Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: CD39L2 selectively releases the β-phosphate of the ADP generated from a kinase reaction. The released free phosphate is detected using phosphate-detection reagents. The rate of free phosphate production reflects the kinetics of the kinase reaction.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Generated

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: A ) pH profile of CD39L2 activity with ADP or ATP at room temperature. CD39L2 has optimal pH at 5.5 and is selectively active on ADP throughout the pH range. B ) First-order-rate constant of CD39L2 for ADP or ATP at pH 7.5. The specific rate constant of CD39L2 for ADP or ATP in the kinase assay buffer at room temperature was determined to be 41.0 or 0.7 nmol·min −1 mM −1 ·µg −1 (the slopes of the curves), respectively. D ) Relative CD39L2 activity at different salt concentrations.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Kinase Assay

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: Kinetic parameters of CD39L2.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques:

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: A ) Rate constants for CD39L2 in the presence of different concentrations of ATP. The reactions were performed with 0.12 µg of CD39L2 in 50 µL of the kinase assay buffer at room temperature. For clarity, only data at 0, 1.25 and 5 mM of ATP are shown. Slopes (s) of the curves represent the rate constants. Elevated phosphate levels in all reactions due to ATP hydrolysis were regarded as background and were subtracted out. B ) ATP inhibition factor ( i ), the ratio of a rate constant in the presence of ATP to the rate constant in the absence of ATP, is plotted versus ATP concentration.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Kinase Assay, Inhibition, Concentration Assay

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: A ) A Glucose curve. All reactions were initiated in the presence of 0.12 mM ATP, 0.2 µg GCK and 0.3 µg CD39L2 in 150 µL assay buffer at room temperature and proceeded for 15 minutes. The reaction that contained no glucose was set as a blank and the OD readings were plotted versus glucose concentration. The curve fit the Michaelis-Menten equation well with K m of 7.23±1.44 mM. B ) An ATP curve. Reactions were performed in the presence of 20 mM glucose, 1 µg GCK and 0.1 µg CD39L2 and variable concentrations of ATP in 50 µL assay buffer at room temperature and proceeded for 25 minutes. The reaction that contained no ATP was set as a blank. For each ATP concentration, a no-kinase negative control was performed for background correction. The OD readings of the reactions (purple) were first corrected with backgrounds subtraction (blue) and further corrected using ATP inhibition factors (red) and finally fit with the Michaelis-Menten equation to obtain a K m around 3.55 mM. C ) A GCK dose curve was performed with 10 mM ATP, 20 mM of glucose and 0.2 µg CD39L2 in 50 µL assay buffer at room temperature. All reactions were proceeded for 20 minutes and the OD was plotted versus GCK input. The reaction that contained no kinase was set as a blank. The slope of the curve, 6.84 OD/µg, corresponded to a specific activity of 2416 pmol/min/µg using Eq.10 ( k 2 = 8.2 nmol·min −1 ·mM −1 ; Vol = 50 µL; t = 25 min; i = 0.271; r = 0.396).

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Negative Control, Inhibition, Activity Assay

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: A ) An APS curve. All reactions were initiated with 0.12 mM ATP, 0.1 µg of APSK and 0.3 µg CD39L2 in 150 µL assay buffer at room temperature and proceeded for 10 minutes. The OD was plotted versus the APS concentration and the apparent K m (designated as K m ′) was visually estimated to be about 10 µM. B ) An ATP curve. All reactions were performed in the presence of 100 µM APS, 0.2 µg of APSK and 0.3 µg of CD39L2 in 150 µL assay buffer at room temperature and proceeded for 15 minutes. The obtained OD readings (purple) were first corrected by background subtraction (blue) and then corrected by the ATP inhibition factors (red) and finally fit with the Michaelis-Menten equation to obtain a K m of 126 µM. C ) An APSK enzyme curve. All reactions were performed with 1 mM ATP, 0.1 mM APS and 0.14 µg CD39L2 in 50 µL assay buffer at room temperature for 20 minutes. The OD was plotted versus APSK input. The slope of curve was converted to a specific activity, 1125 pmol/min/µg, using Eq.10 and r = 0.61.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Inhibition, Activity Assay

