|Home | About | Journals | Submit | Contact Us | Français|
The natural product (−)-dictyostatin is a microtubule stabilizing agent that potently inhibits the growth of human cancer cells including paclitaxel-resistant clones. Extensive structure-activity relationship studies have revealed several regions of the molecule that could be altered without loss of activity. The most potent synthetic dictyostatin analog described to date, 6-epi-dictyostatin, has in vivo antitumor activity against human breast cancer xenografts superior to paclitaxel. Despite their encouraging preclinical activities, the complex chemical structure of the dictyostatins presents a major obstacle in their development into novel antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26 dihydrodictyostatins and found several agents that compared with 6-epi-dictyostatin retained nanomolar activity in cellular microtubule bundling assays but showed cross-resistance to paclitaxel in cells with mutations in beta-tubulin. Extending these studies, we applied the new, highly convergent synthesis to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin. Both compounds were potent microtubule perturbing agents that induced mitotic arrest and microtubule assembly in vitro and in intact cells. In vitro radioligand binding studies showed that 25,26-dihydrodictyostatin and its C-6 epimer were able to displace [3H]paclitaxel and [14C]epothilone B from microtubules with potencies comparable to (−)-dictyostatin and discodermolide. Both compounds inhibited the growth of paclitaxel- and epothilone B-resistant cell lines at low nanomolar concentrations, synergized with paclitaxel in MDA-MB-231 human breast cancer cells, and had antiangiogenic activity in transgenic zebrafish larvae. The data identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as candidates for scale-up synthesis and further preclinical development.
Microtubules (MTs) are an important component in cell division and mitosis. Interference with MT dynamics causes a block in cell cycle progression and eventually programmed cell death (apoptosis), desirable results for treating rapidly dividing cancer cells. MT perturbing agents such as taxanes, epothilones, or vinca alkaloids, which stabilize or destabilize MTs, are successfully used in the treatment of solid or hematologic malignancies (1). The clinical successes of these anticancer agents have made MTs one of the most validated molecular cancer targets. Current, FDA-approved MT stabilizing agents are the taxanes paclitaxel (Taxol™), docetaxel (Taxotere™), cabazitaxel (Jevtana™), an albumin-bound form of paclitaxel (Abraxane™), and a semi-synthetic analog of epothilone B, ixabepilone (Ixempra™). Despite their success, the development of drug resistance reduces the effectiveness of these agents (2), resulting in a continued effort to develop novel MT perturbing agents.
Several MT stabilizing agents are currently under investigation as potential anticancer therapies (3). A particularly promising agent, (+)-discodermolide, a potent microtubule stabilizer with activity superior to paclitaxel, entered into Phase I clinical trials, but disappointingly failed due to pulmonary toxicity (4). Previously overshadowed by (+)-discodermolide, (−)-dictyostatin, a closely related compound, has recently gained attention as a potential anticancer agent. A decade after isolation, the complex structure was finally resolved (5), and two total syntheses (6, 7) provided enough sample for a detailed characterization (7, 8). Extensive structure activity relationship (SAR) studies have provided important information for the development of several (−)-dictyostatin analogs (9–11). These studies culminated in the discovery of 6-epi-dictyostatin, which was shown to have antitumor activity superior to paclitaxel in mice bearing human breast cancer MDA-MB-231 xenografts (12). In spite of these promising preclinical results, the complex structure and difficult synthesis of (−)-dictyostatin and analogs present major obstacles in their further preclinical development.
We recently reported a streamlined synthesis that generated new 16-desmethyl-25,26-dihydrodictyostatins that were considerably easier to make and in preliminary biological studies retained much of the potency of (−)-dictyostatin (13). Based on the biological activity of the series, which suggested reduction of the C25, C26 double bond is well tolerated but removal of the C16 methyl group results in loss of activity against paclitaxel-resistant cells (13), we applied the new streamlined synthesis to generate 25,26-dihydrodictyostatin (1a) and 6-epi-25,26-dihydrodictyostatin (1b). High-content cellular analysis revealed that 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin induced mitotic arrest and stabilized cellular MTs with potencies similar to that of the natural product. In vitro, both agents caused tubulin assembly with potency similar to paclitaxel and displaced [3H]paclitaxel and [14C]epothilone B from preformed MTs. The new analogs inhibited the growth of human cancer cells at low nanomolar concentrations, retained antiproliferative activity in epothilone B- and paclitaxel-resistant cancer cell lines, were able to synergize with paclitaxel, and had antiangiogenic activity in a zebrafish model. The data validate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as bona fide MT stabilizing agents and identify them as candidates for continued preclinical development.
