The recent approvals of anti-cancer therapeutics 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. Herein we demonstrate 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 with non-cancerous lines (NIH 3T3 and HL-60 granulocytes). We also demonstrate that these compounds induced the differentiation of HL-60 and HT29 cells. Finally, these compounds synergistically potentiate 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 represent candidate therapies for this disease.

Protein arginine deiminase activity (PAD) is increased in cancer, rheumatoid arthritis, and ulcerative colitis. Although the link between abnormal PAD activity and disease is clear, the relative contribution of the individual PADs to human disease is not known; there are 5 PAD isozymes in humans. Building on our previous development of F- and Cl-amidine as potent pan-PAD irreversible inhibitors, we describe herein a library approach that was used to identify PAD-selective inhibitors. Specifically, we describe the identification of Thr-Asp-F-amidine (TDFA) as a highly potent PAD4 inactivator that displays ≥15-fold selectivity for PAD4 versus PAD1 and ≥50-fold versus PADs 2 and 3. This compound is active in cells and can be used to inhibit PAD4 activity in cellulo. The structure of the PAD4•TDFA complex has also been solved and the structure and mutagenesis data indicate that the enhanced potency is due to interactions between the side chains of Q346, R374, and R639. Finally, we converted TDFA into a PAD4-selective ABPP and demonstrate that this compound, biotin-TDFA, can be used to selectively isolate purified PAD4 in vitro. In total, TDFA and biotin-TDFA represent PAD4-selective chemical probes that can be used to study the physiological roles of this enzyme.

Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator.

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 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 Nα-(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 probes of PAD function.

The Protein Arginine Deiminases (PAD), which catalyze the hydrolysis of peptidyl-arginine to form peptidyl-citrulline, play important roles in a variety of cell signaling pathways including apoptosis, differentiation, and transcriptional regulation. In addition to these important cellular roles, PAD activity is dysregulated in multiple human diseases (e.g., Rheumatoid Arthritis (RA), Cancer, and Colitis), and, significantly, PAD inhibition with Cl-amidine has been shown to reduce disease severity in the Collagen Induced Arthritis model of RA. Although these enzymes play important roles in human cell signaling and disease, the mechanisms that regulate PAD activity under both physiological and pathological conditions are poorly understood. One possible mechanism for regulating PAD activity is autodeimination, which we and others have shown that PAD4 is subject to in vitro and in vivo. Herein, we demonstrate that PAD4 autodeimination does not alter the activity, substrate specificity, or calcium dependence of this isozyme. However, the results of these studies indicate a novel role for autodeimination in modulating the ability of PAD4 to interact with Histone Deacetylase 1 (HDAC1), citrullinated histone H3 (Cit H3), and Protein Arginine Methyltransferase 1 (PRMT1).

BackgroundBlast-induced neurotrauma (BINT) is the signature life threatening injury of current military casualties. Neuroinflammation is a key pathological occurrence of secondary injury contributing to brain damage after blast injury. We have recently demonstrated that blast-triggered complement activation and cytokine release are associated with BINT. Here, we evaluated if administration of the complement inhibitor recombinant human decay-accelerating factor (rhDAF) is beneficial on neuroinflammation and neurodegeneration in a rat model of moderate BINT. Administration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP. These novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI.ResultAdministration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP.ConclusionThese novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI.

No abstract

The Protein Arginine Deiminases (PAD), which catalyze the hydrolysis of peptidyl-arginine to form peptidyl-citrulline, are potential targets for the development of a Rheumatoid Arthritis (RA) therapeutic, as well as other human diseases including colitis and cancer. Additionally, these enzymes, and in particular PAD4, appear to play important roles in a variety of cell signaling pathways including apoptosis, differentiation, and transcriptional regulation. To better understand the factors that regulate in vivo PAD4 activity, we set out to design and synthesize a series of activity-based protein profiling (ABPP) reagents that target this enzyme. Herein we describe the design, synthesis and evaluation of six ABPPs including (i) FITC conjugated F-amidine (FFA1 and 2) and Cl-amidine (FCA1 and 2), and (ii) biotin conjugated F-amidine (BFA) and Cl-amidine (BCA). We further demonstrate the utility of these probes for labeling PAD4 in cells, as well as for isolating PAD4 and PAD4 binding proteins. These probes will undoubtedly prove to be powerful tools that can be used to dissect the factors controlling the dynamics of PAD4 expression, activity and function.