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Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

ABSTRACT: Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

SUBMITTER: Martin Monreal M

PROVIDER: S-EPMC8560728 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4).

Project description:PurposeCitrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM-along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)-are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age.MethodsWild-type (WT) and Pad4-/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ.ResultsWT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination.ConclusionsBoth PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.

| S-EPMC6074612 | biostudies-literature

Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors.

Project description:The protein arginine deiminases (PADs) catalyze the post-translational deimination of arginine side chains. Multiple PAD isozymes have been characterized, and abnormal PAD activity has been associated with several human disease states. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor and has been implicated in the neurodegenerative response to spinal cord injury. Here, we describe the design, synthesis, and evaluation of conformationally constrained versions of the potent and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series was also demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells.

| S-EPMC5018872 | biostudies-other

Thioredoxin Modulates Protein Arginine Deiminase 4 (PAD4)-Catalyzed Citrullination.

Project description:Protein citrullination is a post-translational modification catalyzed by the protein arginine deiminases (PADs). This modification plays a crucial role in the pathophysiology of numerous autoimmune disorders including RA. Recently, there has been a growing interest in investigating physiological regulators of PAD activity to understand the primary cause of the associated disorders. Apart from calcium, it is well-documented that a reducing environment activates the PADs. Although the concentration of thioredoxin (hTRX), an oxidoreductase that maintains the cellular reducing environment, is elevated in RA patients, its contribution toward RA progression or PAD activity has not been explored. Herein, we demonstrate that hTRX activates PAD4. Kinetic characterization of PAD4 using hTRX as the reducing agent yielded parameters that are comparable to those obtained with a routinely used non-physiological reducing agent, e.g., DTT, suggesting the importance of hTRX in PAD regulation under physiological conditions. Furthermore, we show that various hTRX mutants, including redox inactive hTRX variants, are capable of activating PAD4. This indicates a mechanism that does not require oxidoreductase activity. Indeed, we observed non-covalent interactions between PAD4 and hTRX variants, and propose that these redox-independent interactions are sufficient for hTRX-mediated PAD4 activation.

| S-EPMC6396667 | biostudies-literature

Peptidyl arginine deiminase inhibition alleviates angiotensin II-induced fibrosis.

Project description:ObjectivesThe conversion of protein arginine residues to citrulline by calcium-dependent peptidyl arginine deiminases (PADs) has been implicated in the pathogenesis of several diseases, indicating that PADs are therapeutic targets. A recent study indicated that PAD4 regulates age-related organ fibrosis and dysfunction; however, the specific role of this PAD and its citrullination substrate remains unclear. We investigated whether pharmacological inhibition of PAD activity could affect the progression of fibrosis and restore heart function.MethodsCardiac hypertrophy was induced by chronic infusion of angiotensin (Ang) II. After 2 weeks of AngII infusion, a PAD inhibitor (Cl-amidine hydrochloride) or vehicle (saline) was injected every other day for the next 14 days together with the continued administration of AngII for a total of up to 28 days. Cardiac fibrosis and remodeling were evaluated by quantitative heart tissue histology, echocardiography, and mass spectrometry.ResultsA reverse AngII-induced effect was observed in PAD inhibitor-treated mice (n=6) compared with AngII vehicle-treated mice, as indicated by a significant reduction in the heart/body ratio (AngII: 6.51±0.8 mg/g vs. Cl-amidine: 5.27±0.6 mg/g), a reduction in fibrosis (AngII: 2.1-fold increased vs. Cl-amidine: 1.8-fold increased), and a reduction in left ventricular posterior wall diastole (LWVPd) (AngII: 1.1±0.04 vs. Cl-amidine: 0.78±0.02 mm). Label-free quantitative proteomics analysis of heart tissue indicated that proteins involved in fibrosis (e.g., periostin), cytoskeleton organization (e.g., transgelin), and remodeling (e.g., myosin light chain, carbonic anhydrase) were normalized by Cl-amidine treatment.ConclusionOur findings demonstrate that pharmacological inhibition of PAD may be an effective strategy to attenuate cardiac fibrosis.

| S-EPMC10408542 | biostudies-literature

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Project description:Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

| S-EPMC7073130 | biostudies-literature

Peptidyl Arginine Deiminase 2 (PADI2)-Mediated Arginine Citrullination Modulates Transcription in Cancer.

