Antiproliferative activity

Cellular assays were performed using BT-474, SKBR-3, Calu-3, MCF-7, and MCF-10A cells (ATCC). Antiproliferative activity was measured by CellTiter-Glo^®, [49] a luminescence-based cell viability assay. In a 96-well plate, 1 × 10⁴ cells/well were seeded and incubated overnight at 37°C and 5% CO₂. Stock solutions of peptidomimetics were prepared by dissolving 1.5 mg/mL of compounds in DMSO. Stock solution of the compounds were diluted using the serum-free medium to prepare solutions of different concentrations of compounds. Compounds in the medium were added to the wells in triplicate and incubated for 72 h. As controls, cells treated with 1% sodium dodecyl sulfate and 1% dimethyl sulfoxide were used. At the end of the experiment, CellTiter-Glo^® reagent was added, and luminescence was measured by a plate reader. A dose response curve was generated using luminescence vs concentration of compounds and IC₅₀ values were obtained using Prism^® (GraphPad Software, La Jolla, CA). Experiments were repeated at least three times to obtain standard deviation values. EGFR mutated lung cancer cell lines NCI-H1975 (ATCC^® CRL 5908™) was obtained from ATCC, and antiproliferation assays were carried our as describe above. Erlotinib was from Sigma (Sigma-Aldrich Chemie GmbH). To evaluate the synergistic effect of compound 18 with erlotinib, first a dose response curve was generated for erlotinib, and compound 18 alone in different cell lines including EGFR mutated NCI-H1975 cells. Then cells were treated with different concentrations of compound 18 (0.005–10 μM) along with fixed concentrations of erlotinib. Fixed erlotinib dose of 1 μM, 1 μM, 30 μM, and 10 μM were selected for BT-474, Calu-3, MCF-7 and mutated cell line NCI-H1975 respectively. The isobologram was generated by plotting IC₅₀ values (50% cell growth inhibition) of compound 18 and erlotinib, alone and in combination. From the isobologram, synergistic effect of compound 18 with erlotinib was determined.