PAD

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Introduction

Histones are the essential building blocks of chromatin, which serve as the scaffold of DNA in eukaryotes. Changes in histone structure will directly affect its interaction with DNA (or other nuclear proteins), thereby changing the overall chromatin function. Histones are highly post-translationally modified (PTM), including methylation, phosphorylation, acetylation, ubiquitination, and citrullination. Among them, citrullination (also known as deimination) is the hydrolysis of peptidyl-arginine into peptidyl-citrulline. This process is catalyzed by the protein arginine deiminase (PAD) family of enzymes. PAD overexpression and enzyme activity upregulation have been observed in many diseases, including rheumatoid arthritis (RA), Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis (MS) and cancer, etc.

In humans, the PAD family consists of five calcium-dependent isozymes (PADs 1-4 and 6), which share approximately 50% sequence similarity. PAD is expressed in a large number of cells and tissues, including epidermis and uterus (PAD1), skeletal muscle, brain, inflammatory cells, and secretory glands (PAD2), hair follicles and keratinocytes (PAD3), granulocytes and several types of cancer (PAD4), as well as oocytes and embryos (PAD6). PAD4 is the only isozyme that has been shown to play a role in histone deimination, although recent reports indicate that PAD2 may also deiminate histones. Recent studies have shown that in addition to the cytoplasm and nucleus, PAD may also exist in granules and mitochondria, such as PAD4 and PAD2.

There is currently evidence that fibrinogen, actin, and fillagrin are also substrates of PAD, and citrullination of these proteins is known to occur in rheumatoid arthritis.

Figure 1. PADs catalyze the hydrolysis of peptidyl-arginine to peptidyl-citrulline (Bicker, K.L.; Thompson, P.R. 2014)

PAD Structure and Mechanism

A lot of work has been done to clarify the structure of PAD4 with/without calcium and substrate binding. Studies have shown that PAD4 contains a total of five calcium-binding sites, two of which help bridge the N-terminal and C-terminal domains, and the remaining three calcium-binding sites are located in the N-terminal domain. These three binding sites can be further divided into two immunoglobulin-like subdomains, one of which contains a nuclear localization signal (NLS). Interestingly, residues 158-171 of the N-terminal domain are highly disordered in the calcium-free form, but form a highly ordered α-helix in the presence of calcium. Scientists speculate that this conformational change regulates the interaction between proteins and enzymes. In the C-terminal domain, calcium binding induces a conformational shift, which in turn creates the active site cleft.

Nowadays, lots of work has been done on the catalytic mechanism of PAD. These works revealed that PAD uses a reverse protonation mechanism, in which the active site nucleophile Cys645 in PAD4 exists as a thiolate in the active form of the enzyme. His471, another important residue in the active site, promotes the initial release of ammonia from the guanidine group of the substrate, and then activates a water molecule to complete the hydrolysis of the substrate. Studies on PAD 1 and 3 have also confirmed that they also proceed through a similar reverse protonation mechanism.

Figure 2. The proposed mechanisms of PAD inactivation by halo-acetamidine based inhibitors (Bicker, K.L.; Thompson, P.R. 2014)

Development of New PAD Inhibitors

Although several reversible PAD inhibitors have been identified (for example, taxol, streptomycin, and minocycline), these compounds are relatively weak PAD inhibitors, and the most effective inhibitors described so far irreversibly modify the enzymes. The lack of effectiveness of reversible inhibitors may be related to the small active site cavity, which can only accommodate the side chain of arginine residues when the enzyme binds to calcium. Due to the lack of efficacy of reversible inhibitors, scientists have focused their efforts on the development of Cl-amidine analogs with improved potency, selectivity, and bioavailability. Several new PAD inhibitors have also been identified using the library approaches. For example, a relatively small library of 264 compounds was synthesized on the solid phase and then screened to identify PAD inhibitors. This method identified the tripeptide (Thr-Asp-F-amidine; TDFA) as a highly selective (up to 65-fold) PAD4 inhibitor with excellent in vivo potency.

Figure 3. Structure, potency, and selectivity of the most useful PAD inhibitors (Bicker, K.L.; Thompson, P.R. 2014)