The Experts below are selected from a list of 1341 Experts worldwide ranked by ideXlab platform
Paul R Thompson - One of the best experts on this subject based on the ideXlab platform.
-
Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages.
The Journal of Immunology, 2019Co-Authors: Neha Mishra, Paul R Thompson, Lidja Schwerdtner, Kelly L. Sams, Santanu Mondal, Fareed Ahmad, Reinhold E. Schmidt, Scott A. Coonrod, 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.
-
Thioredoxin Modulates Protein Arginine Deiminase 4 (PAD4)-Catalyzed Citrullination
Frontiers Media S.A., 2019Co-Authors: Paul R Thompson, Mitesh Nagar, Ronak TilvawalaAbstract: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
-
Development of a Selective Inhibitor of Protein Arginine Deiminase 2
2017Co-Authors: Aaron Muth, Venkataraman Subramanian, Sangram S Parelkar, Mitesh Nagar, Edward Beaumont, Hema Srinath, Philip Kerry, Paul Mcewan, Kathleen Clancy, Paul R ThompsonAbstract:Protein Arginine Deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy (30a). Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated
-
detection and identification of Protein citrullination in complex biological systems
Current Opinion in Chemical Biology, 2016Co-Authors: Kathleen W Clancy, Eranthie Weerapana, Paul R ThompsonAbstract:Protein citrullination is a post-translational modification of Arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated Proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated Proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated Proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated Proteins as well as the specific residues modified within those Proteins.
-
Protein Arginine Deiminases and associated citrullination physiological functions and diseases associated with dysregulation
Current Drug Targets, 2015Co-Authors: Erin E. Witalison, Paul R Thompson, Lorne J. HofsethAbstract:Human Proteins are subjected to more than 200 known post-translational modifications (PTMs) (e.g., phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, Nacetylation, and citrullination) and these PTMs can alter Protein structure and function with consequent effects on the multitude of pathways necessary for maintaining the physiological homeostasis. When dysregulated, however, the enzymes that catalyze these PTMs can impact the genesis of countless diseases. In this review, we will focus on Protein citrullination, a PTM catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Specifically, we will describe the roles of the PADs in both normal human physiology and disease. The development of PAD inhibitors and their efficacy in a variety of autoimmune disorders and cancer will also be discussed.
Bryan Knuckley - One of the best experts on this subject based on the ideXlab platform.
-
the development of n α 2 carboxyl benzoyl n5 2 fluoro 1 iminoethyl l ornithine amide o f amidine and n α 2 carboxyl benzoyl n5 2 chloro 1 iminoethyl l ornithine amide o cl amidine as second generation Protein Arginine Deiminase pad inhibitors
Journal of Medicinal Chemistry, 2011Co-Authors: Corey P. Causey, Venkataraman Subramanian, Bryan Knuckley, Jessica L. Slack, Justin E Jones, Daisuke Kamei, Larry E Jones, Pedram Ebrahimi, Alexander A Chumanevich, Yuan LuoAbstract:Protein Arginine Deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1–4 and 6), regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation of F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure–activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical p...
-
n α benzoyl n5 2 chloro 1 iminoethyl l ornithine amide a Protein Arginine Deiminase inhibitor reduces the severity of murine collagen induced arthritis
Journal of Immunology, 2011Co-Authors: Van C Willis, Bryan Knuckley, Corey P. Causey, Nirmal K Banda, Kristen N Cordova, Piyanka E Chandra, Alison M Gizinski, Brandt Levitt, Magdalena J Glogowska, Liudmila KulikAbstract:Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-Proteins. Citrullinated synovial Proteins, which are generated via the actions of the Protein Arginine Deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial Proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.
-
substrate specificity and kinetic studies of pads 1 3 and 4 identify potent and selective inhibitors of Protein Arginine Deiminase 3
Biochemistry, 2010Co-Authors: Bryan Knuckley, Christina J Dreyton, Monica Bhatia, Corey P. Causey, Justin E Jones, Tanesha C Osborne, Hidenari Takahara, Paul R ThompsonAbstract:Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the Protein Arginine Deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD acti...
-
haloacetamidine based inactivators of Protein Arginine Deiminase 4 pad4 evidence that general acid catalysis promotes efficient inactivation
ChemBioChem, 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.
-
Protein Arginine Deiminase 4 pad4 current understanding and future therapeutic potential
Current Opinion in Drug Discovery & Development, 2009Co-Authors: Justin E Jones, Corey P. Causey, Bryan Knuckley, Jessica L Slacknoyes, Paul R ThompsonAbstract:The Protein Arginine Deiminases (PADs), and in particular PAD4, have emerged as potential therapeutic targets for the treatment of rheumatoid arthritis (RA). In this review, evidence linking dysregulated PAD activity to the onset and progression of RA is presented, and the potential role of such aberrant activity in other human diseases, such as multiple sclerosis and cancer, is discussed. The known physiological roles of the PADs, particularly PAD4, and current knowledge regarding PAD structure, catalysis and inhibition are also described.
Yuan Luo - One of the best experts on this subject based on the ideXlab platform.
-
the development of n α 2 carboxyl benzoyl n5 2 fluoro 1 iminoethyl l ornithine amide o f amidine and n α 2 carboxyl benzoyl n5 2 chloro 1 iminoethyl l ornithine amide o cl amidine as second generation Protein Arginine Deiminase pad inhibitors
Journal of Medicinal Chemistry, 2011Co-Authors: Corey P. Causey, Venkataraman Subramanian, Bryan Knuckley, Jessica L. Slack, Justin E Jones, Daisuke Kamei, Larry E Jones, Pedram Ebrahimi, Alexander A Chumanevich, Yuan LuoAbstract:Protein Arginine Deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1–4 and 6), regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation of F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure–activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical p...
-
profiling Protein Arginine Deiminase 4 pad4 a novel screen to identify pad4 inhibitors
Bioorganic & Medicinal Chemistry, 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.
-
activity based Protein profiling reagents for Protein Arginine Deiminase 4 pad4 synthesis and in vitro evaluation of a fluorescently labeled probe
Journal of the American Chemical Society, 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.
-
inhibitors and inactivators of Protein Arginine Deiminase 4 functional and structural characterization
Biochemistry, 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.
-
a fluoroacetamidine based inactivator of Protein Arginine Deiminase 4 design synthesis and in vitro and in vivo evaluation
Journal of the American Chemical Society, 2006Co-Authors: Yuan Luo, Bryan Knuckley, Young Ho Lee, Michael R Stallcup, Paul R ThompsonAbstract:Protein Arginine Deiminase 4 (PAD4) is a calcium-dependent transcriptional corepressor that has been implicated in the onset and progression of rheumatoid arthritis. Herein we describe the synthesis and in vitro evaluation of a fluoroacetamidine-containing compound, N-α-benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide, 1, hereafter referred to as F-amidine, that is the most potent PAD4 inhibitor ever described. Additional studies described herein indicate that F-amidine can also inhibit PAD4 activity in vivo. The bioavailability of this compound suggests that F-amidine will be a powerful chemical probe of PAD4 function that can be used to dissect the roles of this enzyme in both rheumatoid arthritis and transcriptional control. The fact that inhibition is of an irreversible nature suggests that, with appropriate functionalization, F-amidine analogues will be robust activity-based Protein-profiling and proteomic capture reagents.
Corey P. Causey - One of the best experts on this subject based on the ideXlab platform.
-
the development of n α 2 carboxyl benzoyl n5 2 fluoro 1 iminoethyl l ornithine amide o f amidine and n α 2 carboxyl benzoyl n5 2 chloro 1 iminoethyl l ornithine amide o cl amidine as second generation Protein Arginine Deiminase pad inhibitors
Journal of Medicinal Chemistry, 2011Co-Authors: Corey P. Causey, Venkataraman Subramanian, Bryan Knuckley, Jessica L. Slack, Justin E Jones, Daisuke Kamei, Larry E Jones, Pedram Ebrahimi, Alexander A Chumanevich, Yuan LuoAbstract:Protein Arginine Deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1–4 and 6), regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation of F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure–activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical p...
-
n α benzoyl n5 2 chloro 1 iminoethyl l ornithine amide a Protein Arginine Deiminase inhibitor reduces the severity of murine collagen induced arthritis
Journal of Immunology, 2011Co-Authors: Van C Willis, Bryan Knuckley, Corey P. Causey, Nirmal K Banda, Kristen N Cordova, Piyanka E Chandra, Alison M Gizinski, Brandt Levitt, Magdalena J Glogowska, Liudmila KulikAbstract:Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-Proteins. Citrullinated synovial Proteins, which are generated via the actions of the Protein Arginine Deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial Proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.
-
Protein Arginine Deiminase 4: a target for an epigenetic cancer therapy
Cellular and Molecular Life Sciences, 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.
-
substrate specificity and kinetic studies of pads 1 3 and 4 identify potent and selective inhibitors of Protein Arginine Deiminase 3
Biochemistry, 2010Co-Authors: Bryan Knuckley, Christina J Dreyton, Monica Bhatia, Corey P. Causey, Justin E Jones, Tanesha C Osborne, Hidenari Takahara, Paul R ThompsonAbstract:Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the Protein Arginine Deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD acti...
-
haloacetamidine based inactivators of Protein Arginine Deiminase 4 pad4 evidence that general acid catalysis promotes efficient inactivation
ChemBioChem, 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.
Venkataraman Subramanian - One of the best experts on this subject based on the ideXlab platform.
-
development of a selective inhibitor of Protein Arginine Deiminase 2
Journal of Medicinal Chemistry, 2017Co-Authors: Venkataraman Subramanian, Mitesh Nagar, Aaron Muth, Edward Beaumont, Philip S Kerry, Paul A Mcewan, Hema Srinath, Kathleen W Clancy, Sangram S ParelkarAbstract:Protein Arginine Deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy (30a). Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.
-
Development of a Selective Inhibitor of Protein Arginine Deiminase 2
2017Co-Authors: Aaron Muth, Venkataraman Subramanian, Sangram S Parelkar, Mitesh Nagar, Edward Beaumont, Hema Srinath, Philip Kerry, Paul Mcewan, Kathleen Clancy, Paul R ThompsonAbstract:Protein Arginine Deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy (30a). Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated
-
design synthesis and biological evaluation of tetrazole analogs of cl amidine as Protein Arginine Deiminase inhibitors
Journal of Medicinal Chemistry, 2015Co-Authors: Venkataraman Subramanian, Scott A. Coonrod, Jason S Knight, Sangram S Parelkar, Lynne J Anguish, Mariana J Kaplan, Paul R ThompsonAbstract:Protein Arginine Deiminases (PADs) catalyze the post-translational hydrolysis of Arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also b...
-
Design, Synthesis, and Biological Evaluation of Tetrazole Analogs of Cl-Amidine as Protein Arginine Deiminase Inhibitors
Journal of medicinal chemistry, 2015Co-Authors: Venkataraman Subramanian, Scott A. Coonrod, Jason S Knight, Sangram S Parelkar, Lynne J Anguish, Mariana J Kaplan, Paul R ThompsonAbstract:Protein Arginine Deiminases (PADs) catalyze the post-translational hydrolysis of Arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs.
-
insights into the mechanism of streptonigrin induced Protein Arginine Deiminase inactivation
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Christina J Dreyton, Erin D Anderson, Venkataraman Subramanian, Dale L Boger, Paul R ThompsonAbstract:Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the Protein Arginine Deiminases (PADs 1–4 and PAD6 in humans), converts the positively charged guanidinium group of an Arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.