The dictyostatin analogs 1a and 1b were prepared by full syntheses. The Supporting Information contains complete characterization details and copies of NMR spectra. Full experimental details of the synthesis will be published elsewhere. [3H]Paclitaxel was obtained from the Drug Synthesis and Chemistry Branch, NCI. [14C]Epothilone B was a gift from Novartis Pharma.
HeLa human cervical carcinoma cells (ATCC, Manassas, VA), A549 human lung cancer cells, and their epothilone B-resistant counterparts EpoB40/A549 (a gift from Susan Horwitz, Albert Einstein College of Medicine) were maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS, Cellgro), 2 mM L-glutamine (Invitrogen), and 1% penicillin-streptomycin (Invitrogen). Maintenance medium for EpoB40/A549 cells contained 40 nM epothilone B, which was removed prior to experimental setup. The HeLa/DZR cell line was generated as previously described (14) using ethyl methane sulfonate mutagenesis followed by stepwise increased concentrations of the antimitotic, tubulin assembly inhibiting, macrocyclic polyketide disorazole C1 (0.1–10.8 nM), leading to ~30-fold resistance to disorazole C1. These cells were valuable in our studies because they are resistant to natural products at least in part due to the overexpression of the ATP-binding cassette ABCB1 transporter (14). Thus, HeLa/DZR cells are cross resistant to the natural products vinblastine, doxorubicin and paclitaxel but not to cisplatin (14). Cells were cultured as previously described (14).
MDA-MB-231 human breast cancer cells (ATCC), 1A9 human ovarian carcinoma cells and their paclitaxel-resistant clones 1A9/PTX10 and 1A9/PTX22 (a gift from Drs. Tito Fojo and Paraskevi Giannakakou) were maintained in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum. Maintenance medium for 1A9/PTX10 and 1A9/PTX22 cells was further supplemented with 17 nM paclitaxel and 10 µM verapamil. Forty-eight hours prior to test agent analyses, paclitaxel and verapamil were removed and the cells placed into phenol red-free RPMI 1640 medium supplemented with 10% FBS and antibiotics. All cells were maintained in a humidified atmosphere of 95% air-5% CO2 at 37°C. The identities of the HeLa and MDA-MB-231 cell lines were confirmed by The Research Animal Diagnostic Laboratory (RADIL) at the University of Missouri, Columbia, MO (http://www.radil.missouri.edu), using a PCR based method that detects 9 short tandem repeat (STR) loci, followed by comparison of results to the ATCC STR database.
We used our previously reported cell-based immunofluorescence assay (11, 15) for high-content analysis of mitotic arrest and microtubule stabilization. In brief, 7,500 HeLa cells per well were seeded into the wells of two 384-well collagen-coated microplates (Becton Dickinson), allowed to adhere for 5 h, and treated for an additional 21 h with either vehicle control (DMSO) or test agents. Cells were fixed with 4% formaldehyde containing 20 µg/mL Hoechst 33342, permeabilized with 0.2% Triton X-100 and immunostained with the following antibody combinations: anti-α-tubulin (Sigma Aldrich, T9026, mouse monoclonal, 1:3000 dilution)/ fluorescein isothiocyanate (FITC)-labeled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-095-150, 1:500 dilution) and anti-phosphohistone H3 (Millipore, 06-570, rabbit polyclonal, 1:500 dilution)/Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152, 1:500 dilution) for mitotic arrest, or anti-acetylated tubulin (Sigma Aldrich, T7451, mouse monoclonal, 1:1000 dilution)/Cy-3-labeled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-165-150, 1:500 dilution) for quantitation of stabilized cellular MTs. Cells were imaged on the ArrayScan II HCS reader (Thermo Fisher Cellomics, Pittsburgh, PA) using a 20X objective and an Omega XF93 filter set at excitation/emission wavelengths of 350/461 nm (Hoechst), 494/519 nm (FITC), and 556/573 nm (Cy3). For each condition images of 1,000 cells were acquired and analyzed using a Target Activation Bioapplication algorithm (Thermo Fisher Cellomics, Pittsburgh, PA) essentially as described (16). An image mask was generated from the Hoechst-stained nuclei. MT density and acetylation were defined as the average pixel intensity in an area defined by the nuclear mask. For determination of mitotic index and nuclear condensation, thresholds for Hoechst 33342 and phosphohistone-H3 intensities were defined as one S.D. above the average Hoechst 33342 or Cy3 intensity obtained from 28 vehicle-treated wells located in the center of the microplate. Cells were classified as positive if their average Hoechst 33342 or Cy3 intensity exceeded this threshold. Minimal detectable effective concentrations (MDEC) were estimated from concentration-response curves as described (17).
