Activity-guided fractionation identifies CMA2 as an insulin secretion inhibitor in human islets. (A) Natural product subfractions from SNB-022-1 were tested in quadruplicate for inhibitory activity in Ins-GLuc MIN6 β cells stimulated with the diazoxide (Dz) paradigm. Cells were treated 24 h with the original SNB-022-1 fraction, subfractions (10 µg/ml), or DMSO (0.1%). Cells were preincubated in glucose-free KRBH for 1 h followed by 1 h in the presence of either Dz (250 µM) or Dz + 40 mM KCl and 20 mM glucose. Secreted luciferase activity relative to Dz control is shown as the mean ± SEM. (B) Nuclear magnetic resonance spectra of the purified compound from fraction SNB-022-1 identified as CMA2. (C) The spectra in B matched CMA2, and the structure is shown. The structure of the closely related mithramycin is shown for comparison. (D) The Ins-GLuc MIN6 cells were treated 24 h with different doses of CMA2 followed by a glucose-stimulated secretion assay, and the resulting relative luciferase units (RLUs) were used to determine the effective concentration EC₅₀. Data are the mean ± SEM (n = 4). (E) The Ins-GLuc MIN6 cells were treated with CMA2 (100 nM) starting at the beginning of the 1-h KRBH preincubation and during the 1-h glucose (20 mM) stimulation. Secreted luciferase activity was measured at the end of the assay. Data represent the mean ± SEM of three independent experiments performed in triplicate. (F) Human islets were hand-picked into groups of 50 and treated 24 h with CMA2 at the indicated doses in 500 µl complete CMRL medium. The islets were then subjected to static glucose-stimulated insulin secretion assays, and the secreted and total insulin concentrations were measured. Data are the mean ± SEM from three different batches of donor islets.