Identifying a resistance determinant for the antimitotic natural products disorazole C<inf>1</inf> and A<inf>1</inf>

Disorazoles are macrocyclic polyketides first isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. Both the major fermentation product disorazole A1 and its much rarer companion disorazole C1 exhibit potent cytotoxic activity against many human tumor cells. Furthermore, the disorazoles appear to bind tubulin uniquely among known antimitotic agents, promoting apoptosis or premature senescence. It is uncertain what conveys tumor cell sensitivity to these complex natural products. Therefore, we generated and characterized human tumor cells resistant to disorazole C1. Resistant cells proved exceedingly difficult to generate and required single step mutagenesis with chronic stepwise exposure to increasing concentrations of disorazole C1. Compared with wild-type HeLa cells, disorazole C1-resistant HeLa/DZR cells were 34- and 8-fold resistant to disorazole C1 and disorazole A1 growth inhibition, respectively. HeLa/DZR cells were also remarkably cross-resistant to vinblastine (280-fold), paclitaxel (2400-fold), and doxorubicin (47-fold) but not cisplatin, suggesting a multidrug-resistant phenotype. Supporting this hypothesis, MCF7/MDR cells were 10-fold cross-resistant to disorazole C 1. HeLa/DZR disorazole resistance was not durable in the absence of chronic compound exposure. Verapamil reversed HeLa/DZR resistance to disorazole C1 and disorazole A1. Moreover, HeLa/DZR cells expressed elevated levels of the drug resistance ATP-binding cassette ABCB1 transporter. Loss of ABCB1 by incubation with short interfering RNA restored sensitivity to the disorazoles. Thus, the multidrug resistance transporter ABCB1 can affect the cytotoxicity of both disorazole C1 and A1. Disorazole C1, however, retained activity against cells resistant against the clinically used microtubule-stabilizing agent epothilone B. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics

Identifying and quantifying heterogeneity in high content analysis: application of heterogeneity indices to drug discovery.

One of the greatest challenges in biomedical research, drug discovery and diagnostics is understanding how seemingly identical cells can respond differently to perturbagens including drugs for disease treatment. Although heterogeneity has become an accepted characteristic of a population of cells, in drug discovery it is not routinely evaluated or reported. The standard practice for cell-based, high content assays has been to assume a normal distribution and to report a well-to-well average value with a standard deviation. To address this important issue we sought to define a method that could be readily implemented to identify, quantify and characterize heterogeneity in cellular and small organism assays to guide decisions during drug discovery and experimental cell/tissue profiling. Our study revealed that heterogeneity can be effectively identified and quantified with three indices that indicate diversity, non-normality and percent outliers. The indices were evaluated using the induction and inhibition of STAT3 activation in five cell lines where the systems response including sample preparation and instrument performance were well characterized and controlled. These heterogeneity indices provide a standardized method that can easily be integrated into small and large scale screening or profiling projects to guide interpretation of the biology, as well as the development of therapeutics and diagnostics. Understanding the heterogeneity in the response to perturbagens will become a critical factor in designing strategies for the development of therapeutics including targeted polypharmacology

Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1S⃞

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

A simplified synthesis of novel dictyostatin analogues with In Vitro activity against epothilone B-resistant cells and antiangiogenic activity in zebrafish embryos

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 can be altered without loss of activity. The most potent synthetic dictyostatin analogue described to date, 6-epi-dictyostatin, has superior in vivo antitumor activity against human breast cancer xenografts compared with paclitaxel. In spite of their encouraging activities in preclinical studies, the complex chemical structure of the dictyostatins presents a major obstacle for their development into novel antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26-dihydrodictyostatins and found several agents that, when compared with 6-epi-dictyostatin, retained nanomolar activity in cellular microtubule-bundling assays but had lost activity against paclitaxel-resistant cells with mutations in β-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 C6-epimer were capable of displacing [ 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. These data identify 25,26-dihydrodictyostatin and 6-epi-25,26- dihydrodictyostatin as candidates for scale-up synthesis and further preclinical development. ©2011 AACR

Comparison of the activation of STAT3 across 5 cell lines.

<p>Application of the HI's to the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102678#pone-0102678-g002" target="_blank">Figure 2</a>. Left Panel) Activation of pSTAT3 by exposure to IL-6 for 15 min at the indicated concentrations. Right Panel) Activation of pSTAT3 by exposure to Oncostatin M for 15 min at the indicated concentrations. Red Bars) Diversity index (DIV) indicating the relative heterogeneity associated with the activation of pSTAT3. The horizontal red line indicates the selected threshold for classifying populations a heterogeneous. Green Bars) The non-Normality index (nNRM) indicating the extent of deviation from a single, normally distributed population. The green horizontal line indicates the selected threshold for classifying a population as having macro-heterogeneity. Blue Bars) The percent outliers (%OL) indicates the percentage of cells with an activity level that is above the upper inner fence or below the lower inner fence. The horizontal blue line indicates the selected threshold that is used to classify a population as having more than the expected number of outliers.</p

Decision tree for interpreting the Heterogeneity Indices.

<p>Using thresholds established for each index, DIV, nNRM and %OL, a binary decision tree can be used to characterize heterogeneity in a given sample. The thresholds for DIV (0.03) and nNRM (0.05) were selected as the mean +3 SD for each index in replicate negative control wells for Cal33 cells. The threshold for %OL (4.5%) is the percent outliers expected for a normal distribution.</p

Heterogeneity in the activation STAT3 in Cal33 cells.

<p>Cal33 cells were treated with IL-6 (50 ng/ml) for 15 min. then fixed and labeled with an antibody to phospho-STAT3-Y705. A) Pseudocolor image of STAT3 activation shows a high degree of heterogeneity in the intensity of the Cy5-labeled secondary antibody (color scale at lower right indicates mapping of relative fluorescent intensities to colors). Scale bar is 100 um (lower left). B) The standard deviation of the well average STAT3 activity in replicate wells (EC50 = 3.3 ng/ml, error bars are ±1σ, N = 8) indicates the assay is highly reproducible despite the observed cellular heterogeneity (Z’ = 0.54) C) The standard deviation of the cellular STAT3 activity (error bars are ±1σ) indicates the high variability in the cell-to-cell STAT3 Activity consistent with the appearance of the image (A).</p

Heterogeneity analysis applied throughout the early drug discovery process.

<p>Heterogeneity analysis is required to guide decisions throughout the drug discovery process, beginning with defining disease relevant biology in clinical samples, and establishing benchmarks for subsequent analyses. Next disease relevant models, which by necessity will be heterogeneous, are developed and optimized. Heterogeneity is characterized in the models, and thresholds for HI's are established along with potency criteria to select hits. Screening hits are advanced to secondary assays based on their potency and HI profile. Heterogeneity of response to compounds will be model dependent, and assessing heterogeneity in orthogonal secondary assays will provide insights into understanding the MOA. Monitoring the heterogeneity profile during SAR and lead optimization is essential to keeping lead development on target and mechanism of the disease relevant biology.</p

Variation in the cellular distributions of STAT3 activation by IL-6 and OSM in several cell types.

<p>Top series) Histo-box plots of the activation of STAT3 by IL-6 after 15 min exposure to IL-6 at the indicated concentrations in 2 HNSCC cell lines, 1 breast cell line and 2 breast cancer cell lines. Bottom series) The activation of STAT3 by OSM was measured at 15 min. in the same 5 cell lines as above. Note: 686LN cells were found to be much more sensitive to IL-6 and much less sensitive to OSM than the other cell lines, so the concentrations were adjusted appropriately.</p