A gene expression–based approach identifies atovaquone as a novel STAT3 inhibitor. (A) Overview of strategy using the Connectivity Map to identify drugs that are potential STAT3 inhibitors based on gene expression. (B) Results from the Connectivity Map analysis. More negative scores indicate more dissimilarity with respect to the STAT3 signature, with atovaquone being the most negatively scoring compound. (C) Chemical structure of atovaquone and appearance of plasma samples isolated from patients taking trimethoprim-sulfamethoxazole (control) or atovaquone. (D) STAT3 reporter cells were pretreated with drug for 1 hour, then stimulated with IL-6 (10 ng/mL) for 5 hours. Activity of firefly luciferase was measured and normalized by viable cell number (Cell Titer Glo). Data are means ± standard deviation of 2 independent experiments. (E) Effect of atovaquone on STAT3 reporter cells compared with reporter cells for STAT1 (stimulated by interferon-γ), STAT5 (stimulated by prolactin), and NF-κB (stimulated by tumor necrosis factor α). Data are means from 2 independent experiments.