Growth inhibition of A549 and EpoB40/A549 cells was assessed over three days using a modified version of our previously described high-content cytotoxicity assay (18). Cells were plated in 384-well collagen-coated plates at 1,000 cells per well, allowed to adhere overnight, and treated in quadruplicate with 10-point 2-fold serial dilutions of individual test agents or vehicle control (DMSO) for an additional 72 h. After the 72 h treatment period, cells were fixed and nuclei stained with 10 µg/mL Hoechst 33342. Four imaging fields were acquired on the ArrayScan II at excitation/emission wavelengths of 350/461 nm using a 10x objective, and nuclei enumerated as described (18). Cell densities were calculated as objects per imaging field and normalized to vehicle control density at the end of the study.
Growth inhibition of 1A9 human ovarian cancer cells and the paclitaxel-resistant clones 1A9/PTX10 and 1A9/PTX22 was assessed over three days using a previously described colorimetric assay (8). Cells were seeded at a low density into 96-well plates. Following a 48 h attachment and growth period, the cells were treated with a concentration range of individual test agents in quadruplicate or vehicle control (DMSO, n=8) for an additional 72 h. Cell proliferation was assessed spectrophotometrically after exposure to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium and N-methyloxyphenazine methylsulfate (MTS) followed by an absorbance reading at 490 nm minus the absorbance reading at 630 nm. One full microplate was developed at the end of the attachment period to determine cell numbers at the time of treatment. The 50% growth inhibitory concentrations (GI50) of test agents were calculated from the spectrophotometrically determined expansion of the control cells over the 72 h period.
HeLa/DZR cells were transfected with 20 nM ABCB1 siRNA or scrambled siRNA (Stealth siRNA Negative Control Hi GC, both from Invitrogen) as described previously (14). Treatment with this ABCB1 siRNA caused >75% decrease in ABCB1 protein levels at 24- and 72-h after transfection as measured by Western blotting (14). Briefly, HeLa/DZR cells were plated at a density of 7.5 × 104 cells/well into a six-well tissue culture plate and transfected 24 h thereafter with 20 nM ABCB1 siRNA or scrambled siRNA using 5 µL/well Dharmafect 1 reagent (Dharmacon, Lafayette, CO) and 480 µL/well Optimem transfection medium (Invitrogen) in a total volume of 2 mL/well. After 5 h, the transfection medium was replaced with fresh medium. Twenty-four h later cells were detached with 0.05% trypsin, seeded into 96 well plates at a density of 1,000 cells/well, and allowed to attach overnight. Cells were then treated with test agents or vehicle control for 72 h. Growth inhibition was determined by measuring Hoechst 33342-stained nuclei as described above.
Combination cytotoxicity studies were performed essentially as described (19). MDA-MB-231 cells were treated in quadruplicate for 96 h with 10-point 2-fold serial dilutions of paclitaxel, test agents, or a fixed ratio of test agent and paclitaxel based on the GI50 values of the individual agents. Images were acquired on the ArrayScan II and nuclei enumerated as described above. Affected fractions (Fa) were calculated as Fa = cell density of drug treated cells / cell density of vehicle treated cells. The data were analyzed using the median-effect analysis of Chou and Talalay (20), assuming mutually exclusive drug effects. The degree of synergism, additivity, and antagonism was measured by calculating combination indices (CI) over a range of affected fractions exactly as described previously (19).
Experiments were performed as previously described (11) using tubulin purified in our laboratory from bovine brains by the method of Hamel (21). MTs were preformed by incubating 2 µM bovine tubulin with 40 µM ddGTP in 0.75 M MSG, pH 6.6, at 37 °C for 30 min. In separate tubes, a 50 µL solution of 8 µM test agent and 4 µM radiolabeled [3H]paclitaxel or [14C]epothilone B in 0.75 M MSG, pH 6.6, with a final DMSO content of 1%, was incubated for 10 min at 37 °C. An aliquot (50 µL) of the preformed MTs was added to the radioligand/test agent mixture and incubated at 37 °C for an additional 30 min. Final concentrations of tubulin, radioligand and test agents were 1 µM, 2 µM, and 4 µM, respectively. Reaction mixtures were then centrifuged at 17,000 × g for 30 min at room temperature and the amount of unbound radioligand determined by analyzing 50 µL of the supernatant by scintillation spectrometry (Beckman-Coulter LS6500). To account for non-specific radioligand binding, the amount of bound radioligand was calculated by subtracting the amount of radioligand in the supernatant in the presence of test agent from the amount of radioligand in the supernatant in the presence of a large molar excess of the agent with the highest binding affinity (20 µM (−)-dictyostatin) (8, 11). The extent of displacement was then calculated as percent inhibition = (1−(radioligand bound with test agent/radioligand bound with DMSO))*100.
