In vitro activity: Human donor islets obtained from integrated islet distribution program (www.iidp.cohorg. ) were incubated overnight in CMRL media (cat. no. 15–110-CV MediaTech, Inc. Manassas, VA) containing 10% fetal bovine serum, 1 U penicillin, and 1 μg streptomycin (pen/strep). Islets were equilibrated in serum free media (CMRL containing pen/Strep and 1% fatty acid free human serum albumin (cat. no. A1887 Sigma, St. Louis, MO)), for 1 h prior to pretreatment with 10 μM of 35 for 30 min. For 12-HETE induction, islets were treated with 100 μM arachidonic acid (cat. no. BML-FA003–0100, Enzo Life Sciences Plymouth Meeting, PA) and 5 μM A23187 (cat. no. C7522, Sigma, St. Louis, MO) for 4 h at 37 °C. Islets were harvested, centrifuged at 1000 rpm for 5 min with cleared supernatant and islet pellet being stored at –80 °C. For extraction of the supernatants, samples were acidified to pH 3 with 1N HCl for 30 min and spun at 1000 rpm for 5 min. Samples were added to a prepared column (prewashed with 3 mL of EtOH, followed by 3 mL of H2O) and washed with 3 mL of H2O, followed by 3 mL of 15% EtOH and 3 mL of hexane. The samples were eluted with 3 mL of ethyl acetate and dried under nitrogen gas before, reconstitution in 500 mL of 12-HETE ELISA sample buffer (Enzo Life Sciences, Plymouth Meeting, PA). Cell pellets were extracted using CHCl3/MeOH and samples dried under nitrogen gas before reconstitution in 250 μL of ELISA sample buffer. 12-HETE levels in samples were determined using a 12-HETE ELISA kit (cat. no. 901–050, Enzo Life Sciences, Plymouth Meeting, PA).

Kinase Assay: All screening operations were performed on a fully integrated robotic system (Kalypsys Inc., San Diego, CA) as described elsewhere. 3 μL of enzyme (approximately 80 nM 12-LOX, final concentration) were dispensed into 1536-well Greiner black clear-bottom assay plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool equipped with 1536-pin array. The plate was incubated for 15 min at room temperature, and then a 1 μL aliquot of substrate solution (50 μM arachidonic acid final concentration) was added to start the reaction. The reaction was stopped after 6.5 min by the addition of 4 μL of FeXO solution (final concentrations of 200 μM Xylenol Orange (XO) and 300 μM ferrous ammonium sulfate in 50 mM sulfuric acid). After a short spin (1000 rpm, 15 s), the assay plate was incubated at room temperature for 30 min. The absorbances at 405 and 573 nm were recorded using ViewLux high-throughput CCD imager (Perkin-Elmer, Waltham, MA) using standard absorbance protocol settings. During dispensing, enzyme and substrate bottles were kept submerged into a +4 °C recirculating chiller bath to minimize degradation. Plates containing DMSO only (instead of compound solutions) were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decrease in enzyme specific activity. Data were normalized to controls, and plate-based data corrections were applied to filter out background noise.

Cell Assay: Cells were gown to 90% confluency in 24-well plates in DMEM (cat. no. 11885092, Life Technologies Grand Island, NY) +10% FBS. Cells were pretreated with ML355 and stimulated as for human islets. After four hours, the media was removed and spun at 1000 rpm for 5 min. The cleared supernatant was stored at –80 °C prior to analysis. For analysis, supernatants were extracted on SepPak c18 SPE column (cat. no. WAT054945, Waters Corporation, Milford, MA) and dried under nitrogen gas before reconstitution in 500 μL of 12-HETE ELISA buffer and analysis following manufacturers recommendations (cat. no. 901–050, Enzo Life Sciences, Plymouth Meeting, PA).

J Med Chem. 2014 Jan 23; 57(2): 495–506.; Arterioscler Thromb Vasc Biol. 2017 Oct;37(10):1828-1839.