The protein arginine deiminases (PADs) are a family of Ca2+-dependent enzymes that catalyze the conversion of peptidyl-arginine to peptidyl-citrulline in numerous protein substrates. Disruption of normal PAD activity plays a role in the pathogenesis of multiple inflammatory diseases such as rheumatoid arthritis (RA), chronic obstructive pulmonary disease, ulcerative colitis, multiple sclerosis, psoriasis, Alzheimer’s disease, and in various cancers. PAD inhibitors described in the literature have been useful chemical tools to study the role of PAD enzymes in inflammatory diseases and cancer biology. Most published PAD inhibitors are mechanism-based inactivators belonging to the halogen-amidine chemotype. For emerging targets such as the PADs, it can be difficult to distinguish compound-specific effects from those truly resulting from enzyme inhibition. We therefore initiated a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify a second PAD inhibitor chemotype.
The PAD4 HTS campaign identified the natural product streptonigrin (SID 11532976) as an irreversible PAD4-specific inhibitor. We describe herein the medicinal chemistry optimization of streptonigrin to the pan PAD probe ML325 (SID 118043677). ML325 inhibits PAD1, 2, 3, and 4 in vitro with IC50 values of 70 nM, 200 nM 170 nM, and 240 nM respectively. In a kinetic assay of inhibition more appropriate for irreversible inhibitors, ML325 has kinact/KI values of 3500, 7300, 1900, and 5300 M−1min−1 for PAD1, 2, 3 and 4 respectively; indicating it has less than 4-fold selectivity among the four family members. Despite its promiscuity within the PAD family, ML325 exhibits high selectivity vs. more than 20 cysteine-reactive proteins as assayed by activity-based protein profiling. ML325 was also demonstrated to inhibit all four PAD isozymes irreversibly and to be non-cytotoxic to NIH-3T3 cells. The complete properties, characterization, and synthesis of ML325 are detailed in this report.