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 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 G 2 /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 IC 50 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 [ 3 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,

Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity

“…The hydrophobic substituted phenyl ring fits snugly in the hydrophobic (narrow funnel) region of the binding pocket. The docked model for JG-03-14 is qualitatively similar to one reported earlier [18].…”

“…One member of this series (JG-03-14, 3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester), for which NCI tumor panel activity had been obtained [19], was suggested by COMPARE [16] to have an activity profile similar to colchicine. Cellular studies with JG-03-14 further support the contention that these compounds function as microtubule poisons [18]. In addition, JG-03-14 was found to have the capacity to promote both autophagic and apoptotic cell death, albeit in different cell lines, while retaining activity in tumor cells expressing the multidrug resistant pump P-glycoprotein [18,24].…”

“…Cellular studies with JG-03-14 further support the contention that these compounds function as microtubule poisons [18]. In addition, JG-03-14 was found to have the capacity to promote both autophagic and apoptotic cell death, albeit in different cell lines, while retaining activity in tumor cells expressing the multidrug resistant pump P-glycoprotein [18,24]. Because the development of additional synthetic or semi-synthetic pyrrole derivatives in this class is facilitated with their relatively facile syntheses, including modification of the molecule by adding and removing functional groups [19], we have both a rather extensive collection of molecules in-hand (Scheme 1) for building predictive molecular models and the potential for rational design and synthesis of many others.…”

“…These compounds represent an emerging class of agents with potential activity against a variety of human tumors with activity expressed at nM or µM concentrations in human tumor cell lines [18,19], but having advantages over natural products in terms of drug design and development. In particular, we have been exploring a series of brominated pyrroles whose structure suggests that they might interfere with tubulin function.…”

“…Results from a number of assays have previously appeared regarding the antiproliferative and cytotoxic activities of the lead compound JG-03-14 [18,19,24]. However, most of the compounds in this series have not been examined in detail.…”