Tubulin assembly was monitored turbidimetrically at 350 nm in temperature-controlled, multichannel Beckman-Coulter 7400 or Gilford 250 spectrophotometers as described previously (8, 22). Reaction mixtures without test compounds consisted of bovine brain tubulin (1 mg/mL) in 0.1 M (4-morpholino)ethane sulfonate (Mes) and were cooled to 2.5°C to establish baselines. Compounds predissolved in DMSO were added to give the indicated final concentrations and each reaction mixture (0.25 mL final volume) was subjected to a temperature gradient. From the precooled state, the temperature was rapidly raised to 30°C (in approximately 1 min) and maintained for 20 min. The temperature was then rapidly lowered back to 0.25–2.5°C. Absorbance at 350 nm was monitored every 15 s.
The Tg(Fli1:EGFP)y1 transgenic zebrafish line (obtained from Dr. Brant Weinstein) was maintained as described (23). Embryos were collected at 24 h post fertilization (hpf) and staged according to (24). For each condition, five Tg(Fli1:EGFP)y1 transgenic zebrafish embryos were placed in 500 µL E3 medium (5 mM NaCl, 0.33 mM CaCl2, 0.17 mM KCl, 0.33 mM MgSO4) and treated with vehicle (DMSO, 0.5%) or various concentrations of test agents (1 µM to 25 µM) for an additional 24 h. After manual removal of the chorions, single embryos were transferred to wells of a 96-well half area plate (Greiner, Monroe, NC) containing 40 µg/ml MS222 (tricaine methanesulfonate, Sigma) in E3 for imaging.
Photomicrographs of fluorescent ISV were acquired with the ImageXpress Ultra high-content reader (Molecular Devices, Sunnyvale, CA) using a 4X objective and the 488 nm argon laser. Images were uploaded into the Definiens Developer software suite (Definiens AG, Germany) and analyzed with a custom designed Cognition Network Technology (CNT) ruleset as described (25). Thresholding modifications were made to the CNT ruleset to accommodate the higher resolution and pixel depth of the ImageXpress system compared with the previously used ArrayScan (25). Total embryo size and intensity measurements were used to identify dead embryos, plate-loading artifacts, and autofluorescent compounds. Wells that contained no embryos, or embryos in which no dorsal region could be detected were eliminated. For the remaining wells, the ruleset provided numerical measurements of ISV development (area, length, and shape). The parameter that most robustly measured ISV development was the total ISV area (in pixels). Data were normalized to vehicle controls. Experiments were repeated at least three times.
We recently reported a streamlined synthesis of dictyostatin and used it to prepare two 16-desmethyl-25,26-dihydrodictyostatins epimeric at C6 (13). Based on the biological activity of the series, we concluded that the reduction of the C25–C26 double bond is well tolerated but that removal of the C16 methyl group causes loss of activity against paclitaxel resistant cells (13). Accordingly, we selected 25,26-dihydrodictyostatin 1a and 6-epi-25,26-dihydrodictyostatin 1b as target compounds.
The streamlined route, which features high convergence, modularity, a relative ease with which structurally complex new analogs of DCT can be prepared without ambiguity in the C2–C3 configuration, and reliability of the fragment couplings, was used to make the new analogs 1a and 1b. Fragment couplings and completion of the syntheses are summarized in Figure 1. Briefly, a Horner-Wadsworth-Emmons (HWE) reaction (26) was used to couple the known top fragment 4 (13) with new middle fragment 3 to give 5. 1,4-Reduction of the enone, removal of the para-methoxybenzyl (PMB) group, stereoselective ketone reduction and mono-silylation then provided 6. Intermolecular esterification with epimeric acid chlorides 7a,b incorporated the bottom fragment (27) to give 8a,b. Selective removal of the primary tert-butyldimethylsilyl (TBS) group and oxidation provided aldehydes 9a,b that were substrates for an intramolecular Nozaki-Hiyama-Kishi (NHK) reaction (13) to give macrolactone 10a,b. Selectivity in the formation of the new stereocenter at C9 depended on the configuration at C6 with the b isomer being more selective (10b, 10/1; 10b, 3/1). Desilylation and careful purification to remove the C9 epimers provided the target products 1a and 1b. The strategy enabled the total synthesis of both analogs in a total of 39 steps, with a longest linear sequence of 11 steps from commercially available starting material.
