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. 2009 Oct;74(4):358-68.
doi: 10.1111/j.1747-0285.2009.00866.x. Epub 2009 Aug 18.

High-content analysis of cancer-cell-specific apoptosis and inhibition of in vivo angiogenesis by synthetic (-)-pironetin and analogs

Andreas Vogt  1 , Peter A McPherson, Xiaoqiang Shen, Raghavan Balachandran, Guangyu Zhu, Brianne S Raccor, Scott G Nelson, Michael Tsang, Billy W Day
Affiliations

High-content analysis of cancer-cell-specific apoptosis and inhibition of in vivo angiogenesis by synthetic (-)-pironetin and analogs

Andreas Vogt et al. Chem Biol Drug Des. 2009 Oct.

Abstract

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 alpha,beta-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.

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Figures

Figure 1
Figure 1. Design and synthesis of pironetin analogs
A. Covalent tubulin modification by αLys352 conjugate addition to bound (−)-pironetin and a pironetin derivative designed to mimic interaction with αLys352 without covalent binding. B, Synthesis of pironetin derivatives 3 and 4.
Figure 2
Figure 2. (A–C) High-content analysis of mitotic arrest
HeLa cells were treated with vehicle (dashed line) or ten two-fold dilutions of paclitaxel (□), vincristine (■), (−)-pironetin (●), 3 (○), or 4 (▲) for 21 h and analyzed by high-content analysis for (A) microtubule mass, (B) condensed nuclei, and (C) mitotic index. All agents with the exception of 4 caused arrested cells in mitosis. The microtubule-destabilizing agents vincristine, (−)-pironetin, and 3 showed initial increases in tubulin immunostaining that reversed at higher concentrations. In contrast, tubulin immunostaining increased steadily with the microtubule-stabilizing agent, paclitaxel. Synthetic (−)-pironetin was as potent as vincristine and paclitaxel, whereas 3 was about 20-fold less active. Data are the averages from quadruplicate wells from a single experiment that has been repeated twice with similar results. (D–G) Microtubule morphology. Photomicrographs of HeLa cells treated with (D) vehicle (DMSO), (E) vincristine (200 nM), (F) (−)-pironetin (100 nM), or (G) 3 (1.5 μM), and stained for α-tubulin (green), phospho-histone H3 (red), and nuclei (blue). Vehicle-treated cells have highly organized microtubules and a low percentage of mitotic cells. All agents caused a heterogeneous response of microtubule disorganization and possibly loss of microtubule mass, increased numbers of phospho-histone H3 positive cells, as well as chromatin condensation and nuclear fragmentation. Images shown are representative fields from a single experiment that has been repeated twice with similar results. (H and I). Loss of tubulin immunostaining in cells treated with microtubule destabilizing agents occurs in both mitotic and non-mitotic cell populations. Three HCS parameters of cellular activity in 1,000 individual cells were graphically represented using Spotfire Decision Site. (H) Vehicle-treated cells (grey) show baseline tubulin staining (x-axis), a low percentage of mitotic cells (y-axis), and 2N DNA content (z-axis). High concentrations of paclitaxel (100 nM, red) caused mitotic arrest and increased microtubule staining due to formation of bright, stable microtubule bundles. In contrast, mitotic and non-mitotic cell populations in vincristine-treated cells (yellow) were shifted to lower tubulin staining intensity compared with paclitaxel, although the loss was more pronounced in the mitotic cells. (I) Pironetin (100 nM, green) and 3 (2.5 μM, black) show mitotic arrest with reduced microtubule staining in the mitotic cells, similar to vincristine (yellow) and different from paclitaxel (red). All axes are logarithmic scale.
Figure 3
Figure 3. Inhibition of tubulin assembly in vitro
Electrophoretically homogenous bovine brain tubulin (final concentration 10 μM; 1 mg/mL) was preincubated with test agents dissolved in DMSO (4% v/v final concentration) and monosodium glutamate (0.8 M final concentration) for 15 min at 30 °C. The reaction mixture was cooled to 0 °C and GTP (0.4 mM final concentration) was added. Reaction mixtures were transferred to cuvettes held at 2.5 °C in a temperature controlled multichannel spectrophotometer reading absorbance (turbidity) at 350 nm. Baselines were established and temperature was quickly raised to 30 °C (in approximately 1 min). After 20 min, the temperature was returned to 2.5 °C. (A) The known microtubule destabilizer, vinblastine (VBL) prevented tubulin assembly in a concentration-dependent manner; (B) (−)-Pironetin delayed the onset but increased the extent of tubulin assembly. (C) Effect of compound on tubulin assembly in the absence of GTP. Experimental conditions were as above except that to some samples no GTP was added. (D) Effect of (−)-pironetin on preformed microtubules. Instead of pre-incubation with test agent, drug in DMSO was added 7 min after initiating GTP-induced assembly.
Figure 4
Figure 4. (−)-Pironetin and 3 caused cancer-cell specific apoptosis and cell cycle arrest
HeLa and IMR-90 cells (10,000 per well) were plated on the left and right halves, respectively, of a 384-well microplate. Wells were treated from right to left with 10 two-fold serial dilutions of drugs as shown. The two columns in the center of the plate show 14 replicates of vehicle treated control wells for each cell line. Starting concentrations for each agent are shown in parentheses. (A) Cell cycle arrest. Total Hoechst 33342 staining intensities from a minimum of 1,000 cells are presented as DNA content density distributions for each individual well on the microplate. Highlighted are the lowest concentrations where a shift in DNA content could be detected by visual inspection. (B) High-content analysis of apoptosis. HeLa, IMR-90, or A-549 cells treated with vehicle or ten two-fold dilutions of paclitaxel (□), vincristine (■), (−)-pironetin (●), 3 (○), or 4 (▲) were analyzed by high-content analysis for cell density, chromatin condensation, caspase cleavage, and p53 induction. Data are the averages of quadruplicate wells from a single experiment that has been repeated at least once with similar results. (HeLa, n=1).
Figure 5
Figure 5. Pironetin and 3 inhibit angiogenesis in zebrafish embryos
Lateral views of 32hpf (upper panel) or 48 hpf (lower panel) Tg(fli1:EGFP)y1 treated with compounds indicated. (A) In vehicle treated embryos, intersegmental vessels (ISV) sprout from the dorsal aorta (DA) and connect to the dorsal longitudinal anastomotic vessel (DLAV). (B) (−)-pironetin (2.5 μM) prevented the normal outgrowth of ISV and formation of the DLAV. (C) At 50μM 3, ISV growth was stunted in a similar fashion to 2.5 μM (−)-pironetin. These phenotypes are similar to embryos treated with 40μM SU11652, a known VEGFR2 inhibitor (D). Fluorescence images are shown inverted to improve visibility.
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