High-throughput screen for GSDMD inhibitors.

Liposome leakage was detected by an increase in fluorescence when Tb³⁺ bound to DPA in Buffer C. Human GSDMD (0.3 μM), dispensed into 384-well plates (Corning 3820) containing PC/PE/CL liposomes (50 μM liposome lipids), was incubated with compounds from the ICCB-Longwood Screening Facility collection for 1 hr before addition of caspase-11 (0.15 μM) to each well. The fluorescence intensity of each well was measured at 545 nm with an excitation of 276 nm 1 hr after addition of caspase-11 using a Perkin Elmer EnVision plate reader. The final percent inhibition was calculated as [(fluorescence_{test compound} − fluorescence_{negative control})/(fluorescence_{positive control} − fluorescence_{negative control})] × 100, where wells with GSDMD without inhibitors was used as positive control and with caspase-11 as negative control. 50% inhibition was arbitrarily chosen as a threshold. The hits were evaluated in concentration-response experiments in a dose range of 0.008–50 μM to determine IC₅₀. For liposome leakage assay using GSDMD-NT, human GSDMD (0.3 μM), incubated with caspase-11 (0.03 μM) at 4 °C for 24 hrs, was incubated with disulfiram at indicated concentrations for 1 hr before addition of liposomes (50 μM) and fluorescence measurements.