We first characterized the novel agents for mitotic arrest and microtubule perturbation using our multiparameter high-content analysis assay (11, 15) as described in the Materials and Methods Section. Immunofluorescence images of HeLa cells treated with test agents for 21 h show that the new analogs, like 6-epi-dictyostatin, caused MT bundling (shown in green), chromatin condensation (blue), and elevated levels of phosphohistone H3 (red) at nanomolar concentrations (Figure 1B). All agents showed concentration-dependent changes (Figure 1C). From the range of concentrations tested, a minimum detectable effective concentration (MDEC) value was determined (28). The data indicate that the new agents were equipotent to 6-epi-dictyostatin and paclitaxel. A detailed summary of the mitotic arrest assay results can be found in Table S1 in the Data Supplements Section.
We next asked if the new agents stabilized MTs in cells and caused MT assembly of isolated tubulin in vitro. It was previously shown that acetylated tubulin is a marker for stabilized cellular MTs (29). Cells were stained with antibodies against alpha tubulin or acetylated tubulin, respectively, to visualize cellular MTs and MT acetylation. Figure 2A shows distinct differences in the concentration-response curves of tubulin and acetylated tubulin staining obtained with (−)-dictyostatin, a known MT stabilizer, or vincristine, a known MT destabilizer. In cells treated with (−)-dictyostatin, we observed a steady increase in cellular MT density as well as acetylated MTs that plateaued at high concentrations. In contrast, vincristine caused an initial increase in cellular MT density and MT acetylation at low concentrations that was lower in magnitude and that reversed at higher concentrations. This bimodal response is characteristic for MT destabilizing agents: the initial increase results from morphological changes (i.e., cell rounding); the subsequent decrease is due to extraction of monomeric tubulin into the permeabilization buffer during cell processing and staining (15). Both the shape and the magnitude of MT and acetylated MT density curves caused by the dictyostatin analogs (Figure 2A) were identical to that elicited by (−)-dictyostatin, suggesting 25,26-dihydrodictyostatin (1a) and 6-epi-25,26-dihydrodictyostatin (1b) caused MT stabilization. Immunofluorescence micrographs of acetylated MTs confirmed the results of the automated analysis (Figure 2B).
To further confirm the MT stabilizing activity of the new analogs, we performed in vitro tubulin assembly studies using a turbidity assay (22) and paclitaxel as a positive control. Isolated tubulin from bovine brain was incubated with vehicle (DMSO) or various concentrations of test agents and subjected to a temperature gradient as shown in Figure 2C. The new agents induced rapid and vigorous tubulin assembly with potency similar to paclitaxel and (−)-dictyostatin (Figure 2C). Assembly was concentration-dependent and the resulting polymer was cold-stable, similar to paclitaxel and consistent what we had previously observed with 6-epi-dictyostatin (11).
We previously showed that (−)-dictyostatin competes with [3H]paclitaxel and [14C]epothilone B for binding to tubulin polymer formed in the presence of ddGTP (8). We therefore tested whether the new analogs retained this ability. Discodermolide, (−)-dictyostatin, and the new analogs were incubated with preformed MTs labeled with [3H]paclitaxel and [14C]epothilone, and the amount of unbound tracer measured by scintillation spectrometry. Table 1 shows that the new analogs displaced [3H]paclitaxel and [14C]epothilone B with similar potency to discodermolide or (−)-dictyostatin. These experiments provided conclusive evidence that the new dictyostatin analogs bind the taxoid site on tubulin polymer with affinities similar to that of (−)-dictyostatin.
