BB-Cl-Amidine

Inducement of ER Stress by PAD Inhibitor BB-Cl-Amidine to Effectively Kill AML Cells

Objective: Acute myeloid leukemia (AML) is a highly heterogeneous and recurrent hematological malignancy. Despite the emergence of novel chemotherapy drugs, AML patients' complete remission (CR) remains unsatisfactory. Consequently, it is imperative to discover new therapeutic targets or medications to treat AML. Such epigenetic changes like DNA methylation and histone modification play vital roles in AML. Peptidylarginine deminase (PAD) is a protein family of histone demethylases, among which the PAD2 and PAD4 expression have been demonstrated to be elevated in AML patients, thus suggesting a potential role of PADs in the development or maintenance of AML and the potential for the identification of novel therapeutic targets. Methods: AML cells were treated in vitro with the pan-PAD inhibitor BB-Cl-Amidine (BB-Cl-A). The AML cell lines were effectively induced into apoptosis by BB-Cl-A. However, the PAD4-specific inhibitor GSK484 did not. Results: PAD2 played a significant role in AML. Furthermore, we found that BB-Cl-A could activate the endoplasmic reticulum (ER) stress response, as evidenced by an increase in phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). As a result of the ER stress activation, the BB-Cl-A effectively induced apoptosis in the AML cells. Conclusion: Our findings indicated that PAD2 plays a role in ER homeostasis maintenance and apoptosis prevention. Therefore, targeting PAD2 with BB-Cl-A could represent a novel therapeutic strategy for treating AML.

BB-Cl-Amidine as a novel therapeutic for canine and feline mammary cancer via activation of the endoplasmic reticulum stress pathway

Background: Mammary cancer is highly prevalent in dogs and cats and results in a poor prognosis due to critically lacking viable treatment options. Recent human and mouse studies have suggested that inhibiting peptidyl arginine deiminase enzymes (PAD) may be a novel breast cancer therapy. Based on the similarities between human breast cancer and mammary cancer in dogs and cats, we hypothesized that PAD inhibitors would also be an effective treatment for mammary cancer in these animals.
Methods: Canine and feline mammary cancer cell lines were treated with BB-Cl-Amidine (BB-CLA) and evaluated for viability and tumorigenicity. Endoplasmic reticulum stress was tested by western blot, immunofluorescence, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Canine and feline mammary cancer xenograft models were created using NOD scid gamma (NSG) mice, and were treated with BB-CLA for two weeks.
Results: We found that BB-CLA reduced viability and tumorigenicity of canine and feline mammary cancer cell lines in vitro. Additionally, we demonstrated that BB-CLA activates the endoplasmic reticulum stress pathway in these cells by downregulating 78 kDa Glucose-regulated Protein (GRP78), a potential target in breast cancer for molecular therapy, and upregulating the downstream target gene DNA Damage Inducible Transcript 3 (DDIT3). Finally, we established a mouse xenograft model of both canine and feline mammary cancer in which we preliminarily tested the effects of BB-CLA in vivo.
Conclusion: We propose that our established mouse xenograft models will be useful for the study of mammary cancer in dogs and cats, and furthermore, that BB-CLA has potential as a novel therapeutic for mammary cancer in these species.

Neutrophil Extracellular Traps Augmented Alveolar Macrophage Pyroptosis via AIM2 Inflammasome Activation in LPS-Induced ALI/ARDS

Background: Uncontrollable inflammation is a critical feature of gram-negative bacterial pneumonia-induced acute respiratory distress syndrome (ARDS). Both neutrophils and alveolar macrophages participate in inflammation, but how their interaction augments inflammation and triggers ARDS is unclear. The authors hypothesize that neutrophil extracellular traps (NETs), which are formed during neutrophil NETosis, partly cause alveolar macrophage pyroptosis and worsen the severity of ARDS.
Methods: The authors first analysed whether NETs and caspase-1 are involved in clinical cases of ARDS. Then, the authors employed a lipopolysaccharide (LPS)-induced ARDS model to investigate whether targeting NETs or alveolar macrophages is protective. The AIM2 sensor can bind to DNA to promote AIM2 inflammasome activation, so the authors studied whether degradation of NET DNA or silencing of the AIM2 gene could protect alveolar macrophages from pyroptosis in vitro.
Results: Analysis of aspirate supernatants from ARDS patients showed that NET and caspase-1 levels were correlated with the severity of ARDS and that the levels of NETs and caspase-1 were higher in nonsurvivors than in survivors. In vivo, the NET level and proportion of pyroptotic alveolar macrophages in bronchoalveolar lavage fluid (BALF) were obviously higher in LPS-challenged mice than in control mice 24 h after injury. Administration of DNase I (a NET DNA-degrading agent) and BB-Cl-amidine (a NET formation inhibitor) alleviated alveolar macrophage pyroptosis, and Ac-YVAD-cmk (a pyroptosis inhibitor) attenuated NET levels in BALF and neutrophil infiltration in alveoli. All treatments markedly attenuated the severity of ARDS. Notably, LPS causes NETs to induce alveolar macrophage pyroptosis, and degradation of NET DNA or silencing of the AIM2 gene protected against alveolar macrophage pyroptosis.
Conclusion: These findings shed light on the proinflammatory role of NETs in mediating the neutrophil-alveolar macrophage interaction, which influences the progression of ARDS.

Protein Arginine Deiminases (PADs): Biochemistry and Chemical Biology of Protein Citrullination

Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Objectives: An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE.
Methods: NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice.
Results: Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease.
Conclusions: PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.