Development of Next-Generation Fluoroacetamidine-Containing Activity-Based Probes for the Selective Labeling of the...

The post-translational conversion of an arginine into citrulline, known as protein citrullination (PC), is catalyzed by the protein arginine deiminases (PADs). These calcium-dependent enzymes have emerged as promising therapeutic targets for multiple diseases, including rheumatoid arthritis, sepsis, and systemic lupus erythematosus. BB-Cl-amidine, the most widely used pan-PAD inhibitor, features a highly reactive chloroacetamidine warhead that modifies numerous off-target proteins. Thus, there is a need to develop more selective pan-PAD inhibitors. Herein, we report the development of LB665, a novel fluoroacetamidine-based pan-PAD inhibitor that demonstrates cellular efficacy and potency comparable to BB-Cl-amidine, while maintaining markedly improved proteome-wide selectivity for the PADs. From LB665, we generated a variety of activity-based probes that selectively label and enrich active PADs from cell and tissue lysates, demonstrating their utility for profiling PAD activity and target engagement studies. Together, LB665 and its derivatives constitute a powerful chemical toolkit that will aid in the further exploration of PAD biology and support the future development of PAD-targeted therapeutics.