Brianne S. Raccor

Structure–activity and High‐content Imaging Analyses of Novel Tubulysins

The synthesis and biological evaluation of three tubulysin analogs provides the first structure–activity relationship in this family of potent cytotoxic myxobacteria metabolites. Most importantly, the labile N,O‐acetal at N14 is not essential for biological activity. Further, structural simplifications are possible without abolishing biological activities. The N‐terminal amino acid can be replaced with N‐methylsarcosine, and the configuration at the acetoxy‐bearing stereocenter at C11 is important but not critical for almost all aspects of the biological profile. Our data encourage further development of these compounds as potential therapeutic agents in cancer treatment.

High-Content Analysis of Cancer-Cell-Specific Apoptosis and Inhibition of in Vivo Angiogenesis by Synthetic (−)-Pironetin and Analogs

The natural product (−)‐pironetin is a structurally simple small molecule microtubule‐perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic α,β‐unsaturated lactone moiety. We used alkaloid‐catalyzed acyl halide‐aldehyde cyclocondensation reactions in asymmetric total syntheses of (−)‐pironetin and three synthetic analogs, and evaluated their biological activities by high‐content analysis in cell culture and in a zebrafish model. Synthetic (−)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi‐drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (−)‐pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide‐aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (−)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule‐perturbing agents in the context of a whole organism or under conditions of multi‐drug resistance.

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

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 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. 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 (Ki) determinations using [14C]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 [14C]epothilone B with Ki values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (Ki 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 β-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.

Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization, with an IC50 of 11.8 ± 0.4 μM, and inhibited [3H]vinblastine binding noncompetitively, with a Ki of 4.5 ± 0.6 μM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1, with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.