Proxi MaZiQ array Target Discovery Platform: cell-free identification of drug-interacting proteins using Drug~AirID2C and recombinant human protein arrays

Abstract Most small-molecule drugs exert their biological effects by binding to proteins, making unbiased target identification essential for evaluating drug specificity and potential off-target effects. Herein, we developed the Proxi MaZiQ array Target Discovery Platform, a cell-free drug-protein interaction detection system that combines recombinant human protein arrays with Drug~AirID 2 C, a proximity biotinylation enzyme chemically conjugated to small-molecule compounds. AirID 2 C was engineered from AirID by replacing surface-exposed lysine residues with arginine residues while retaining catalytically important lysines and introducing an N-terminal 5K3D tag (KKKDKKDD) for controlled drug conjugation. Drug~AirID 2 C molecules prepared using NHS ester chemistry or click chemistry selectively biotinylated known drug-binding proteins, including cereblon for thalidomide and pomalidomide, BET family proteins for JQ1, and HSP90 for geldanamycin, both in solution and on recombinant protein arrays. Biotinylated proteins were detected using AlphaScreen assays and horseradish peroxidase-based array detection. Application of AP-C5~AirID 2 C to a kinase- and phosphatase-focused protein array identified candidate AP-C5-interacting proteins, including MINK1, whose kinase activity was inhibited by AP-C5. These results demonstrate that the Proxi MaZiQ array Target Discovery Platform enables biochemical identification of candidate drug-interacting proteins and provides a useful approach for target identification and target-profile analysis of small-molecule compounds in a defined in vitro setting.