Fig 1: Small-molecule inhibitors bind to PD-L1 with no impact on cell viability. (A) Thermal shifts indicate the stabilization of PD-L1 by compounds 5 (black), 42 (green), 47 (yellow), 69 (yellow), 75 (black) and 84 (black). Curves represent the fraction of unfolded recombinant human PD-L1 protein, where 0 represents the folded PD-L1 and 1 the unfolded, in the presence of 1% DMSO (light gray), indicated compounds (green, yellow and black) and BMS202 (dark gray) at 100 µM. (B) Different cell lines were incubated with increased concentrations of compounds for 48 hours. Cell viability was normalized to untreated cells. All cell lines, MDA-MB-231 (ATCC# HTB-26), A375 (ATCC# CRL. 1619), and HMEC (ATCC# CRL-3243) showed tolerance to the compounds. Three different concentrations 100 µM (blue), 10 µM (green) and 1 µM (gray) were tested. Data are presented as mean±SD, N=3 and N=1, n=9 or n=3 from three or one independent experiment(s) performed in triplicate. (C–D) Compounds inhibit PD-1/PD-L1 interaction on melanoma and breast cancer cell lines. (C) MDA-MB-231 breast cancer cells (gray) or (D) A375 melanoma cells (gray) were treated with 10 µM of compounds (green, yellow and black), BMS202 (dark gray) and anti-PD-L1 (αPD-L1) (red) for 72 hours. A375 cells were stimulated with 200 ng.mL−1 interferon-ɣ (gray) for 18 hours before treatments to enhance PD-L1 levels. The remaining % of accessible PD-L1 was determined in live cells by flow cytometry. Data are presented as mean±SD, N=3, n=9, or N=1, n=3, from three or one independent experiments performed in triplicate. Statistical analysis: one-way analysis of variance and Tukey’s post test. PD-L1, programmed cell death ligand 1. ATCC, American Type Culture Collection.