Peptidylarginine deiminases 4 as a promising target in drug discovery.

Peptidylarginine deaminase 4 (PAD4) is a crucial post-translational modifying enzyme catalyzing the conversion of arginine into citrulline residues, and mediating the formation of neutrophil extracellular traps (NETs). PAD4 plays a vital role in the occurrence and development of cardiovascular diseases, autoimmune diseases, and various tumors. Therefore, PAD4 is considered as a promising drug target for disease diagnosis and treatment. More and more efforts are devoted to developing highly efficient and selective PAD4 inhibitors via high-throughput screening, structure-based drug design and structure-activity relationship study. This article outlined the physiological and pathological functions of PAD4, and corresponding representative small molecule inhibitors reported in recent years.

PAD4 inhibitor promotes DNA damage and radiosensitivity of nasopharyngeal carcinoma cells.

Peptidylarginine deiminases 4 (PAD4), a kind of enzyme capable of converting protein arginine or mono-methylarginine into citrulline, has been identified to display a key role in diverse diseases. Radiotherapy is frequently used in nasopharyngeal carcinoma (NPC) treatment and induces DNA double strand breaks. In this study, whether PAD4 inhibitor YW3-56 affects the radiosensitivity of NPC cells was explored. RT-qPCR, immunofluorescence, western blot, clonogenic survival, and flow cytometry assays were used to assess the function of PAD4 and YW3-56 in NPC. We found the upregulation of PAD4 expression in NPC cells. PAD4 overexpression suppressed NPC cell apoptosis and promoted cell cycle, while PAD4 depletion had an opposite result. Moreover, the survival of NPC cells after irradiation was increased by overexpression of PAD4. PAD4 overexpression inhibited DNA damage and sensitivity of NPC cells to irradiation. Functional assays showed that YW3-56 treatment promoted DNA damage, apoptosis, and radiosensitivity of NPC cells. Importantly, YW3-56 treatment inhibited tumor growth in vivo. Overall, this study revealed the efficacy of PAD4 inhibitor YW3-56 in promoting sensitivity of NPC cells to irradiation.

The regulation of macrophage polarization by hypoxia-PADI4 coordination in Rheumatoid arthritis.

BACKGROUND: Hypoxia, a common feature of rheumatoid arthritis (RA), induces the over-expression of peptidyl arginine deiminase 4 (PADI4) in fibroblast-like synoviocytes (FLSs) and macrophages. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the role of hypoxia-PADI4 for macrophage polarization in RA patients. METHODS: Synovial tissue (ST) and synovial fluid (SF) were collected from 3 OA patients and 6 RA patients. The distribution of M1 and M2 in ST and cytokines in SF were examined by immunohistochemical analysis and Bio-Plex immunoassays. THP-1 macrophages and BMDM polarization were determined under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions. The effects of PADI4 on macrophages were determined by transfection of adenovirus vector-coated PADI4 (AdPADI4) and the use of PADI4 inhibitor. Finally, the roles of PADI4 in joint synovial lesions on macrophage polarization were investigated in collagen-induced arthritis (CIA) rats. RESULTS: We found increased macrophage polarization of M1 and M2 in the RA ST, compared with OA ST. The ratio of M1/M2 for RA and OA was 1.633 ± 0.1443 and 2.544 ± 0.4429, respectively. The concentration of M1- and M2-type cytokines was higher in RA than that in OA patients. Hypoxia contributed to the increase of the gene and protein expression of M1 and M2 markers. M1- but not M2-type gene expression showed a positive relationship with PADI4 expressionwhile the level of expression of M2-type genes showed no significant difference. The degree of joint swelling and destruction was effectively alleviated, and the number of macrophages especially M1 decreased in CIA rats after down-regulating PADI4 expression. CONCLUSION: Hypoxia is responsible for the co-polarization of M1 and M2. Hypoxia-associated PADI4 is responsible for M1 macrophage activation, implying that the inflammatory environment can be eased by decreasing PADI4 expression and improving the hypoxic environment.

Antithrombin Together with NETs Inhibitor Protected Against Postoperative Adhesion Formation in Mice.

BACKGROUND/AIMS: Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. METHODS: Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. RESULTS: AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. CONCLUSION: Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.

Targeted delivery of protein arginine deiminase-4 inhibitors to limit arterial intimal NETosis and preserve endothelial integrity.

