discodermolide and Carcinoma

Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G(2)/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K(i)) determinations using [(14)C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [(14)C]epothilone B with K(i) values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K(i) values of 4.55 and 4.47 muM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of beta-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.

Topics: Alkanes; Animals; Benzimidazoles; Binding Sites; Brain Chemistry; Carbamates; Carcinoma; Cattle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Epothilones; Female; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; G2 Phase; HeLa Cells; Histones; Humans; Kinetics; Lactones; Macrolides; Microtubules; Molecular Structure; Ovarian Neoplasms; Paclitaxel; Phosphorylation; Protein Binding; Pyrones; Quantitative Structure-Activity Relationship; Radioligand Assay; Tubulin; Tubulin Modulators

(+)-Discodermolide, a C24:4, trihydroxylated, octamethyl, carbamate-bearing fatty acid lactone originally isolated from a Caribbean sponge, has proven to be the most potent of the microtubule-stabilizing agents. Recent studies suggest that it or its analogues may have advantages over other classes of microtubule-stabilizing agents. (+)-Discodermolide's complex molecular architecture has made structure-activity relationship analysis in this class of compounds a formidable task. The goal of this study was to prepare simplified analogues of (+)-discodermolide and to analyze their biological activities to expand structure-activity relationships. A small library of analogues was prepared wherein the (+)-discodermolide methyl groups at C-14 and C-16 and the C-7 hydroxyl were removed, and the lactone was replaced by simple esters. The library components were analyzed for microtubule-stabilizing actions in vitro, antiproliferative activity against a small panel of human carcinoma cells, and cell signaling, microtubule architecture and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even drastic structural simplification can lead to analogues with actions related to microtubule targeting and signal transduction, but that these subtle effects were illuminated only through the high information content cell-based screen.

Topics: Alkanes; Antineoplastic Agents; Binding Sites; Carbamates; Carbon; Carcinoma; Cell Division; Dose-Response Relationship, Drug; Gene Library; HeLa Cells; Humans; Lactones; Microtubules; Models, Chemical; Paclitaxel; Phosphorylation; Pyrones; Signal Transduction; Spindle Apparatus; Tubulin; Tumor Cells, Cultured