Design, Synthesis, and Molecular Evaluation of S_NAr‐Reactive <i>N</i>‐(6‐Fluoro‐3‐Nitropyridin‐2‐yl)Isoquinolin‐3‐Amines as Covalent USP7 Inhibitors Reveals an Unconventional Binding Mode

The cysteine protease ubiquitin-specific protease 7 (USP7), also known as herpes-associated ubiquitin-specific protease (HAUSP), has gained increasing attention in recent years due to its proven overexpression in several cancer types and its role in tumorigenesis. Herein, after a design based on molecular docking experiments, we report the synthesis of a series of mildly electrophilic compounds that covalently modify the catalytic cysteine 223 in USP7 through a nucleophilic aromatic substitution (S_NAr) reaction. The compounds were first evaluated using differential scanning fluorimetry (DSF) to describe their influence on the melting temperature of native and mutant USP7 variants. Furthermore, the possible formation of a covalent bond was analyzed using intact protein mass spectrometry (MS). For promising derivatives, IC₅₀ values were determined in an enzyme activity assay to confirm an inhibitory effect on USP7. Finally, a co-crystal structure revealed that the prototype compound (7a) arylates the catalytic cysteine in the apo form of USP7 via an unconventional binding mode near the catalytic triad. The synthesis and biological evaluation of this compound series provides valuable structure-activity relationships (SAR) and reveals an interesting and unprecedented binding mode, thus providing a basis for improving USP7 inhibitors.