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: All reactions were initiated with 0.2 mM of ATP, 0.2 mM of myelin basic protein peptide and 0.2 µg of CD39L2 in 50 µL assay buffer and proceeded for 15 minutes. The OD was plotted versus ERK1 input. A reaction containing all components except kinase served as a blank. The slope of the curve (1.215 OD/µg) was converted to a specific activity, 482 pmol/min/µg, using Eq.10 and r = 0.597.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

doi: 10.1371/journal.pone.0023172

Figure Lengend Snippet: Blue triangles represent the background. Background reactions contained all components except the kinases. A ) Trials for GCK. Reactions were performed with 20 mM Glucose, 0.1 mM ATP, 0.64 µg GCK and 0.1 µg CD39L2 in 150 µL kinase assay buffer at room temperature for 20 minutes. B ) Trials for APSK. Reactions were performed with 0.0625 mM APS, 0.125 mM ATP, 1 µg APSK, 0.15 µg CD39L2 in 150 µL assay buffer at room temperature for 15 minutes. C ) Trials for ERK1. Reactions were performed with 0.05 mM MBP, 0.1 mM ATP, 0.2 µg ERK1, 0.1 µg CD39L2 in 150 µL assay buffer at room temperature for 20 minutes.

Article Snippet: Recombinant mouse CD39L2 (ENTPD6), human ERK1, and the Malachite Green Phosphate Detection Kit were from R&D Systems (Minneapolis, MN).

Techniques: Kinase Assay

Journal: Nature Chemical Biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Figure 3 | ENPP1 is the dominant hydrolase activity for 2′3′-cGAMP. (a) Knockdown of ENPP1 in MDA-MB231 cells diminished hydrolase activity. Four siRNA oligos against ENPP1 with a control siRNA sequence against PDE12 were used. Four days after siRNA transfection, cells were lysed and assayed for activity (upper panel) and blotted for ENPP1 level (lower level). (b) Hydrolase activity in plasma from Enpp1−/− mice and their littermates. (c) Western blot characterization of Enpp1−/− mice. (d) Hydrolase activity in livers and spleens from Enpp1−/− mice and their littermates. Livers and spleens were minced and Dounce homogenized in lysis buffer containing 1% NP-40, 20 mM Tris-HCl, pH 7.5, and protease inhibitor cocktail. The assay was conducted in 0.2% NP-40, 20 mM Tris-HCl, 150 mM KCl, 2 mM Ca2+, 2 mM Mg2+ and 200 μM Zn2+ at the indicated pH. NaOAc buffer was used for pH 5.0–6.0; PIPES buffer was used for pH 6.5 and 7.0; Tris-HCl buffer was used for pH 7.5–9.0, and borate buffer was used for pH 9.5. These buffer conditions were also used in liver and spleen extract studies. 2′3′-cGAMP runs at a lower Rf in PIPES buffer.

Article Snippet: Recombinant ENPP1 was purchased from R&D Systems (6136-EN-010).

Techniques: Activity Assay, Knockdown, Control, Sequencing, Transfection, Clinical Proteomics, Western Blot, Lysis, Protease Inhibitor

Journal: Nature Chemical Biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Figure 2 | ENPP1 is an efficient hydrolase for 2′3′-cGAMP. (a,b) Ion dependency (a) and pH preference (b) of the dominant hydrolase activity in MDA-MB231 cells. (c,d) Activity of recombinant ENPP1 alone (c) or coupled to alkaline phosphatase (d) in buffer containing 0.2% (v/v) NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca2+, 200 μM Zn2+. (e) Kinetics of 2′3′- cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as c and d. Data are presented as mean ± s.e.

Article Snippet: Recombinant ENPP1 was purchased from R&D Systems (6136-EN-010).

