Analysis of reproducibility and robustness of a human microfluidic four-cell liver acinus microphysiology system (LAMPS).

A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 µM), Troglitazone (150 µM), and caffeine (600 µM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 µM), Tolcapone (88 µM), Trovafloxacin (150 µM with or without 1 µg/mL lipopolysaccharide), Troglitazone (28 µM), Rosiglitazone (0.8 µM), Pioglitazone (3 µM), and caffeine (600 µM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.

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.

Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay.

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

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 ß-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.

Identifying a resistance determinant for the antimitotic natural products disorazole C1 and A1.

Disorazoles are macrocyclic polyketides first isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. Both the major fermentation product disorazole A(1) and its much rarer companion disorazole C(1) 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 C(1). Resistant cells proved exceedingly difficult to generate and required single step mutagenesis with chronic stepwise exposure to increasing concentrations of disorazole C(1). Compared with wild-type HeLa cells, disorazole C(1)-resistant HeLa/DZR cells were 34- and 8-fold resistant to disorazole C(1) and disorazole A(1) 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 C(1) and disorazole A(1). 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 C(1) and A(1). Disorazole C(1), however, retained activity against cells resistant against the clinically used microtubule-stabilizing agent epothilone B.

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 A(1), 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 C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) 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 C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.