Direct Inhibition Of Enzymatic Activity Research Articles

New perspective on the regulation of acetylcholinesterase via the aryl hydrocarbon receptor.

Acetylcholinesterase (AChE, EC 3.1.1.7) plays important roles in cholinergic neurotransmission and has been widely recognized as a biomarker for monitoring pollution by organophosphate (OP) and carbamate pesticides. Dioxin is an emerging environmental AChE disruptor and is a typical persistent organic pollutant with multiple toxic effects on the nervous system. Growing evidence has shown that there is a significant link between dioxin exposure and neurodegenerative diseases and neurodevelopmental disorders, most of which involve AChE and cholinergic dysfunctions. Therefore, an in-depth understanding of the effects of dioxin on AChE and the related mechanisms of action might help to shed light on the molecular bases of dioxin impacts on the nervous system. In the past decade, the effects of dioxins on AChE have been revealed in cultured cells of different origins and in rodent animal models. Unlike OP and carbamate pesticides, dioxin-induced AChE disturbance is not due to direct inhibition of enzymatic activity; instead, dioxin causes alterations of AChE expression in certain models. As a widely accepted mechanism for most dioxin effects, the aryl hydrocarbon receptor (AhR)-dependent pathway has become a research focus in studies on the mechanism of action of dioxin-induced AChE dysregulation. In this mini-review, the effects of dioxin on AChE and the diverse roles of the AhR pathway in AChE regulation are summarized. Additionally, the involvement of AhR in AChE regulation during different neurodevelopmental processes is discussed. These AhR-related findings might also provide new insight into AChE regulation triggered by diverse xenobiotics capable of interacting with AhR.

Abstract 1476: A therapeutic antibody that inhibits CD73 activity by dual mechanisms

Abstract CD73 has a central role in dictating the adenosine concentration within the tumor as it is the final step in converting extracellular ATP to adenosine. Thus, substantial reduction of CD73 enzymatic activity has the potential to reduce immunosuppression of effector immune cells within the tumor. We present data describing an anti-human CD73 antibody that suppresses CD73 by two mechanisms: 1. direct inhibition of enzymatic activity upon binding to CD73 and 2. rapid, near-complete internalization of the enzyme. Durable reduction of cell-surface CD73 was observed in multiple tumor cell lines both in vitro and in vivo. The unique properties of this antibody are a result of the use of a human IgG2-IgG1 hybrid antibody with effector function eliminated by specific mutations of the Fc. The IgG2 sequence of this antibody drives superior internalization of CD73 and enhanced CD73 inhibition. Syngeneic tumor models demonstrate that CD73 contributes to resistance to anti-tumor therapy. Combination therapy with PD-1 blockade and a surrogate anti-mouse-CD73 antibody resulted in a better anti-tumor efficacy than either treatment alone. Finally, we demonstrate a novel technique for assessing CD73 enzymatic activity in situ that has potential for clinical application. These data support antibody-based anti-CD73 therapy in cancer and highlight a novel mechanism for inhibition of CD73 enzymatic activity. Citation Format: Bryan C. Barnhart, Emanuela Sega, Aaron Yamniuk, Sandra Hatcher, Ming Lei, Haben Ghermazien, Anne Lewin, Xi-Tao Wang, Haichun Huang, Pingping Zhang, Alan Korman. A therapeutic antibody that inhibits CD73 activity by dual mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1476.

Structure-activity relationship studies of QS11, a small molecule Wnt synergistic agonist.

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11.

CO-74 – Évaluation du dépistage du diabète chez les enfants atteints de mucoviscidose

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11’s Wnt synergy is also not clear. Here we present structure–activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11.

Gamma secretase modulators.

Notes The authors declare no competing financial interest. Title: Gamma Secretase Modulators Application Number: WO 2013066740A1 Publication date: 10 May 2013 Priority Application: 61/553,384 Priority date: 31 October 2011 Inventors: W. Greenlee, D. Pissarnitski, Z. Zhao, Z. Zhu Assignee Company: Merck Sharpe & Dohme Disease Area: Neurodegeneration Biological Target: Gamma secretase Summary: Gamma secretase is an aspartic protease complex involved in the biosynthesis of Aβ peptide, a potential neurotoxic contributor in the progression of Alzheimer’s disease. Direct inhibition of enzymatic activity introduces potential selectivity issues associated with other biological functions of gamma secretase including Notch and Eph processing. As an alternative approach to gamma secretase inhibition, modulation of enzyme activity using, e.g., NSAIDs is a potentially druggable concept. This patent describes the synthesis and evaluation of a novel chemical series that demonstrates inhibition of gamma secretase activity in vitro and reduction of Aβ42 CSF levels following oral administration of a standard dose of 30 mg/kg. Primary Markush:

