A FluoPol-ABPP PAD2 High-Throughput Screen Identifies the First Calcium Site Inhibitor Targeting the PADs

Abstract

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 μM), to preferentially bind the apoenzyme.

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation

“…Secondly, we show that inhibitors can be developed against a low calcium PAD enzyme conformation, consistent with a recent report 25 but in marked contrast with inactivation of ‘active’ PAD4 by Cl-amidine 10 . Crystal structures of our inhibitor series show that they bind to a novel conformation of the PAD4 active site where part of the site is re-ordered to form a β-hairpin.…”

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

“…17 This suggests the potential for the chemoproteomic probe-enzyme pairs described to may provide a universal entry into biological profiling, functional enzyme analysis, and inhibitor development, as has been demonstrated for other probe classes. [29][30][31] Finally, we note several limitations of our approach as currently constituted. In contrast to previous studies of clickable photoaffinity probes that used enantiomeric probe pairs as a physical discriminant, 16 our CoA-based probes are unable to clearly distinguish direct and indirect interactors due to the non-covalent nature of pulldown method.…”

Chemical Biology of Protein Arginine Modifications in Epigenetic Regulation

“…49 This compound preferentially binds the apoenzyme with a K i of 17 μM for PAD2 and was shown to be competitive with calcium, presumably binding at the calcium 3, 4, and 5 sites. Ruthenium red is also a potent inhibitor for the other PAD isozymes with apparent K i values of 30 μM for PAD1, 25 μM for PAD3, and 10 μM for PAD4.…”