Functional Proteomics of Quinazolin‐4‐One Derivatives Targeting the Proteasome

: Quinazolinones have been recently recognized as valuable scaffolds for developing novel therapeutic opportunities. They indeed exhibit structural versatility and a wide range of biological activities, including antifungal, antitubercular, antihypertensive, anticancer, and antiviral ones. In this work, a focused library of new bioactive 4-(3-H)-quinazolinones has been synthesized, their cytotoxic action against DU-145 prostate cancer cells has been detailed, and compound 4k has been revealed as the most active one. Consequently, its interactome has been characterized by a label-free functional proteomics-based platform coupling drug affinity responsive target stability (DARTS) and targeted limited proteolysis-multiple reaction monitoring-mass spectrometry (t-LiP-MRM-MS). This multifaced strategy has been employed to reveal few subunits of the 26S proteasome machinery as the most reliable compound 4k biological targets. This paved the way for the deepening of the protein-ligand interaction using in vitro and in silico bio-orthogonal techniques. Finally, the analysis of its function in living DU-145 cells prompted compound 4k as a novel quinazolinone-bearing inhibitor of the chymotrypsin-like activity of the proteasomal β-5 subunit, stirring this framework for the development of new anticancer drugs.