(−)-Dictyostatin has antiproliferative activity in paclitaxel-resistant cells (11). To assess if the analogs remained active in drug resistant cancer cell lines, we tested 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin in paclitaxel-resistant 1A9 human ovarian cancer cells with beta-tubulin mutations (Phe270 –> Val) and (Ala364 –>Thr) (30) induced by long-term culture with paclitaxel, and in epothilone B-resistant A549 human lung cancer cells that harbor a point mutation in beta-tubulin (292Gln–>Glu) as a result of long-term exposure to epothilone (31). Table 2 shows that cross-resistance to paclitaxel in the 1A9/PTX10 cells was reduced from 49-fold, to 15-fold with (−)-dictyostatin and further reduced with the new analogs (7- and 8-fold for 1a and 1b, respectively). Similarly, cross resistance to epothilone B was reduced with (−)-dictyostatin (from 94-fold for epothilone B and 18-fold for paclitaxel to 10-fold with (−)-dictyostatin), and further diminished with the new analogs (5-fold and 3-fold, respectively, for 1a and 1b). Diminished cross-resistance was also observed in a recently described disorazole C1-resistant human cervical carcinoma cell line that overexpresses the ABCB1 P-glycoprotein pump (14). Consistent with previously published data (14), these cells were 1395- and 502-fold resistant to paclitaxel and vinblastine, respectively (Table 2). In contrast, the new dictyostatin analogs showed greatly reduced cross-resistance to disorazole C1 compared with paclitaxel and vinblastine, with a residual 12- and 18-fold resistance respectively, for 1a and 1b. To investigate further if the new analogs were affected by multidrug transport proteins, we performed siRNA knockdown of ABCB1, which reversed the residual cross-resistance in the disorazole C1 resistant cells (Table 2).
Discodermolide and paclitaxel represent a synergistic drug combination in human cancer cells (32). We therefore examined the novel dictyostatin analogs in combination with paclitaxel to determine if they also resulted in synergy. We used our previously described growth inhibition assay (18) together with median effect analysis (20) to quantify synergism, additivity, and antagonism. MDA-MB-231 cells were treated with comprehensive concentration gradients of paclitaxel, discodermolide, 6-epi-dictyostatin, 25,26-dihydrodictyostatin 1a, 6-epi-25,26-dihydrodictyostatin 1b, or equipotent, fixed mixtures thereof with paclitaxel for four days, and cell densities quantified by counting Hoechst 33342-stained nuclei. Median effect (Dm), slopes (m), and correlation coefficients (r) for the individual agents and the combinations can be found in Table S2 in the Supporting Information Section. Combination indices were then calculated for various effect levels by the method of Chou and Talalay (20, 33) as described previously (18). As shown in Figure 3, we reproduced the results of Martello et al. (32), who found the combination of paclitaxel and discodermolide to be synergistic at lower effect levels and antagonistic at high effect levels. The dictyostatins had combination index profiles similar to that of discodermolide, although the degree of synergism was lower. The least potent combination was with 6-epi-25,26-dihydrodictyostatin 1b (Figure 3D), which was additive over much of the effect range. The data consistently repeated over the course of multiple independent experiments. The data suggest that (−)-dictyostatin and the new analogs share the ability of discodermolide to synergize with paclitaxel, a feature that is potentially favorable for clinical use.
Some MT perturbing agents have antiangiogenic activity that contributes to in vivo anticancer activity (34). Solid tumors require an adequate supply of blood vessels to survive, grow, and metastasize (reviewed in (35)), and agents targeting tumor angiogenesis are now FDA-approved anti-cancer medicines (e.g., bevacizumab, Avastin ®). We therefore asked if the dictyostatin analogs had antiangiogenic activity. We used the Tg(fli1:EGFP)y1 zebrafish line that expresses EGFP under the control of the Fli1 promoter, thereby labeling all blood vessels and providing a live visual marker for vascular development (36). Zebrafish have a stereotypical vertebrate vasculature that develops in response to the same signals that guide mammalian blood vessel development (37, 38). Zebrafish vasculature recruitment also occurs in response to human glioma xenografts (39, 40), mimicking conditions found in mammals.
Tg(fli1:EGFP)y1 zebrafish embryos at 24 hpf were treated for 24 h with vehicle or various concentrations of test agents and imaged. Figure 4A shows that, as expected, vehicle-treated embryos had well-established intersegmental vessels (ISV) that extended from the dorsal aorta (DA) and connected to the dorsal longitudinal anastomotic vessel (DLAV) (Figure 4A, (Isogai et al., 2001)). Visually, all of the dictyostatin analogs stunted ISV outgrowth and prevented the establishment of the DLAV (Figure 4A, upper panels). Our previously described image analysis algorithm (25) quantified the antiangiogenic phenotype (Figure 4A, lower panels). All agents concentration-dependently inhibited angiogenesis (Figure 4B), with concentrations required to reduce ISV area by 50% compared with control (IC50) of 8.8, 6.1, and 6.7 µM for 6-epi-dictyostatin, 25,26-dihydrodictyostatin 1a, and 6-epi-25,26-dihydrodictyostatin 1b, respectively. Importantly, at concentrations that were antiangiogenic, we observed no obvious signs of toxicity such as the appearance of necrotic opaque cells. At the highest concentration tested (25 µM, data not shown), the test agents caused a bent-tail phenotype, suggesting that the compounds at this concentration would likely cause developmental defects in the embryo.
