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

Journal: Molecules

Article Title: Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

doi: 10.3390/molecules25051116

Figure Lengend Snippet: Figure 1. (a) Schematic illustration of ATP conversion pathways occurring in biological synovial fluid differentiated using PPi‐specific Kyoto Green; (b) Selectivity of Kyoto Green (1 M) towards various ions (2 M), hyaluronic acid (3 mg/mL), glucose (3 mg/mL), albumin (20 mg/mL), PPase (0.4 ng/L), apyrase (4 ng/L), and ENPP1 enzymes (1 ng/L) commonly found in biological synovial fluids. Measurement conditions: 50 mM HEPES buffer, 10 mM NaCl, 1 mM MgCl2 (pH 7.4, 25 °C, ex = 488 nm); (inset, top) Fluorescence emission change of Kyoto Green upon addition of PPi; (inset, bottom) chemical structure of Kyoto Green.

Article Snippet: Recombinant Human ENPP1 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Fluorescence

Journal: Molecules

Article Title: Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

doi: 10.3390/molecules25051116

Figure Lengend Snippet: Figure 2. (a) Conversion of ATP (5 M) to PPi catalyzed by ENPP1; (b) Degradation of PPi (1 M) to Pi catalyzed by inorganic PPase; (c) Hydrolysis of ATP (2 M) to AMP catalyzed by apyrase monitored real‐time in a 0.7‐mL cuvette by 1 M Kyoto Green. Measurement conditions: 50 mM HEPES buffer, 10 mM NaCl, 1 mM MgCl2 (pH 7.4, 25 °C, ex = 488 nm).

Article Snippet: Recombinant Human ENPP1 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Molecules

Article Title: Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

doi: 10.3390/molecules25051116

Figure Lengend Snippet: Figure 3. (a) Competitive catalytic activity of ENPP1 (0.734 ng/L) and apyrase (5.0, 7.5, 10.0 and 12.5 ng/L) toward 5 M ATP monitored in real time by 1 M Kyoto Green; (b) Catalytic activity of ENPP1 (1.1 ng/L) when 50 M of ATP was used as a substrate. PPase (0.03 ng/L) was added at 0 (blue square) and 1 h (red circle) after ENPP1 addition. Measurement conditions: 50 mM HEPES buffer, 10 mM NaCl, 1 mM MgCl2 (pH 7.4, 25 °C, ex = 488 nm).

Article Snippet: Recombinant Human ENPP1 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: Virtual depiction of CdnP and ENPP1 enzyme ribbon structures with inhibitors docked at the active site. (A) Low and (B) high magnification of inhibitor C-13 (green) docking to the active site of CdnP. (C) Low and (D) high magnification of inhibitor E-3 (blue) docking to the active site of ENPP1.

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques:

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: IC 50 values of 16 CdnP inhibitors that showed >30% CdnP inhibition

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques:

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: Structures of selected M.tb CdnP inhibitors identified in the in silico virtual screen. (A–D) Structures, NCI reference numbers, and CdnP IC 50 values of four selected lead compounds that inhibit M.tb CdnP. Inhibitor C-29 showed dual activity against both CdnP and ENPP1. (E) Compounds were tested for their inhibitory potential against purified CdnP protein. AMP, the end-product of the PDE reaction against c-di-AMP, was detected by measuring relative light units (RLU) using our luminescence-based, AMP detection assay.

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques: In Silico, Activity Assay, Purification, Detection Assay

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: IC 50 values of ENPP1 inhibitors that showed >30% ENPP1 inhibition

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques:

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: Structures of selected inhibitors of host ENPP-1 identified in the in silico virtual screen. (A–D) Structures, NCI reference numbers, and ENPP1 IC 50 values of four selected lead compounds that inhibit mammalian ENPP1.

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques: In Silico

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: Predicted molecular interactions of the four lead inhibitors with human ENPP1. Molecular docking of inhibitors E-3 (IC 50 26.4 µM or 10 µg/mL), E-27 (IC 50 16.3 µM or 5 µg/mL), E-37 (IC 50 44.6 µM or 10 µg/mL), and E-60 (IC 50 9.8 µM or 4 µg/mL) with the active site of ENPP1. Predicted hydrogen bonds are shown with purple arrows, while predicted salt bridges are shown with black arrows. Predicted pi-stacking interactions are shown by green lines, with hydrophobic surfaces of the inhibitors being shaded in gray. Predicted pi-cation interactions are shown with red lines. For the enzyme active site, acidic residues and surfaces are colored orange, basic residues are purple, hydrophobic residues are green, and hydrophilic residues and surfaces are blue.

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques:

Journal: Microbiology Spectrum

Article Title: Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

doi: 10.1128/spectrum.02012-23

Figure Lengend Snippet: Lead ENPP1 inhibitor, compound E-3, elicits enhanced cGAS-STING-IRF3 pathway activation and type I interferon release in macrophages. (A) Compound E-3 (NCI 14465) elicits enhanced IRF3 activation in RAW-Blue IRF3-SEAP reporter mouse macrophages at 24 h following 2′,3′-cGAMP transfection. Briefly, reporter macrophages were pre-treated with compound E-3 at concentrations varying from 0 to 165 mM (165 mM is 6.2× the ENPP1 IC 50 and 20.5 mM is 0.75× the IC 50 ) and were subsequently transfected with 1 nM 2′,3′-cGAMP using the X-tremeGENE9 transfection reagent. Culture supernatants were collected 24 h after transfection, and SEAP activity was measured by colorimetry in the presence of the QUANTI-Blue detection reagent. (B) Compound E-3 elicits elevated interferon-β (IFN-β) responses in human monocyte-derived macrophages (hMDMs) stimulated with 2′,3′-cGAMP. hMDMs were pre-treated with compound E-3 at 165 µM and subsequently transfected with 2′,3′-cGAMP (1 nM) using the X-tremeGENE9 transfection reagent. At 24 h post-transfection, culture supernatants were evaluated for IFN- β levels via ELISA. All data are presented as mean values ± S.E.M. ( n = 3 independent biological replicate experiments). Statistical analyses were done using a two-tailed Student’s t -test. P -values are shown for relevant comparisons.

Article Snippet: Human ENPP1 protein was purchased from Origene.

Techniques: Activation Assay, Transfection, Activity Assay, Colorimetric Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test