Techniques: Activity Assay, Recombinant

Journal: Nature Chemical Biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Figure 4 | Development of hydrolysis-resistant hSTING agonists. (a) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 ml buffer containing 20 mM Tris-HCl, pH 8.0, and 20 mM MgCl2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1–10 μM mouse cGAS (residues 147–507) was incubated with 1 mM ATP, 1 mM GTP and 0.1 mg ml−1 DNA from herring testes (HT DNA) in 1 ml of the same buffer for 12 h at room temperature. (b) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM) with or without the presence of alkaline phosphatase (AP). (c) SPA to measure the binding affinity of the analogs toward hSTING (residues 139–379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin-coated SPA plates. Neat 35S-labeled 2′3′-cGAsMP (500 pM) was used as the probe. Data are presented as mean ± s.e.; n = 3. (d) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the medium was measured using a HEK-SEAP cell line. Representative data from biological triplicates are depicted. AU, arbitrary units.

Article Snippet: Recombinant ENPP1 was purchased from R&D Systems (6136-EN-010).

Techniques: Incubation, Binding Assay, Labeling

Journal: Nature Chemical Biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Figure 5 | ENPP1 dampens 2′3′-cGAMP signaling. Lung fibroblast cells from wild-type and Enpp1−/− female mice were incubated with 2′3′-cGAMP analogs (a−c) and HSV-60 (d) at the indicated concentrations for 24 h. IFN-β in the medium was measured using a B16-SEAP cell line. Data are presented as mean ± s.e.; n = 3. AU, arbitrary units.

Article Snippet: Recombinant ENPP1 was purchased from R&D Systems (6136-EN-010).

Techniques: Incubation

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Scientific reports

Article Title: A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression.

doi: 10.1038/s41598-024-80677-8

Figure Lengend Snippet: Fig. 1. Colorectal cancer cells exhibit low or absent Enpp1 expression and require Enpp1 transfection to degrade cGAMP. (a) RNA expression of (i) Enpp1, (ii) cGas, and (iii) Sting1 in a panel of breast, colorectal, and lung adenocarcinoma cell lines present in DepMap portal. Each symbol represents one cell line, with expression as Log2 transcripts per million. (b) Analysis of scRNASeq of a panel of murine tumors highlighting Enpp1 and Sting1 expression in (i) Cancer cells and (ii) stromal cells in MC38 tumors. Scale shows degree of expression by color and the circle size shows the percentage of each cell type that expresses the gene. (c) Analysis of scRNASeq of CD45 + cells in MC38 tumors gated on myeloid cells. (i) TSNE plot showing 6 clustered myeloid populations from MC38 tumors. (ii) Enpp1 expression (red) in clustered populations. Iii) identification of clusters based on key gene expression. (d) (i) Flow cytometry for Enpp1 versus isotype control in murine CT26 and MC38 colorectal carcinoma cell lines, and bone marrow-derived macrophages (BMM0). (ii) Summary of MFI of (i) across replicates. (iii) Recovery of spiked cGAMP from MC38 cells (NT), MC38 cells stably transfected with human Enpp1 (huEnpp1), human Enpp1 T256A mutant (*huEnpp1), or murine Enpp1 (muEnpp1). Key: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Scientific Reports | (2024) 14:29913 12| https://doi.org/10.1038/s41598-024-80677-8 VIR3 Enpp1 inhibitor characterization Enzymatic inhibition assays were performed by incubating 1.5 nM ENPP1 (R&D Systems cat# 6136-EN-010) with serial dilutions of Vir-3 for 15 minutes at 37oC in buffer (50mM Tris pH 8.0, 250mM NaCl, 0.5mM CaCl2, 1uM ZnCl2, 1% DMSO) before adding 20 uM 2’3’-cGAMP (Invivogen cat.# tlrl-nacga23).