The complex chemical structure and difficult synthesis of the dictyostatins is a major impediment to their development into novel antineoplastic agents. This work validates that our recently described synthetic route (13) can be used to rapidly make new analogs. The streamlined route features a bimolecular esterification to make the C1–O21 bond in place of the usual macrolactonization. This bypasses a major problem of Z/E isomerization of the C2–C3 alkene that has plagued the macrolactonization. In turn, the large ring is closed by a mild Nozaki-Hiyama-Kishi reaction to make the C9–C10 bond. It should be possible to access many more analogs thanks to the modularity of this route and the reliability of the fragment couplings and end game steps.
Consistent with prior findings, removal of the C16 methyl moiety did not dramatically affect antiproliferative activity in human tumor cells expressing wild-type tubulin but diminished the ability of the compounds to inhibit the growth of paclitaxel-resistant clones harboring mutations within beta-tubulin (10). We therefore reasoned that retaining the C16 methyl group would preserve the lack of cross-resistance to paclitaxel and selected 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as target compounds. Consistent with existing SAR, both new agents showed low nanomolar antiproliferative activity in HeLa, A-549, and MDA-MB-231 cells, and reduced cross-resistance to paclitaxel and epothilone B in cells with mutant tubulin.
To confirm that the new analogs directly interact with their proposed target, we performed radioligand binding studies. These experiments show the new analogs have affinities for the taxane site similar to paclitaxel, epothilone B, or discodermolide. The precise location of the dictyostatin binding site has not been established, because the interaction of the dictyostatins or discodermolide with tubulin has not been solved by cryoelectron microscopy as it has for paclitaxel and epothilone A (41, 42). Furthermore, two binding sites have been described for taxanes: an internal luminal binding site and an external transient binding site of unknown structure. The radioligand competition studies are unable to distinguish the two sites. However, growth inhibition studies of the natural product (8) and on the 16-desmethyl analogs using 1A9/PTX10 ovarian cancer cells with the Phe270 —>Val mutation that we performed previously (13) are consistent with dictyostatin and analogs binding to the internal site.
The new analogs retained some but not all of the ability of discodermolide to synergize with paclitaxel in human breast cancer cells. Modeling studies based on NMR structures have suggested that the bound conformer of dictyostatin resembles that of discodermolide and provides similar contacts with tubulin (43). Because it is unusual for two drugs that bind to identical sites on the same target to show synergy, the combination cytotoxicity data do support the previously proposed model of overlapping binding sites for paclitaxel and the dictyostatins (43). The extent of synergy varied with the analogs; the least potent agent was 1b, although all of them showed a trend towards higher synergy at lower effect levels. Therefore, our results confirmed a synergistic relationship specifically at the lower concentrations of the two drugs as reported by Horwitz’s group (32). The reasons for the differential activity of the analogs in this assay are unknown. The fact that the dictyostatins were essentially equivalent in all of our assays, including the in vitro radioligand binding studies, makes it seem unlikely that differences in binding affinity or cellular distribution would account for the observed differences. To formulate a valid hypothesis based on structural terms, however, physical evidence such as a high resolution cryoelectron microscopy structure of the dictyostatins and discodermolide is needed. Alternatively, the different degree of synergy of the dictyostatins compared with discodermolide may be a result of off-target effects. As pointed out by Martello et al. (32), discodermolide induces apoptosis by mechanisms unrelated to MT binding, and it is currently not known whether the dictyostatins share these activities. The data do suggest, however, that the combination of paclitaxel with either 6-epi-dictyostatin or 1a merits exploration in in vivo antitumor studies.
Drug resistance is a major problem with MT perturbing agents in clinical use. One clinically important resistance mechanism is overexpression of p-glycoprotein efflux pumps (44). In cultured cells, additional resistance mechanisms have been observed that involve tubulin mutations induced by long-term culture of cell lines in the presence of MT perturbing agents (31, 45), although such drug-induced mutations have not been found in clinical samples. In three such cellular models with mutant tubulin, the new analogs retained activity against both paclitaxel- and epothilone B-resistant cells, and appeared less cross-resistant than the natural product. The 1A9/PTX10 cell line harbors a Phe270 —> Val mutation that is located within the taxane binding site (42) and confers 49-fold resistance to paclitaxel. Consistent with our previous studies with (−)-dictyostatin and 6-epi-dictyostatin (13), cross-resistance was reduced to <10-fold with the new analogs (Table 2). As expected, no cross-resistance was found in the 1A9/PTX22 cell line, which has a Ala364 —> Thr mutation that is adjacent to the taxane binding pocket. In epothilone B resistant A-549 cells with a 292Gln —> Glu mutation, which is located at the periphery of the taxane pocket and makes contact with epothilone but not paclitaxel (42), the analogs showed only a 12–18-fold cross resistance compared with epothilone B (94-fold resistance). The data indicate that reduction of the terminal double bond does not alter the mode of tubulin binding. They are consistent with a mode of binding to tubulin as proposed by Canales et al. (43) that involves the taxane binding pocket but not residues outside the pocket that make contact with the taxane side chain.
