Protein Arginine Deiminases (PADs): Biochemistry and Chemical Biology of Protein Citrullination

Abstract

Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS) and certain cancers. In this Accounts, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate-binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate.Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and leadoptimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various

Post-Translational Protein Deimination Signatures in Serum and Serum-Extracellular Vesicles of Bos taurus Reveal Immune, Anti-Pathogenic, Anti-Viral, Metabolic and Cancer-Related Pathways for Deimination

“…Calcium is a key modulator in a range of immunological, metabolic and developmental functions [199][200][201][202]. Calcium signalling is linked to a wide range of physiological and pathological processes [203][204][205], is a key driver for post-translational deimination [206][207][208] and plays an important role in the regulation of EVs [11,48]. In cattle, calcium is linked to many immunological pathways including inflammatory diseases [209], virus replication [210], parasitic infection [211], wound healing [212], reproduction [213,214] and adrenal function [215].…”

An Efficient Chemo‐Selective N‐Alkylation Methodology for the Structure Diversification of Indolylbenzimidazoles

“…Using this strategy, we achieved the needed selectivity and synthesized a small selection of asymmetrically alkylated 2‐indolylbenzimidazoles (Scheme , 9 a – 9 d) . A series of asymmetrically alkylated 2‐indolybenzimidazoles were recently discovered and reported in literature as potent reversible inhibitors of PADs . Based on our understanding, large scale synthesis of the reported compounds involves a multi‐step synthetic sequence from commercial reagents, including various protecting group manipulation steps.…”

“…Based on our understanding, large scale synthesis of the reported compounds involves a multi‐step synthetic sequence from commercial reagents, including various protecting group manipulation steps. Herein, we show that structural analogs of reported PAD‐inhibitors can be accessed in a one‐pot, two‐step process to accelerate the drug discovery efforts (Scheme ). Furthermore, from a medicinal chemistry stance, our method enables one to quickly diversify the indolylbenzimidazole core to generate novel analogs as potential drug leads.…”

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors

“…Ca 2+ ‐binding reorients key catalytic residues into catalytically competent positions. Catalysis is mediated by a nucleophilic cysteine (C645 in PAD1, 4; C647 in PAD2; C646 in PAD3), a histidine (H471), and two aspartates (D350 and D473) that properly orient the substrate guanidium group for nucleophilic attack by the cysteine . In addition to their important physiological roles, aberrant protein citrullination leads to several autoimmune diseases (e.g., rheumatoid arthritis, lupus, and multiple sclerosis) as well as certain cancers.…”

“…In addition to their important physiological roles, aberrant protein citrullination leads to several autoimmune diseases (e.g., rheumatoid arthritis, lupus, and multiple sclerosis) as well as certain cancers. These disease links have established multiple PADs as promising therapeutic targets …”