AIMS: Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. METHODS AND RESULTS: We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. CONCLUSIONS: NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Inhibition of PAD4 enhances radiosensitivity and inhibits aggressive phenotypes of nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a tumor deriving from nasopharyngeal epithelium. Peptidyl-arginine deiminase 4 (PAD4) is a vital mediator of histone citrullination and plays an essential role in regulating disease process. Radiotherapy is an essential method to treat NPC. In this research, we explored the effect of PAD4 on NPC radiosensitivity. METHODS: We enrolled 50 NPC patients, established mice xenograft model, and purchased cell lines for this study. Statistical analysis and a series of experiments including RT-qPCR, clonogenic survival, EdU, Transwell, and wound healing assays were done. RESULTS: Our data manifested that PAD4 (mRNA and protein) presented a high expression in NPC tissues and cells. GSK484, an inhibitor of PAD4, could inhibit activity of PAD4 in NPC cell lines. PAD4 overexpression promoted the radioresistance, survival, migration, and invasion of NPC cells, whereas treatment of GSK484 exerted inhibitory effects on radioresistance and aggressive phenotype of NPC cells. Additionally, GSK484 could attenuate the effect of PAD4 of NPC cell progression. More importantly, we found that GSK484 significantly inhibited tumor size, tumor weight and tumor volume in mice following irradiation. CONCLUSIONS: PAD4 inhibitor GSK484 attenuated the radioresistance and cellular progression in NPC.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

Stromal-driven and Amyloid ß-dependent induction of neutrophil extracellular traps modulates tumor growth.

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid ß, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and ß-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid ß as the protagonist and potential therapeutic target.

TAT-Modified Gold Nanoparticles Enhance the Antitumor Activity of PAD4 Inhibitors.

PURPOSE: Histone citrullination by peptidylarginine deiminases 4 (PAD4) regulates the gene expression of tumor suppressor. In our previously study, YW3-56 (356) was developed as a potent PAD4 inhibitor for cancer therapy with novel function in the autophagy pathway. To enhance the antitumor activity, the PAD4 inhibitor 356 was modified by the well-established cationic penetrating peptide RKKRRQRRR (peptide TAT) and gold nanoparticles to obtain 356-TAT-AuNPs which could enhance the permeability of chemical drug in solid tumor. METHODS: 356-TAT-AuNPs were prepared, and their morphology were characterized. The antitumor activity of 356-TAT-AuNPs was evaluated in vitro and in vivo. RESULTS: 356-TAT-AuNPs exhibited higher anticancer activity against HCT-116, MCF-7 and A549 cells than 356 and 356-AuNPs. Compared with 356 and 356-AuNPs, 356-TAT-AuNPs entered the cytoplasm and nuclear, exhibited stronger anticancer activity by increasing apoptosis, inducing autophagy and inhibiting of histone H3 citrullination, and in HCT-116 xenograft mouse model, 356-TAT-AuNPs could improve the antitumor activity. CONCLUSION: The modified AuNPs with peptide TAT as drug delivery system are potent in delaying tumor growth and could be a powerful vehicle for profitable anticancer drug development. We believe that peptide TAT modification strategy may provide a simple and valuable method for improving antitumor activity of PAD4 inhibitors for clinical use.

PAD4 selective inhibitor TDFA protects lipopolysaccharide-induced acute lung injury by modulating nuclear p65 localization in epithelial cells.

Protein arginine deiminase 4 (PAD4) serves a critical role in differentiation, development and apoptosis through gene regulation and has emerged as a potential therapeutic target for the treatment of various diseases. However, the roles of PAD4 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remain largely unknown. To investigate the roles of PAD4 during LPS-induced ALI, the present study detected the trend of PAD4 expression in the lung tissues of ALI mice. Subsequently, the efficiency of TDFA on PAD4 and citrullinated H3 histone were detected. And then, histology, the wet/dry weight ratio, survival rate, activated cells infiltration, oxidative stress levels, tight junction proteins and proinflammatory cytokine expression were detected. In addition, the level of transepithelial electrical resistance (TEER) was assessed. Finally, the level of nuclear P65, total phosphorylated P65 and P65 were measured in vivo and in vitro. The results showed that PAD4 expression was upregulated in the lung tissues of LPS-induced ALI. TDFA efficiently decreased the severity of the lung edema, attenuated the severity of pulmonary injury and improved the survival rate following lethal LPS administration. Besides, TDFA reduced activated cells infiltration and suppressed inflammation related parameters, including proinflammatory cytokines production (TNF-α, IL-6 and IL-1ß) and oxidative stress (MDA, GSH and SOD). Furthermore, TDFA reversed the TEER downregulation tendency and tight junction proteins (ZO-1, Occludin, Claudin-4) levels that represent the integrity of alveolar epithelium. Eventually, TDFA exerts its protective roles through modulating nuclear localization of transcription factor NF-κB P65 in epithelial cells. Taken together, these results indicate that PAD4 inhibition may serve as a promising therapeutic approach for LPS-induced ALI.

