Received for publication October 11, 2007.
Revised December 7, 2007.
Accepted for publication December 10, 2007.

Cell-Based and Biochemical Structure-Activity Analyses of Analogues of the Microtubule Stabilizer Dictyostatin

Abstract

Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought due to the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G₂/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. Here we examined a series of dictyostatin analogues 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 analogues, namely 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 [¹⁴C]epothilone B, which has a three-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [¹⁴C]epothilone B with K_i values of 480 nM and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K_i values of 4.55 and 4.47 µM, 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 -tubulin. Finally, we developed a set of QSAR equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogues.

Key words: Structure-activity relationships and modeling, Fluorescence techniques, Cytoskeletal targets