The analogs showed a unique behavior toward cells with acquired resistance against the natural product disorazole C1 (14), which owe their resistance phenotype at least in part to overexpression of the ABCB1 p-glycoprotein pump. All agents were subnanomolar inhibitors of wild-type HeLa cells. Paclitaxel and vinblastine were 1395- and 502-fold less active, respectively, in the resistant cells (HeLa/DZR, Table 2). Knockdown of the P-glycoprotein pump, ABCB1, restored most, of their activity (HeLa/DZR/ABCB1 siRNA, Table 2). In contrast, the HeLa/DZR cells showed only minor cross-resistance to the dictyostatin analogs (12- to 18-fold, HeLa/DZR, Table 2) that was fully reversed by ABCB1 knockdown. The data suggest that the dictyostatins may be only weak substrates for ABCB1. Moreover, because the HeLa/DZR cells were generated by a single exposure to the mutagen ethyl methane sulfonate followed by a stepwise increased disorazole C1 exposure, it is likely that resistance mechanisms other than elevated ABCB1 exist, but these do not appear to influence cellular sensitivity to the dictyostatin analogs.
We had previously shown that microtubule-perturbing agents inhibit angiogenesis in Tg(fli1:EGFP)y1 transgenic fluorescent zebrafish embryos (15). Here we demonstrate that the new analogs also have this property, which is thought to be beneficial for clinical activity (34, 46). In the Tg(fli1:EGFP)y1 model, the agents appeared to have antiangiogenic rather than antivascular activity. During development, intersegmental vessels (ISV)s sprout from the dorsal aorta (DA) at 24 hpf, and at 48 hpf are fully established and connected to the dorsal longitudinal anastomotic vessel (DLAV). To assess the effect of test agents on new vessel outgrowth (angiogenesis), embryos were treated at 24 hpf (when ISV are just beginning to sprout and are barely visible (15)), and analyzed for ISV formation 24 h thereafter. While the analogs caused a concentration-dependent inhibition of new vessel growth, they did not affect existing blood vessels as the head and large trunk vessels were intact. Furthermore, heart beat, circulation, and twitch response (assessed visually) were all normal (data not shown). We also did not observe tissue necrosis, which would show as opaque cells in the fluorescence micrographs (see Figure 4). Test agent-treated embryos also showed little difference in gross morphology when compared with control embryos (Figure 4), although we did observe a bent tail phenotype at the highest concentration tested (25 µM). While the model is currently not well enough characterized to suggest therapeutic safety in the context of angiogenesis inhibition, the data indicate the new dictyostatins have antiangiogenic activity in a zebrafish model of angiogenesis at nontoxic concentrations.
In summary, we have used our previously reported, highly convergent, streamlined synthesis (13) to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin, two new analogs of the highly complex natural product, (−)-dictyostatin. Consistent with existing SAR studies and a mode of action involving high affinity binding to the taxane site on tubulin, the new analogs retained essentially all of the biological activities of (−)-dictyostatin and 6-epi-dictyostatin, the only analog whose activity in adult mammals has been described to date (12). While the new analogs do not represent a significant simplification from a structural standpoint, reduction of the exposed double bond eliminates chemical reactivity and a potential metabolic “soft spot”, as has been shown for discodermolide (47). Future experiments should focus on this issue. The results identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as candidates for scale-up using the improved synthesis procedure and for further preclinical development.
We thank Dr. Brant Weinstein for the transgenic Tg(fli1:EGFP)y1 line, Dr. Susan B. Horwitz for the epothilone B-resistant cells, Drs. Tito Fojo and Paraskevi Giannakakou for the paclitaxel-resistant clones, the National Cancer Institute for [3H]paclitaxel, and Novartis Pharma for [14C]epothilone B.
This work was supported by the National Institutes of Health [Grants CA78039 to A.V and J.S.L., HD053287 to N.A.H.], and the Fiske Drug Discovery Fund.