Paradoxical Activation of Endothelial Nitric Oxide Synthase by NADPH Oxidase

Increased formation of reactive oxygen species (ROS) has been identified as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling endothelial nitric oxide synthase (eNOS). However, the specific contribution of ROS to endothelial function is not well understood. A major source of intracellular ROS is the NADPH oxidase (Nox) family of enzymes. The goal of the current study was to directly assess the contribution of NADPH oxidase derived superoxide to eNOS function by expressing Nox5, a single gene product that constitutively produces superoxide within cells. Paradoxically, we found that instead of inhibiting eNOS, coexpression of Nox5 increased NO release from both bovine and human endothelial cells. To establish the functional significance of this observation in intact blood vessels, the endothelium of mouse aorta was transduced with Nox5 or control adenoviruses. Nox5 potently inhibited Ach-induced relaxation and potentiated contractile responses to phenylephrine. In precontracted aortae, acute exposure to superoxide dismutase induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. These findings suggest that ROS inhibit eNOS function via consumption of NO rather than direct inhibition of enzymatic activity.

Dual mechanisms for telomerase inhibition in DLD‐1 human colorectal adenocarcinoma cells by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) have been reported to have antitumor activity. In this study, we have tested whether telomerase might be a target for the antitumor effect of fatty acids using DLD-1 colorectal adenocarcinoma cells. In a cell-free approach, fatty acids were added directly to cell lysates, and we confirmed that increasing fatty acid unsaturation correlates with increased inhibition of telomerase activity. Using a cell culture approach, DLD-1 cells were cultured with fatty acids. In a time and dose dependent manner, EPA and DHA suppressed cellular telomerase activity and the mRNAs encoding hTERT (human telomerase reverse transcriptase) and c-myc. Based on these observations, we suggest that PUFAs inhibit telomerase activity through dual mechanisms: direct inhibition of enzymatic activity and down regulation of hTERT, one of the telomerase components.

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.

Aspirin inhibits expression of the interleukin-1β-inducible group II phospholipase A 2

Nonsteroidal anti-inflammatory drugs (NSAIDs) clearly inhibit the synthesis and release of prostaglandins. However, these actions are not sufficient to explain all the anti-inflammatory effects of these drugs. Recently, it has been shown that aspirin and sodium salicylate inhibit the activation of the transcription factor NF-κB. Group II phospholipase A 2 (sPLA 2) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin-1β (IL-1β) and this induction is attenuated by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). We now report that aspirin inhibits the IL-1β-induced sPLA 2 activity in rat mesangial cells in a dose-dependent manner. The IC 50 value of aspirin for sPLA 2 inhibition was 6.5 mM. This decrease in sPLA 2 activity was not due to direct inhibition of enzymatic activity but rather to the fact that aspirin inhibits the expression of IL-1β-induced sPLA 2 protein and mRNA. Furthermore, by electrophoretic mobility shift analysis we demonstrate reduced DNA binding of the nuclear factor κB, an essential component of the IL-1β-dependent upregulation of sPLA 2 gene transcription, after treatment of the cells with aspirin. The study described in this report indicates that the inhibition of sPLA 2 expression as induced by pro-inflammatory cytokines potentially represents an additional mechanism of action for aspirin.

Nonsteroidal Anti-inflammatory Drugs Inhibit Expression of the Inducible Nitric Oxide Synthase Gene

Increased nitric oxide production is associated with acute and chronic inflammatory processes. Accordingly, we tested the hypothesis that the therapeutic action of nonsteroidal anti-inflammatory drugs could be attributed at least in part to inhibition of excess nitric oxide production. We report here that sodium salicylate, aspirin, ibuprofen, and indomethacin markedly inhibited the appearance of the inducible inflammatory nitric oxide synthase in rat alveolar macrophages activated with lipopolysaccharide and interferon gamma. We attribute the mechanism of nitric oxide synthase inhibition by nonsteroidal anti-inflammatory drugs to pretranslational control of enzyme expression and not to direct inhibition of enzymatic activity. These observations indicate that the chronic anti-inflammatory action of nonsteroidal anti-inflammatory drugs may be due not only to inhibition of prostaglandin synthesis but also to inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis.