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Paul R. Thompson - One of the best experts on this subject based on the ideXlab platform.
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Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages.
2019Co-Authors: Neha Mishra, Lidja Schwerdtner, Kelly L. Sams, Santanu Mondal, Fareed Ahmad, Reinhold E. Schmidt, Scott A. Coonrod, Paul R. Thompson, Markus M. Lerch, Lukas BossallerAbstract:Protein Arginine Deiminase (PAD) enzymes catalyze the conversion of protein-bound Arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein Arginine Deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1β release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1β release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/- macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1β release, a finding of importance for autoimmune diseases and inflammation.
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Protein Arginine Deiminase 4: a target for an epigenetic cancer therapy
2011Co-Authors: Jessica L. Slack, Corey P. Causey, Paul R. ThompsonAbstract:The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases, and suggested that the factors controlling gene expression are novel drug targets. Protein Arginine Deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine, two potent PAD4 inhibitors, display micromolar cytotoxic effects towards several cancerous cell lines (HL-60, MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally, these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together, these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer, and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease.
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haloacetamidine based inactivators of protein Arginine Deiminase 4 pad4 evidence that general acid catalysis promotes efficient inactivation
2010Co-Authors: Bryan Knuckley, Corey P. Causey, Perry J Pellechia, Paul F Cook, Paul R. ThompsonAbstract:Dysregulated protein Arginine Deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including rheumatoid arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 by the covalent modification of an active-site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation was determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds by a multistep mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active-site cysteine, that is, Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding-that protonation of the tetrahedral intermediate is important for enzyme inactivation-also suggests that, during catalysis, protonation of the analogous intermediate is required for efficient substrate turnover.
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profiling protein Arginine Deiminase 4 pad4 a novel screen to identify pad4 inhibitors
2008Co-Authors: Bryan Knuckley, Yuan Luo, Paul R. ThompsonAbstract:Protein Arginine Deiminase 4 (PAD4) has emerged as a leading target for the development of a Rheumatoid Arthritis (RA) pharmaceutical. Herein, we describe the development of a novel screen for PAD4 inhibitors that is based on a PAD4-targeted Activity-Based Protein Profiling reagent, denoted Rhodamine-conjugated F-Amidine (RFA). This screen was validated by screening 10 Disease Modifying Anti-Rheumatic Drugs (DMARDs) and identified streptomycin, minocycline, and chlortetracycline as micromolar inhibitors of PAD4 activity.
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protein Arginine Deiminase 4 evidence for a reverse protonation mechanism
2007Co-Authors: Bryan Knuckley, Monica Bhatia, Paul R. ThompsonAbstract:The presumed role of an overactive protein Arginine Deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of paramount importance. Toward the goal of developing such inhibitors, we initiated efforts to characterize the catalytic mechanism of PAD4 and thereby identify important mechanistic features that can be exploited for inhibitor development. Herein we report the results of mutagenesis studies as well as our efforts to characterize the initial steps of the PAD4 reaction, in particular, the protonation status of Cys645 and His471 prior to substrate binding. The results indicate that Cys645, the active site nucleophile, exists as the thiolate in the active form of the free enzyme. pH studies on PAD4 further suggest that this enzyme utilizes a reverse protonation mechanism.
John S Bomalaski - One of the best experts on this subject based on the ideXlab platform.
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exploiting Arginine auxotrophy with pegylated Arginine Deiminase adi peg20 to sensitize pancreatic cancer to radiotherapy via metabolic dysregulation
2019Co-Authors: Pankaj Singh, John S Bomalaski, Amit Deorukhkar, Bhanu Prasad Venkatesulu, Ramesh C Tailor, Sunil KrishnanAbstract:Distinct metabolic vulnerabilities of cancer cells compared with normal cells can potentially be exploited for therapeutic targeting. Deficiency of argininosuccinate synthetase-1 (ASS1) in pancreatic cancers creates auxotrophy for the semiessential amino acid Arginine. We explored the therapeutic potential of depleting exogenous Arginine via pegylated Arginine Deiminase (ADI-PEG20) treatment as an adjunct to radiotherapy. We evaluated the efficacy of treatment of human pancreatic cancer cell lines and xenografts with ADI-PEG20 and radiation via clonogenic assays and tumor growth delay experiments. We also investigated potential mechanisms of action using reverse-phase protein array, Western blotting, and IHC and immunofluorescence staining. ADI-PEG20 potently radiosensitized ASS1-deficient pancreatic cancer cells (MiaPaCa-2, Panc-1, AsPc-1, HPAC, and CaPan-1), but not ASS1-expressing cell lines (Bxpc3, L3.6pl, and SW1990). Reverse phase protein array studies confirmed increased expression of proteins related to endoplasmic reticulum (ER) stress and apoptosis, which were confirmed by Western blot analysis. Inhibition of ER stress signaling with 4-phenylbutyrate abrogated the expression of ER stress proteins and reversed radiosensitization by ADI-PEG20. Independent in vivo studies in two xenograft models confirmed significant tumor growth delays, which were associated with enhanced expression of ER stress proteins and apoptosis markers and reduced expression of proliferation and angiogenesis markers. ADI-PEG20 augmented the effects of radiation by triggering the ER stress pathway, leading to apoptosis in pancreatic tumor cells.
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a phase ii study of Arginine Deiminase adi peg20 in relapsed refractory or poor risk acute myeloid leukemia patients
2017Co-Authors: Hui Jen Tsai, Shih Sheng Jiang, Wen Chun Hung, Gautam Borthakur, Sheng Fung Lin, Naveen Pemmaraju, Elias Jabbour, John S BomalaskiAbstract:Exogenous Arginine is required for growth in some argininosuccinate synthetase (ASS)-deficient cancers. Arginine Deiminase (ADI) inhibits growth in various ASS-deficient cancers by depleting Arginine. The efficacy of pegylated ADI (ADI-PEG20) in relapsed/refractory/poor-risk acute myeloid leukemia (AML) was evaluated in 43 patients in a prospective, phase II trial (NCT01910012 (10/07/2013), https://clinicaltrials.gov/ct2/show/NCT01910012?term = ADI-PEG20&rank = 12 ). Despite almost all pre-treatment tumor samples showing ASS deficiency, the best response among 21 evaluable patients was complete response (CR) in 2 (9.5%) and stable disease in 7 (33.3%), yielding a disease control rate (DCR) of 42.9%. The response durations of the two patients with CR were 7.5 and 8.8 months. DCR was correlated with a median of 8 weeks of Arginine depletion to ≤10 μM. Using whole transcriptome sequencing, we compared gene expression profiling of pre- and post-treatment bone marrow samples of the two responders and three non-responders. The expression levels of some markers for AML subtypes and c-MYC regulated genes were considered potential predictors of response to ADI-PEG20. These results suggest that ASS deficiency is a prerequisite but not a sufficient condition for response to ADI-PEG20 monotherapy in AML. Predictive biomarkers and mechanistic explorations will be critical for identifying appropriate patients for future AML trials of ADI-PEG20.
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phase i ii study of pegylated Arginine Deiminase adi peg 20 in patients with advanced melanoma
2013Co-Authors: Achim A Jungbluth, John S Bomalaski, Patrick A Ott, Richard D Carvajal, Neeta Pandittaskar, Eric W Hoffman, Ralph Venhaus, Linda Pan, Lloyd J OldAbstract:Background Arginine Deiminase (ADI) is an enzyme that degrades Arginine, an amino acid that is important for growth and development of normal and neoplastic cells. Melanoma cells are auxotrophic for Arginine, because they lack argininosuccinatesynthetase (ASS), a key enzyme required for the synthesis of Arginine. Patients and methods Patients with advanced melanoma were treated with 40, 80 or 160 IU/m2 ADI-PEG 20 i.m. weekly. Primary endpoints were toxicity and tumor response, secondary endpoints included metabolic response by 18FDG-PET, pharmacodynamic (PD) effects upon circulating Arginine levels, and argininosuccinate synthetase tumor expression by immunohistochemistry. Results 31 previously treated patients were enrolled. The main toxicities were grade 1 and 2 adverse events including injection site pain, rash, and fatigue. No objective responses were seen. Nine patients achieved stable disease (SD), with 2 of these durable for >6 months. Four of the 9 patients with SD had uveal melanoma. PD analysis showed complete plasma Arginine depletion in 30/31 patients by day 8. Mean plasma levels of ADI-PEG 20 correlated inversely with ADI-PEG 20 antibody levels. Immunohistochemical ASS expression analysis in tumor tissue was negative in 24 patients, whereas 5 patients had <5 % cells positive. Conclusions ADI-PEG 20 is well tolerated in advanced melanoma patients and leads to consistent, but transient, Arginine depletion. Although no RECIST responses were observed, the encouraging rate of SD in uveal melanoma patients indicates that it may be worthwhile to evaluate ADI-PEG 20 in this melanoma subgroup.
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Arginine Deiminase peg20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase
2012Co-Authors: Marcus Kelly, Achim A Jungbluth, John S Bomalaski, L J Old, Gerd RitterAbstract:Arginine Deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase
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pegylated Arginine Deiminase treatment of patients with unresectable hepatocellular carcinoma results from phase i ii studies
2004Co-Authors: Francesco Izzo, Gerardo Beneduce, Giuseppe Castello, Vincenzo De Rosa, Frederick W Holtsberg, Mark C Ensor, Paolo Marra, Paolo Vallone, Franco Cremona, John S BomalaskiAbstract:Purpose Recently, we reported that a large number of human hepatocellular cancer (HCC) cell lines were auxotrophic for Arginine. Here we report the results obtained with the amino acid–degrading enzyme Arginine Deiminase (ADI) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) as a means of lowering plasma Arginine to treat HCC. The study was a cohort dose-escalation phase I/II study. Patients and Methods Pharmacodynamic studies indicated an ADI-SS PEG 20,000 mw dose level of 160 U/m2 was sufficient to lower plasma Arginine from a resting level of approximately 130 μmol/L to below the level of detection (< 2 μmol/L) for more than 7 days, a dose later defined as the optimal biologic dose. All patients were to receive three cycles at the optimum biologic dose. Results This therapy was well tolerated, even in patients who had no detectable plasma Arginine for 3 continuous months of therapy. Of the 19 patients enrolled, two had a complete response, seven had a partial response, seven had stable disease,...
Thomas H Lee - One of the best experts on this subject based on the ideXlab platform.
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peptidyl Arginine Deiminase 4 exacerbates ischemic aki by finding nemo
2019Co-Authors: May M Rabadi, Sangjun Han, Mihwa Kim, Vivette D Dagati, Thomas H LeeAbstract: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 re...
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peptidyl Arginine Deiminase 4 activation exacerbates kidney ischemia reperfusion injury
2014Co-Authors: Ahrom Ham, May M Rabadi, Mihwa Kim, Vivette D Dagati, Kevin M Brown, Thomas H LeeAbstract:Peptidyl Arginine Deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of Arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.
Yael David - One of the best experts on this subject based on the ideXlab platform.
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protein Arginine Deiminase 4 antagonizes methylglyoxal induced histone glycation
2020Co-Authors: Qingfei Zheng, Adewola Osunsade, Yael DavidAbstract:Protein Arginine Deiminase 4 (PAD4) facilitates the post-translational citrullination of the core histones H3 and H4. While the precise epigenetic function of this modification has not been resolved, it has been shown to associate with general chromatin decompaction and compete with Arginine methylation. Recently, we found that histones are subjected to methylglyoxal (MGO)-induced glycation on nucleophilic side chains, particularly Arginines, under metabolic stress conditions. These non-enzymatic adducts change chromatin architecture and the epigenetic landscape by competing with enzymatic modifications, as well as changing the overall biophysical properties of the fiber. Here, we report that PAD4 antagonizes histone MGO-glycation by protecting the reactive Arginine sites, as well as by converting already-glycated Arginine residues into citrulline. Moreover, we show that similar to the deglycase DJ-1, PAD4 is overexpressed and histone citrullination is upregulated in breast cancer tumors, suggesting an additional mechanistic link to PAD4's oncogenic properties.
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Protein Arginine Deiminase 4 antagonizes methylglyoxal-induced histone glycation
2020Co-Authors: Qingfei Zheng, Adewola Osunsade, Yael DavidAbstract:Protein Arginine Deiminase 4 (PAD4) facilitates the posttranslational citrullination of histones H3 and H4. Here, the authors provide evidence that PAD4 antagonizes histone methylglyoxal-glycation by rewriting the glycated Arginine into citrulline and protecting the reactive sites from further glycation
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protein Arginine Deiminase 4 antagonizes methylglyoxal induced histone glycation
2019Co-Authors: Qingfei Zheng, Adewola Osunsade, Yael DavidAbstract:Abstract Protein Arginine Deiminase 4 (PAD4) facilitates the post-translational citrullination of the core histones H3 and H4. While the precise epigenetic function of this modification has not been resolved, it was shown to associate with general chromatin decompaction and to compete with Arginine methylation. Recently, we showed that histones are subjected to methylglyoxal (MGO)-induced glycation on nucleophilic side chains, particularly Arginines, under metabolic stress conditions. These non-enzymatic adducts change chromatin architecture and the epigenetic landscape by competing with enzymatic modifications. Here we report that PAD4 antagonizes histone MGO-glycation by protecting the reactive sites with oxygen substitution, as well as by converting already-glycated Arginine residues into citrulline. Moreover, we show that similar to the deglycase DJ-1, PAD4 is overexpressed and histone citrullination is upregulated in breast cancer tumors, suggesting an additional mechanistic link to PAD4’s oncogenic properties. Significance Metabolic syndromes and diabetes increase the risk for certain diseases such as cancer. However, the mechanism behind this correlation is poorly understood. Methylglyoxal (MGO), a reactive dicarbonyl sugar metabolite found in cells under metabolic stress, can non-enzymatically modify Arginine and lysine residues in histone proteins, making it a new epigenetic marker linking metabolism and disease. Histone MGO-glycation induces changes in chromatin architecture and the epigenetic landscape, and abrogates gene transcription. In this study, we found that protein Arginine Deiminase 4 (PAD4) exhibits dual functions to antagonize histone MGO-glycation: removing glycation adducts from Arginines and converting the unmodified side chains into citrulline, which protects them from undergoing glycation. This unprecedented biochemical mechanism demonstrates a potential function of PAD4 in cancer cells.