Project description:Protein arginine deimination leading to the non-coded amino acid citrulline remains a key question in the field of post-translational modifications ever since its discovery by Rogers and Simmonds in 1958. Citrullination is catalyzed by a family of enzymes called peptidyl arginine deiminases (PADIs). Initially, increased citrullination was associated with autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, as well as other neurological disorders and multiple types of cancer. During the last decade, research efforts have focused on how citrullination contributes to disease pathogenesis by modulating epigenetic events, pluripotency, immunity and transcriptional regulation. However, our knowledge regarding the functional implications of citrullination remains quite limited, so we still do not completely understand its role in physiological and pathological conditions. Here, we review the recently discovered functions of PADI2-mediated citrullination of the C-terminal domain of RNA polymerase II in transcriptional regulation in breast cancer cells and the proposed mechanisms to reshape the transcription regulatory network that promotes cancer progression.

| S-EPMC7072959 | biostudies-literature

Peptidyl arginine deiminase-4 exacerbates ischemic AKI by finding NEMO.

Project description:Peptidyl arginine deiminase-4 (PAD4) catalyzes the conversion of peptidylarginine residues to peptidylcitrulline. We have previously shown that kidney ischemia-reperfusion (I/R) injury increases renal proximal tubular PAD4 expression and activity. Furthermore, kidney PAD4 plays a critical role in ischemic acute kidney injury (AKI) by promoting renal tubular inflammation, neutrophil infiltration, and NF-?B activation. However, the mechanisms of PAD4-mediated renal tubular inflammation and NF-?B activation after I/R remain unclear. Here, we show that recombinant PAD4 preferentially citrullinates recombinant IKK? [also called NF-?B essential modulator (NEMO)] over recombinant IKK? or IKK?. Consistent with this finding, PAD4 citrullinated renal proximal tubular cell IKK? and promoted NF-?B activation via I?B? phosphorylation in vitro. NEMO inhibition with a selective NEMO-binding peptide attenuated PAD4-mediated proinflammatory cytokine mRNA induction in HK-2 cells. Moreover, NEMO inhibition did not affect proximal tubular cell survival, proliferation, or apoptosis, unlike global NF-?B inhibition. In vivo, NEMO-binding peptide treatment protected against ischemic AKI. Finally, NEMO-binding peptide attenuated recombinant PAD4-mediated exacerbation of ischemic AKI, renal tubular inflammation, and apoptosis. Taken together, our results show that PAD4 exacerbates ischemic AKI and inflammation by promoting renal tubular NF-?B activity and inflammation via NEMO citrullination. Targeting NEMO activation may serve as a potential therapy for this devastating clinical problem.

| S-EPMC6620589 | biostudies-literature

An improved synthesis of haloaceteamidine-based inactivators of protein arginine deiminase 4 (PAD4).

Project description:Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form citrulline and ammonia. This enzyme has been implicated in several disease states, e.g. rheumatoid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solution-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been developed. This synthesis proceeds in 80% yield over 4 steps at a significantly (12-fold) lower cost.

| S-EPMC2597826 | biostudies-literature

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening.

Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays.ResultsTwenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment.ConclusionThree compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.

| S-EPMC3521205 | biostudies-literature

Small Molecule Inhibitors of Protein Arginine Methyltransferases.

Project description:IntroductionArginine methylation is an abundant posttranslational modification occurring in mammalian cells and catalyzed by protein arginine methyltransferases (PRMTs). Misregulation and aberrant expression of PRMTs are associated with various disease states, notably cancer. PRMTs are prominent therapeutic targets in drug discovery.Areas coveredThe authors provide an updated review of the research on the development of chemical modulators for PRMTs. Great efforts are seen in screening and designing potent and selective PRMT inhibitors, and a number of micromolar and submicromolar inhibitors have been obtained for key PRMT enzymes such as PRMT1, CARM1, and PRMT5. The authors provide a focus on their chemical structures, mechanism of action, and pharmacological activities. Pros and cons of each type of inhibitors are also discussed.Expert opinionSeveral key challenging issues exist in PRMT inhibitor discovery. Structural mechanisms of many PRMT inhibitors remain unclear. There lacks consistency in potency data due to divergence of assay methods and conditions. Physiologically relevant cellular assays are warranted. Substantial engagements are needed to investigate pharmacodynamics and pharmacokinetics of the new PRMT inhibitors in pertinent disease models. Discovery and evaluation of potent, isoform-selective, cell-permeable and in vivo-active PRMT modulators will continue to be an active arena of research in years ahead.

| S-EPMC4929062 | biostudies-literature

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