotein, Molecular dynamics, Cytoskeletal targets

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