Techniques: Expressing, Transfection, RNA Expression, Gene Expression, Flow Cytometry, Control, Derivative Assay, Stable Transfection, Mutagenesis

Journal: Scientific reports

Article Title: A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression.

doi: 10.1038/s41598-024-80677-8

Figure Lengend Snippet: Fig. 3. Enpp1 knockout results in increased responses to cGAMP and improved responses to radiation treatment. (a) (i) Bone marrow macrophages (BMM0) were generated from wt, Enpp1+/- or Enpp1-/- mice and cell lysates were western blotted for Enpp1 and GAPDH protein expression. Original blots are presented in Supplementary Figure 6. (ii) wt or Enpp1-/- BMM0 were left untreated or treated with cGAMP and type I IFN secretion was determined by bead assay. (b) (i) MC38 cells were injected into wt or Enpp1-/- mice and tumors were allowed to develop for 10-14 days. Mice were randomized to receive no further treatment or 12Gy focal RT to the tumor. (ii) Mice were followed for survival. Key: NS = not significant * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Scientific Reports | (2024) 14:29913 12| https://doi.org/10.1038/s41598-024-80677-8 VIR3 Enpp1 inhibitor characterization Enzymatic inhibition assays were performed by incubating 1.5 nM ENPP1 (R&D Systems cat# 6136-EN-010) with serial dilutions of Vir-3 for 15 minutes at 37oC in buffer (50mM Tris pH 8.0, 250mM NaCl, 0.5mM CaCl2, 1uM ZnCl2, 1% DMSO) before adding 20 uM 2’3’-cGAMP (Invivogen cat.# tlrl-nacga23).

Techniques: Knock-Out, Generated, Western Blot, Expressing, Injection

Journal: Scientific reports

Article Title: A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression.

doi: 10.1038/s41598-024-80677-8

Figure Lengend Snippet: Fig. 4. Development of VIR3 Enpp1 Inhibitor(a) Structure of VIR3. (b) Representative data from biochemical assay monitoring recombinant ENPP1 cGAMP hydrolysis activity and inhibition by VIR3. (c) Representative data from cellular assay monitoring cGAMP hydrolysis on HepG2 cells in the presence of VIR3. (d) In vivo target engagement was measured by administering 5ug of 2’3’-cGAMP IV to mice and then collecting blood samples 3 minutes later into a tube containing ENPP1 inhibitors. The level of cGAMP remaining the plasma at the time of collection was measured by ELISA both before VIR3 oral administration (open circles) or after (closed circles) (n = 3-5 mice per group, data is plotted as mean +/-SEM).

Article Snippet: Scientific Reports | (2024) 14:29913 12| https://doi.org/10.1038/s41598-024-80677-8 VIR3 Enpp1 inhibitor characterization Enzymatic inhibition assays were performed by incubating 1.5 nM ENPP1 (R&D Systems cat# 6136-EN-010) with serial dilutions of Vir-3 for 15 minutes at 37oC in buffer (50mM Tris pH 8.0, 250mM NaCl, 0.5mM CaCl2, 1uM ZnCl2, 1% DMSO) before adding 20 uM 2’3’-cGAMP (Invivogen cat.# tlrl-nacga23).

Techniques: Recombinant, Activity Assay, Inhibition, In Vivo, Drug discovery, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression.

doi: 10.1038/s41598-024-80677-8

Figure Lengend Snippet: Fig. 5. Treatment with VIR3 Enpp1 inhibitor improves tumor control by RT. (a) (i) MC38 cells were injected into wt mice and tumors were allowed to develop for 14 days. Mice were randomized to receive 21 daily doses of VIR3 or vehicle by oral gavage starting on d13, and further randomized to no further treatment or 12 Gy focal RT to the tumor on d14. (ii) Mice were followed for survival. (b) (i) Treatment as per a) but tumors were CT26 tumors injected into BALB/c mice (ii) Mice were followed for survival. Key: NS = not significant * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Scientific Reports | (2024) 14:29913 12| https://doi.org/10.1038/s41598-024-80677-8 VIR3 Enpp1 inhibitor characterization Enzymatic inhibition assays were performed by incubating 1.5 nM ENPP1 (R&D Systems cat# 6136-EN-010) with serial dilutions of Vir-3 for 15 minutes at 37oC in buffer (50mM Tris pH 8.0, 250mM NaCl, 0.5mM CaCl2, 1uM ZnCl2, 1% DMSO) before adding 20 uM 2’3’-cGAMP (Invivogen cat.# tlrl-nacga23).

Techniques: Control, Injection