Figure 1.

Screening strategy and summary of inhibitors of secretion. A–F, ANF-EGFP (A and D) localized to secretory granules detected by VMAT2 (B) and RMCP2 (E) immunofluorescence. Merge panels (C and F) indicate co-localization. G, ANF-EGFP secretion was stimulated by ionomycin treatment. EFGP fluorescence was measured in overlying medium and detergent-solubilized cells after 15 min of incubation without or with ionomycin. Maximal percent secretion was normalized to 1.0. Values shown (mean ± S.D.) are from a screen for controls without ionomycin (1065 wells), ionomycin-treated without compounds (1065 wells), or from ionomycin-treated (24,992 wells) with 4 μm compounds. Hits in the screen reduced secretion by ≥50% at 4 μm. H, DataWarrior was used to sort 129 confirmed hits into five major classes (A–E) based on ring structure plus a diverse sixth class (F). The representative class structures shown correspond to F5128-0085 (A), F2927-0504 (B), F2902-0553 (C), F5448-0530 (D), F2297-0090 (E), and F5024-0157 (F).