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Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

ABSTRACT: Protein arginine deiminases (PADs) are involved in a number of cellular pathways, and they catalyze the transformation of peptidyl arginine residue into a citrulline as part of post-translational modifications. To understand ligand preferences, a group of probe molecules were investigated against PAD1, PAD2, and PAD4. These probe molecules carried a well-known covalent modifier of the catalytic cysteine residue, 2-chloroacetamidine moiety, which was tethered to an ?-amino acid via a carbon linker. The chain length for the linker varied from 0 to 4. Time-dependent assays indicated that 2-chloroacetamidine (2CA) with no linker inhibited all PAD enzymes with a similar trend in the second-order rate constants, although with poor affinity. Among the other three probe molecules, compound 3 with a three-carbon linker exhibited the best second-order rate constants for optimal ligand reactivity with the binding site. These analyses provide insights into the relative patterns of covalent inactivation of PAD isozymes and the design of novel inhibitors targeting PAD enzymes as potential therapeutic targets.

SUBMITTER: Bello AM

PROVIDER: S-EPMC4027443 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

Bello Angelica M AM Wasilewski Ewa E Wei Lianhu L Moscarello Mario A MA Kotra Lakshmi P LP

ACS medicinal chemistry letters 20130115 2

Protein arginine deiminases (PADs) are involved in a number of cellular pathways, and they catalyze the transformation of peptidyl arginine residue into a citrulline as part of post-translational modifications. To understand ligand preferences, a group of probe molecules were investigated against PAD1, PAD2, and PAD4. These probe molecules carried a well-known covalent modifier of the catalytic cysteine residue, 2-chloroacetamidine moiety, which was tethered to an α-amino acid via a carbon linke ...[more]

PMID: 24900657

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Project description: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 Account, 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 lead-optimization 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 animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.

Dataset Information

Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

Publications

Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

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