Identification and Characterization of a New Tubulin-Binding

We studied the mechanism of action of 3,5-dibromo-4-(3,4dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester (JG03-14) 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 poly(ADP-ribose) polymerase (PARP). JG-03-14 has antiproliferative activity against a wide range of cancer cell lines, with an average IC50 value of 62 nM, and it is a poor substrate for transport by P-glycoprotein. JG-03-14 inhibited 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 [H]colchicine to tubulin, suggesting that it bound 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 indicated 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, interacted 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. Microtubules are cellular structures that play a central role in metabolism, intracellular transport, and cell division. A wide range of chemicals have been identified that interrupt microtubule function. These compounds can be divided into microtubule stabilizers and microtubule depolymerizers. Microtubule stabilizers include paclitaxel, discodermolide, the epothilones, and the laulimalides. Microtubule stabilizers cause an increase in the density of cellular microtubules, and they stimulate the assembly of purified tubulin. In contrast, microtubule depolymerizers cause a loss of cellular microtubules, and they inhibit the assembly of purified tubulin. Microtubule depolymerizing compounds can be further subdivided into those that bind to tubulin within the colchicine site and those that bind within the vinca domain. Agents acting upon the colchicine site include 2ME2, combretastatin A-4, and podophyllotoxin. The phenotypic effects of microtubule stabilizing and depolymerizing agents are quite disparate when they are used at high concentrations in cells, but at their lowest antiproliferative concentrations, both classes of agents inhibit microtubule dynamics (Jordan and Wilson, 2004). In due course, inhibition of microtubule dynamics is believed to hinder the normal function of the mitotic spindle, This work was supported by grants from the William Randolph Hearst Foundation, the Amon Carter Foundation (to S.L.M.) and the National Institutes of Health Area Program R15-CA67236 (to J.T.G.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.034876. ABBREVIATIONS: 2ME2, 2-methoxyestradiol; Pgp, P-glycoprotein; PDB, Protein Database; DMA-colchicine, N-deacetyl-N-(2-mercaptoacetyl)colchicine; RMSD, root mean square difference; MD, molecular dynamics; FBS, fetal bovine serum; SRB, sulforhodamine B; RR, relative resistance; PARP, poly(ADP-ribose) polymerase; DMSO, dimethyl sulfoxide; R123, rhodamine 123; DiOC2(3), 3 -diethyloxacarbocyanine iodide; NCI, National Cancer Institute. 0026-895X/07/7201-132–140 MOLECULAR PHARMACOLOGY Vol. 72, No. 1 U.S. Government work not protected by U.S. copyright 34876/3225054 Mol Pharmacol 72:132–140, 2007 Printed in U.S.A.