Efficient colorimetric assay promotes the virtual screen of granzyme B inhibitors

Granzyme B (GrmB) is a key biomarker for immune activation and tumor cell eradication, as well as the therapeutic target for autoimmune and chronic inflammatory disorders. Recent bioinformatic methods have been extensively applied to discover new sites of action, thereby enabling the screening of corresponding inhibitors. However, verification of silico predictions requires efficient experimental tests in vitro, and this work aims to provide an efficient assay method that enables the colorimetric detection of GrmB activity with high specificity and sensitivity (0.01 units) within a short time frame (30 min). With the design of a mirror peptide substrate that induces the aggregation of gold nanoparticle probes (AuNPs), a high-throughput color change can be quantified by a microplate reader. It has thus been applied to detect T-cell-secreted GrmB, as well as for inhibitor screening. Four candidate inhibitors have been screened out from a large-scale library pool containing 1.5 million small molecules. Combined with the computational simulation analysis, the Morgan fingerprints analysis, 2D binding mode analysis, etc. our study has further shown a shared interaction feature of the four inhibitors, indicating a new dominant inhibitory site of GrmB at Asn218. Therefore, this work provides a complementary tool for the virtual screening of GrmB inhibitors and reveals a new inhibitory site of GrmB. It may promote diagnostic and therapeutic applications of GrmB in immunotherapy and disease management.