High-throughput screening for the identification of new therapeutic options for metastatic pheochromocytoma and paraganglioma

Abstract

Drug repurposing or repositioning is an important part of drug discovery that has been growing in the last few years for the development of therapeutic options in oncology. We applied this paradigm in a screening of a library of about 3,800 compounds (including FDA-approved drugs and pharmacologically active compounds) employing a model of metastatic pheochromocytoma, the most common tumor of the adrenal medulla in children and adults. The collection of approved drugs was screened in quantitative mode, testing the compounds in compound-titration series (dose-response curves). Analysis of the dose-response screening data facilitated the selection of 50 molecules with potential bioactivity in pheochromocytoma cells. These drugs were classified based on molecular/cellular targets and signaling pathways affected, and selected drugs were further validated in a proliferation assay and by flow cytometric cell death analysis. Using meta-analysis information from molecular targets of the top drugs identified by our screening with gene expression data from human and murine microarrays, we identified potential drugs to be used as single drugs or in combination. An example of a combination with a synergistic effect is presented. Our study exemplifies a promising model to identify potential drugs from a group of clinically approved compounds that can more rapidly be implemented into clinical trials in patients with metastatic pheochromocytoma or paraganglioma.

Figure 3. Drugs-gene targets correlation and network.

A) Drug name and number of high correlated genes mapped from the human PHEO and the murine MTT microarray data set. B) Connected node network representation in which all the targets of each drug are connected to the targets of all other drugs in the network.

Figure 1. Validation of primary screening.

Panel A and B: Dose response curves of the selected compound in MPC, MTT and PC12 PHEO cell lines. The Y-axis represents signal intensity as a percent of the maximal value. The X-axis represents the log 10 concentrations of the respective compound (inside square brackets).

Figure 2. Apoptosis induced by screened drugs.

A) To quantify viable cells, the membrane impermeable dye 7-amino actinomycin D (7-ADD) was added to a cell suspension of PC12 cells, after overnight treatment with the respective compound. The bar graph represents relative live cell percentage. B) MTT cells were cultured with the indicated drug and dose for 20 hr and total cell lysates were subjected to Western blot for full-length PARP, cleaved PARP and actin.

Figure 4. Drug combination analysis.

A and B) Dose response curves for SAHA and Epirubicin; C) Median-effect plot; D) Algebraic estimate of the combinational index (CI) for the combination of epirubicin with SAHA relative to the fraction of affected cells.