Mechanism of Action of Cytotoxic Macrolides Amphidinolide X and J.

Abstract

Microtubules and actin filaments play important biological roles in mitosis, cytokinesis, cell signaling, intracellular transport, and cell motility of eukaryotic cells.1, 2 Molecules that target these cytoskeleton proteins are potential antitumor3 or anti-HIV agents.4 In fact, there are several clinical drugs that target the stabilization (paclitaxel-like behavior) or destabilization (vinca-like or colchicine-like behavior) of microtubules, specifically their heterodimeric component, α,β-tubulin.3 On the other hand, no actin-targeting drug has yet entered clinical studies.

Amphidinolides are a series of structurally dissimilar cytotoxic macrolides isolated from dinoflagellates (Amphidinium sp.).5–7 Their mechanisms of action are unknown, except that for one that has one of the largest rings, the 26-membered macrolide amphidinolide H (Amp-H, MW=562.73); this shows cytotoxicity in the nanomolar range against several carcinoma cell lines.8 Amp-H drastically and irreversibly deformed actin fibers;9 the actin fibers completely disappeared, and only a few disorganized aggregates remained in the cells. Amp-H induced multinucleated cells by disrupting actin organization (polyploid cells). In vitro assays on purified actin indicated that Amp-H stimulates actin polymerization, and stabilizes the actin filaments (F-actin).10, 11

In contrast, most of the smallest amphidinolides are cytotoxic in the micromolar range. For example, amphidinolide X (1, MW=448.59)12 and amphidinolide J (4, MW=390.56)13 have IC50 values of 1.3 and 6.9 μM, respectively, against the lymphocytic leukemia cell line L1210,12–14 although their mechanisms of action have not been reported. As these small amphidinolides are easier to synthesize than the larger molecules,15, 16 it would be desirable to identify their binding sites. Appropriate chemical modifications of these natural products might afford leads with activities below 0.1 μM that might eventually give rise to new antitumor agents. We report here biological studies of 1, the structurally related synthetic diolides 2 and 3, and 4 (Scheme 1). We examined their effect on the proliferation of A2780 (human ovarian carcinoma) and of LoVo (human colon carcinoma) cell lines, as well as on the cytoskeleton proteins tubulin, actin, and intermediate filaments in A549 (lung carcinoma) and PtK2 cells. Their effects on actin polymerization was then studied in vitro