Evaluation of protein arginine deiminase-4 inhibitor in TNBS- induced colitis in mice.

BACKGROUND AND AIM: Many evidences indicated that neutrophil extracellular traps (NETs) are involved in the pathogenesis of inflammatory bowel disease (IBD). Citrullination of histones by Protein Arginine Deiminase-4 (PAD4) is central for NETs formation. This paper aimed to explore the definite role of NETs in mouse model of Crohn's disease (CD) with 2,4,6-trinitrobenzene sulfonic acid (TNBS). METHODS: The expression of NETs-associated proteins and mRNAs in colon tissue were detected by immunohistochemistry and Real-time Quantitative PCR (QPCR) respectively. Neutrophils were isolated and stimulated in vitro to form NETs. In addition, we also administered Cl-amidine, PAD4 inhibitor, resulting in less NETs formation to investigate protective effect by measuring weight loss, gross bleeding, colon length, myeloperoxidase (MPO) activity, and cytokine expression in mice. RESULTS: The results showed enhanced expression of Ly6G, citrullinated histone H3 (CitH3), and PAD4 in TNBS-induced colitis mice and higher ability of neutrophil to produce NETs in vitro. Blocking NETs formation through Cl-amidine effectively alleviated the clinical colitis index and tissue inflammation in TNBS mice, regulated the expression of pro- or anti-inflammatory cytokines. In addition, Cl-amidine reduced the gene expression of PAD4 and the expression of NETs-associated proteins in the colon of TNBS mice and inhibited the formation of NETs in vitro. CONCLUSIONS: Our data showed that Cl-amidine could alleviate the clinical colitis index in TNBS mice to some extend and suggested blocking NETs formation through inhibition of PAD4 as therapeutic targets for the treatment of CD.

A Novel Peptidylarginine Deiminase 4 (PAD4) Inhibitor BMS-P5 Blocks Formation of Neutrophil Extracellular Traps and Delays Progression of Multiple Myeloma.

Multiple myeloma is a plasma cell malignancy, which grows in the bone marrow (BM). The major population of cells in the BM is represented by neutrophils and they can form neutrophil extracellular traps (NET). Here, we investigated whether multiple myeloma cells induce NET formation and whether targeting this process would delay multiple myeloma progression. We demonstrated that murine and human multiple myeloma cells stimulate citrullination of histone H3 and NET formation by neutrophils and that this process is abrogated by pharmacological targeting of peptidylarginine deiminase 4 (PAD4) with a novel-specific small molecule inhibitor BMS-P5. Administration of BMS-P5 to multiple myeloma-bearing mice delays appearance of symptoms and disease progression. Taken together, our data demonstrate that targeting PAD4 may be beneficial for treatment of multiple myeloma.

Peptidylarginine Deiminase IV Regulates Breast Cancer Stem Cells via a Novel Tumor Cell-Autonomous Suppressor Role.

Peptidylarginine deiminases (PADI) catalyze posttranslational modification of many target proteins and have been suggested to play a role in carcinogenesis. Citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells. Here, we investigated a possible role for PADI4 in regulating breast cancer stem cells. PADI4 activity limited the number of cancer stem cells (CSC) in multiple breast cancer models in vitro and in vivo. Mechanistically, PADI4 inhibition resulted in a widespread redistribution of histone H3, with increased accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 downregulated expression of master transcription factors of stemness, NANOG and OCT4, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci; this effect was not seen in the non-stem cells. A gene signature reflecting tumor cell-autonomous PADI4 inhibition was associated with poor outcome in human breast cancer datasets, consistent with a tumor-suppressive role for PADI4 in estrogen receptor-positive tumors. These results contrast with known tumor-promoting effects of PADI4 on the tumor stroma and suggest that the balance between opposing tumor cell-autonomous and stromal effects may determine net outcome. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells and provide insight into context-specific effects of PADI4 in epigenetic modulation. SIGNIFICANCE: These findings demonstrate a novel activity of the citrullinating enzyme PADI4 in suppressing breast cancer stem cells through epigenetic repression of stemness master transcription factors NANOG and OCT4.

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

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.