Fenbufen

High-throughput screening for inhibitors of human caspase-4 was performed using the 1280 compound Prestwick Chemical Library at the University of Colorado High-throughput Screening Core Facility. The screen was designed in a 384-well format (Greiner 781207), using 33 μM compound, 1 μM recombinant human caspase-4 in high citrate buffer (50 mM Tris-HCl, pH 7.5, 1 M sodium citrate, 10 mM DTT, 10% sucrose), and 20 μM Promega Caspase-Glo® 9 Assay substrate (O’Brien et al., 2005 (link); Roschitzki-Voser et al., 2012 (link)). While designed for use with caspase-9, the LEHD substrate is also recognized by caspase-4 (Roschitzki-Voser et al., 2012 (link); Talanian et al., 1997 (link)). The assay Z’ factor is 0.66, recommending this method for high-throughput experimentation (Figure S1) (Zhang et al., 1999 (link)). Plates were read with endpoint luminescent analysis 20 minutes and 60 minutes after substrate addition. Compounds were considered to be hits if caspase-4 activity was inhibited to less than 25% relative to the solvent control. Using these criteria, we identified 27 hits, which are summarized in Table 1. Fenbufen, indoprofen, ketoprofen, ketorolac, felbinac, naproxen, ibuprofen, and tiaprofenic were purchased from Sigma Aldrich. Aspirin was purchased from TCI America. z-VAD-FMK was obtained from InvivoGen.