Bryan Knuckley - One of the best experts on this subject based on the ideXlab platform.
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haloacetamidine based inactivators of protein Arginine Deiminase 4 pad4 evidence that general acid catalysis promotes efficient inactivation
2010Co-Authors: Bryan Knuckley, Corey P. Causey, Perry J Pellechia, Paul F Cook, Paul R. ThompsonAbstract:Dysregulated protein Arginine Deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including rheumatoid arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 by the covalent modification of an active-site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation was determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds by a multistep mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active-site cysteine, that is, Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding-that protonation of the tetrahedral intermediate is important for enzyme inactivation-also suggests that, during catalysis, protonation of the analogous intermediate is required for efficient substrate turnover.
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profiling protein Arginine Deiminase 4 pad4 a novel screen to identify pad4 inhibitors
2008Co-Authors: Bryan Knuckley, Yuan Luo, Paul R. ThompsonAbstract:Protein Arginine Deiminase 4 (PAD4) has emerged as a leading target for the development of a Rheumatoid Arthritis (RA) pharmaceutical. Herein, we describe the development of a novel screen for PAD4 inhibitors that is based on a PAD4-targeted Activity-Based Protein Profiling reagent, denoted Rhodamine-conjugated F-Amidine (RFA). This screen was validated by screening 10 Disease Modifying Anti-Rheumatic Drugs (DMARDs) and identified streptomycin, minocycline, and chlortetracycline as micromolar inhibitors of PAD4 activity.
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protein Arginine Deiminase 4 evidence for a reverse protonation mechanism
2007Co-Authors: Bryan Knuckley, Monica Bhatia, Paul R. ThompsonAbstract:The presumed role of an overactive protein Arginine Deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of paramount importance. Toward the goal of developing such inhibitors, we initiated efforts to characterize the catalytic mechanism of PAD4 and thereby identify important mechanistic features that can be exploited for inhibitor development. Herein we report the results of mutagenesis studies as well as our efforts to characterize the initial steps of the PAD4 reaction, in particular, the protonation status of Cys645 and His471 prior to substrate binding. The results indicate that Cys645, the active site nucleophile, exists as the thiolate in the active form of the free enzyme. pH studies on PAD4 further suggest that this enzyme utilizes a reverse protonation mechanism.
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activity based protein profiling reagents for protein Arginine Deiminase 4 pad4 synthesis and in vitro evaluation of a fluorescently labeled probe
2006Co-Authors: Yuan Luo, Bryan Knuckley, Monica Bhatia, Perry J Pellechia, Paul R. ThompsonAbstract:Protein Arginine Deiminase 4 (PAD4), which catalyzes the post-translational conversion of peptidyl Arginine to peptidyl citrulline, is widely regarded as one of the best new targets for the development of a novel rheumatoid arthritis therapeutic. In addition to its presumed role in this disease, PAD4 is also a calcium-dependent histone Deiminase that acts as a transcriptional co-repressor. Herein we describe the design, synthesis, and in vitro evaluation of two fluorescently labeled activity-based protein profiling (ABPP) reagents that specifically and irreversibly modify the active, that is, calcium-bound, form PAD4 with equal affinity to previously described small molecule chemical probes of PAD4 function. These fluorescently tagged ABPPs will be useful for identifying the conditions under which this enzyme is activated in vivo and may prove to be useful RA diagnostics.
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inhibitors and inactivators of protein Arginine Deiminase 4 functional and structural characterization
2006Co-Authors: Yuan Luo, Bryan Knuckley, Monica Bhatia, Kyouhei Arita, Young Ho Lee, Michael R Stallcup, Mamoru Sato, Paul R. ThompsonAbstract:Protein Arginine Deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific Arginine residues in proteins to citrulline. Recently, we reported the synthesis and characterization of F-amidine, a potent and bioavailable irreversible inactivator of PAD4. Herein, we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation, the structure of the PAD4-F-amidine x calcium complex, and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition, they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder.