Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors

doi: 10.1080/14756366.2022.2119566

Figure Lengend Snippet: (a) Representative ENPP1 inhibitors. (b) Design of novel ENPP1 inhibitors possessing pyrrolopyrimidines and pyrrolopyridines core scaffolds.

Article Snippet: Serially diluted ENPP1 inhibitors (usually range from 10 μM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5–10 min at room temperature (RT).

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors

doi: 10.1080/14756366.2022.2119566

Figure Lengend Snippet: Enzymatic inhibitory activities of 4-piperidine substituted sulfamides 18 and sulfone amides 20 against ENPP1.

Article Snippet: Serially diluted ENPP1 inhibitors (usually range from 10 μM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5–10 min at room temperature (RT).

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors

doi: 10.1080/14756366.2022.2119566

Figure Lengend Snippet: Enzymatic inhibitory activities of sulfamides 25 against ENPP1.

Article Snippet: Serially diluted ENPP1 inhibitors (usually range from 10 μM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5–10 min at room temperature (RT).

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors

doi: 10.1080/14756366.2022.2119566

Figure Lengend Snippet: (a) Docking model of 18p bound to ENPP1. 18p (light gold) is represented in stick model and other protein domains are briefly shown in cartoon model. Two zinc (gray) and their coordinating Histidines (cyan) are shown. Several key residues interacting with 18p are shown in blue. (b) The ligand (purple) and protein (brown) were represented in ball (C: black; N: blue; O: red; Zn: cyan) and stick (bond) model, green dash shows hydrogen bonding or metal coordination. Hydrophobic intermolecular interactions are described by half whisker shapes with its amino acid residues.

Article Snippet: Serially diluted ENPP1 inhibitors (usually range from 10 μM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5–10 min at room temperature (RT).

Techniques: Whisker 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

Journal: iScience

Article Title: CD38 restrains the activity of extracellular cGAMP in a model of multiple myeloma

doi: 10.1016/j.isci.2024.109814

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for flow cytometry: anti-CD138/FITC (clone MI15, BD Biosciences); anti-CD38/APC (clone HIT2, BD Biosciences); anti-ENPP1/APC (polyclonal sheep IgG, R&D), polyclonal sheep IgG/APC (R&D).

Techniques: Virus, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Labeling, Sequencing, Plasmid Preparation, Software, Staining

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Chemical structures of cGAMP (top) and single isotopically-labeled cGAMP (bottom) used as an internal standard at a concentration of 0.5-1 μM. b-c , Full (7-8 point) standard curves and LC traces of lowest cGAMP standards in 50/50 acetonitrile/water spiked in directly (LOQ = 4 nM) ( b ) or after concentrating and extracting 12.5x from complete cell culture media (LOQ = 0.3 nM from original sample, 4 nM in concentrated sample) ( c ). IS = internal standard. Data are representative of > 10 independent experiments. d , Calibration of cell number to ATP concentration measured by LC-MS/MS. Mean ± SEM ( n = 2). e , cGAS expression of 293T, 293T cGAS ENPP1 -/- , and 293T cGAS ENPP1 low cell lines analyzed by western blot (left). ENPP1 hydrolysis activity of 32 P-cGAMP in whole cell lysates from 1 million each of 293T cGAS, 293T cGAS ENPP1 -/- , and 293T cGAS ENPP1 low cells, measured by TLC and autoradiography (right). Lysate data are representative of two independent experiments. f , Intracellular concentrations of cGAMP from 293T WT and 293T cGAS ENPP1 -/- cells without exogenous stimulation at steady state measured using LC-MS/MS. Mean ± SEM ( n = 2). BQL = below quantification limit. g , Expression of cGAS in 293T WT, 293T stably expressing mouse cGAS, and 293T stably expressing human cGAS assessed by western blot. h , Intracellular and extracellular concentrations of cGAMP from 293T stably expressing mouse cGAS and 293T stably expressing human cGAS. Cells were mock transfected with only lipid or transfected with lipid + 0.5 µg/mL empty vector pcDNA6. After 24 hours, cells were refreshed with serum-free media and incubated for another 24 hours before analysis. Mean ± SEM ( n = 2).

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Labeling, Concentration Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Activity Assay, Autoradiography, Stable Transfection, Transfection, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a-b , Intracellular and extracellular concentrations of cGAMP from 293T cGAS ENPP1 -/- cells without exogenous stimulation measured using LC-MS/MS. At time 0, cells were replenished with serum-free media. Mean ± SEM ( n = 2) with some error bars too small to visualize. Data are representative of three independent experiments. c , The amount of cGAMP exported per cell over time calculated from data in ( b ). The export rate was calculated using linear regression. d , The fraction of extracellular/total cGAMP molecules (left y-axis) calculated from data in ( a ) and ( b ) compared to the fraction of extracellular/total lactate dehydrogenate (LDH) activity as a proxy for cell death (right y-axis). e , Potential mechanisms of cGAMP export, including release in extracellular vesicles, exocytosis, ATP-driven transporter, and electrochemical gradient-driven transporter (facilitated diffusion transporter or anti/symporter). f , Intracellular and extracellular cGAMP concentrations produced by 293T cGAS ENPP1 -/- cells measured before and after passing the media through 10 kDa filters. Mean ± SEM ( n = 2). Data are representative of two independent experiments. g , Schematic of the conditioned media transfer experiment for ( h ). 293T cGAS ENPP1 low cells were transfected with 0.5 μg/mL empty pcDNA6 vector and treated ± 20 nM recombinant mouse ENPP1 (mENPP1). Conditioned media from these cells were transferred to primary CD14 + human PBMCs. h , IFNB1 mRNA levels were normalized to CD14 and the fold induction was calculated relative to untreated CD14 + cells. Mean ± SEM ( n = 2). * P = 0.04 (one-way ANOVA). cGAMP concentrations were measured in the conditioned media by LC-MS/MS. Mean ± SEM ( n = 2). ** P = 0.002 (one-way ANOVA). Data are representative of two independent experiments. i , 293T cGAS ENPP1 low cells were incubated with serum-free ATP depletion media (no glucose, 6 mM 2-deoxy-D-glucose, 5 mM NaN 3 ) or serum-free complete media for 1 hour. cGAMP and ATP concentrations were measured by LC-MS/MS. Mean ± SEM ( n = 2-3). *** P < 0.001 (Student’s t test). j-k , The amount of total ( j ), intracellular (exponential fit), and extracellular (polynomial fit) ( k ) cGAMP over time produced by 293T cGAS ENPP1 -/- cells after transfection with empty plasmid pcDNA6 (1.5 μg/mL). Data are representative of two independent experiments. l , cGAMP export rates plotted against intracellular concentration of cGAMP after transfection with empty plasmid pcDNA6 (1.5 μg/mL), calculated from data in ( j ) and ( k ).

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Liquid Chromatography with Mass Spectroscopy, Activity Assay, Diffusion-based Assay, Produced, Transfection, Plasmid Preparation, Recombinant, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Schematic of experiment for ( b ). Human CD14 + PMBCs stimulated with increasing concentrations of extracellular cGAMP for 16 h. b , IFNB1 mRNA levels were normalized to indicated gene and fold induction was calculated relative to untreated CD14 + cells. Mean ± SEM ( n = 2 technical qPCR replicates). c , Schematic of the conditioned media transfer experiment for ( d ). cGAS-null 293T or 293T cGAS ENPP1 low cells were transfected with 0.5 μg/mL empty pcDNA6 vector. Conditioned media from these cells was transferred to primary CD14 + human PBMCs and IFNB1 expression was assessed after 16 h. d , IFNB1 mRNA levels were normalized to CD14 and the fold induction was calculated relative to untreated CD14 + cells. Mean ± SEM ( n = 4). *** P = 0.0003 (one-way ANOVA). cGAMP concentrations were measured in the conditioned media by LC-MS/MS. Mean ± SEM ( n = 2). *** P = 0.0002 (one-way ANOVA). Data are representative of two independent experiments. e , Coomassie gel of recombinant mouse ENPP1 purified from media; elution fractions were pooled before use (left). 32 P-cGAMP degradation by mouse ENPP1 analyzed by TLC (right). f , 293T cGAS ENPP1 low cells were incubated with serum-free ATP depletion media (no glucose, 6 mM 2-deoxy-D-glucose, 5 mM NaN 3 ) or serum-free complete media for 1 hour. Total protein content was measured by BCA and cell death was measured by lactate dehydrogenase activity. Mean ± SEM ( n = 2-3). BQL = below quantitation limit. g , Data from fit with the allosteric sigmoidal model v export = V max [ substrate ] n / ( K half ) n + [ substrate ] n , where V max = the maximal transporter velocity, n = the Hill slope, and K half = the substrate concentration at half V max . Here, we have constrained V max < 5000 molecules cell -1 s -1 , and the resulting fit shows that K m > 60 μM.

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Transfection, Plasmid Preparation, Expressing, Liquid Chromatography with Mass Spectroscopy, Recombinant, Purification, Incubation, Activity Assay, Quantitation Assay, Concentration Assay

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Three possible cellular locations of ENPP1 activity. b , 293T cGAS ENPP1 -/- cells were transfected with empty pcDNA6 vector or vector containing human ENPP1 . Whole cell lysates were analyzed after 24 h for ENPP1 protein expression using western blot (top) and for ENPP1 32 P-cGAMP hydrolysis activity at pH 9.0 using thin layer chromatography (TLC) (bottom). Data are representative of two independent experiments. c , Intracellular and extracellular cGAMP concentrations measured using LC-MS/MS. BQL = below quantification limit. Mean ± SEM ( n = 2). ** P = 0.002 (Student’s t test). Data are representative of three independent experiments. d , Chemical structure of ENPP1 inhibitor STF-1084. e , Inhibitory activity of STF-1084 in vitro against purified mouse ENPP1 with 32 P-cGAMP as the substrate at pH 7.5 ( K i,app = 110 ± 10 nM) and in cells against human ENPP1 transiently expressed in 293T cGAS ENPP1 -/- cells (IC 50 = 340 ± 160 nM). Extracellular cGAMP levels were analyzed by LC-MS/MS after 24 hours. Mean ± SEM ( n = 3 independent experiments for in vitro assay, n = 2 for in cells assay), with some error bars too small to visualize. f , Intracellular and extracellular cGAMP concentrations for 293T cGAS ENPP1 -/- cells transfected with empty pcDNA6 vector or vector containing human ENPP1 in the presence or absence of 10 μM STF-1084 after 24 hours. cGAMP levels were analyzed by LC-MS/MS. BQL = below quantification limit. Mean ± SEM ( n = 3). **** P < 0.0001 (one-way ANOVA). Data are representative of two independent experiments. g , Schematic of the conditioned media experiment for ( h) . 293T cGAS ENPP1 low cells were transfected with vector containing human ENPP1 and incubated in the presence or absence of STF-1084. Conditioned media from these cells was transferred to primary CD14 + human PBMCs. h , IFNB1 mRNA levels were normalized to CD14 and the fold induction was calculated relative to untreated CD14 + cells. Mean ± SEM ( n = 2). ** P = 0.007 (one-way ANOVA). cGAMP concentrations were measured in the conditioned media by LC-MS/MS. Mean ± SEM ( n = 2). ** P = 0.006 (one-way ANOVA). Data are representative of two independent experiments.

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Thin Layer Chromatography, Liquid Chromatography with Mass Spectroscopy, In Vitro, Purification, Incubation

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Structure of QS1. b , QS1 inhibitory activity (compared to STF-1084) against purified mouse ENPP1 with 32 P-cGAMP as the substrate at pH 7.5 (QS1 K i,app = 6.4 ± 3.2 μM). Mean ± SEM ( n = 2 independent experiments). c , Intracellular, extracellular, and total cGAMP for 293T cGAS ENPP1 -/- cells transfected with empty vector or vector containing human ENPP1 in the presence or absence of QS1. cGAMP levels were measured after 24 hours by LC-MS/MS. Mean ± SEM ( n = 2). * P < 0.05. ** P < 0.01 (one-way ANOVA).

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Activity Assay, Purification, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Experimental setup to assess the role of extracellular cGAMP in vivo. b , Coomassie gel of the cytosolic domain of recombinant mouse WT and R237A STING. c , Binding curves for the cytosolic domain of mouse WT STING (neutralizing) and R237A STING (non-binding) determined by a membrane binding assay using radiolabeled 35 S-cGAMP as the probe. Mean ± SEM ( n = 2 from two independent experiments). d , Crystal structure of mouse WT STING in complex with cGAMP with R237 highlighted in pink (PDB ID 4LOJ). e , IFNB1 mRNA fold induction in CD14 + PBMCs treated with 2 μM cGAMP in the presence of neutralizing or non-binding STING (2 μM to 100 μM, 2.5-fold dilutions). Mean ± SEM ( n = 2 technical qPCR replicates). f , WT or Cgas -/- E0771 cells (1×10 6 ) were orthotopically injected into WT, Cgas -/- , or Sting -/- C57BL/6J mice on day 0. Neutralizing STING or non-binding STING was intratumorally injected on day 2. Tumors were harvested and analyzed by FACS on day 3. Samples were gated on cells in FSC-A/SSC-A, singlets (FSC-W), living cells, CD45 + , MHC II + (APCs), CD11c + populations. (Non-binding STING: WT mice n = 5; Cgas -/- mice n = 4; Cgas -/- cells n = 5; Sting -/- mice n = 5. Neutralizing STING: WT mice n = 5; Cgas -/- mice n = 5; Cgas -/- cells n = 5; Sting -/- mice n = 4). Mean ± SD. ** P < 0.01. (Welch’s t test). g , WT or Cgas -/- 4T1-luc cells (1×10 6 ) were orthotopically injected into WT BALB/cJ mice, and the same procedure was performed as in ( f ). Percent CD11c + cells of total MHC II + (APCs). (Non-binding STING: WT cells n = 2; Cgas -/- cells n = 5; neutralizing STING: WT cells n = 3; Cgas -/- cells n = 5). * P < 0.05. (Welch’s t test). h , 4T1-luc cells (1×10 6 ) were orthotopically injected into WT BALB/cJ mice on day 0. PBS ( n = 5) or recombinant mouse ENPP1 (mENPP1) ( n = 6) was intratumorally injected on day 2. Tumors were harvested and analyzed by FACS on day 3. Mean ± SD. * P < 0.05. (Welch’s t test). i , E0771 cells (5×10 4 ) were orthotopically injected into WT ( n = 10) or Enpp1 -/- ( n = 6) C57BL/6J mice. Tumor volume and survival were monitored. ** P < 0.01. P value for tumor volume determined by pairwise comparisons using post hoc tests with a Tukey adjustment and for Kaplan Meier curve determined using the Log-rank Mantel-Cox test. j , E0771 cells (5×10 4 ) were orthotopically injected into WT ( n = 10) or Sting - /- ( n = 9) C57BL/6J mice. The tumors were treated with IR (8 Gy) when they reached 100 ± 20 mm 3 . Tumor volume and survival were monitored without further treatment. k , E0771 cells (5×10 4 ) were orthotopically injected into WT C57BL/6J mice. The tumors were treated with IR (8 Gy) when they reached 100 ± 20 mm 3 and injected with non-binding ( n = 8) or neutralizing STING ( n = 10) every other day for the duration of the experiment. Tumor volume and survival were monitored. l , E0771 cells (5×10 4 ) were orthotopically injected into WT C57BL/6J mice. The tumors were treated with IR (8 Gy) when they reached 100 ± 20 mm 3 and injected with non-binding ( n = 5) or neutralizing STING ( n = 5) on days 2 and 4 after IR. Tumors were harvested and analyzed by FACS on day 5. Mean ± SD. * P = < 0.05. (Welch’s t test).

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: In Vivo, Recombinant, Binding Assay, Membrane, Injection

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , ENPP1 activity in 4T1-luc, E0771, and MDA-MB231 cells using the 32 P-cGAMP degradation assay. Data are representative of three independent experiments. b , CXCL10 production by cancer cell lines 4T1-luc and E0771 measured by ELISA. At time 0, cells were left untreated or treated with IR (8 Gy or 20 Gy) and refreshed with media supplemented with 50 μM STF-1084. Media was collected and cells were counted at indicated time points. Mean ± SEM ( n = 2). c , FACS gating scheme for live dead analysis in established tumors. E0771 cells (5×10 4 ) were orthotopically injected into WT C57BL/6J mice. The tumors were treated with IR (8 Gy) when they reached 100 ± 20 mm 3 and harvested and analyzed by FACS 24h after IR. For caspase activity, a single cell suspension was incubated for 1h with the FAM-FLICA Poly Caspase substrate before FACS stain and analysis. Mean ± SD. d , FACS gating scheme for experiments in , . e , E0771 cells (5×10 4 ) were orthotopically injected into WT C57BL/6J mice. The tumors were treated with IR (8 Gy) when they reached 100 ± 20 mm 3 and injected with non-binding ( n = 5) or neutralizing STING ( n = 5) on days 2 and 4 after IR. Tumors were harvested and analyzed by FACS on day 5. Mean ± SD.

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Activity Assay, Degradation Assay, Enzyme-linked Immunosorbent Assay, Injection, Suspension, Incubation, Staining, Binding Assay

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Validating Enpp1 -/- 4T1-luc clones (11 clones were pooled) using the 32 P-cGAMP degradation assay. Lysates were normalized by protein concentrations. b , Established 4T1-luc WT (harboring scrambled sgRNA) ( n = 10) or Enpp1 -/- tumors ( n = 10) (100 ± 20 mm 3 ) were monitored without treatment. Tumor volumes are shown. c , Established 4T1-luc WT (harboring scrambled sgRNA) ( n = 10) or Enpp1 -/- tumors ( n = 10) (100 ± 20 mm 3 ) were treated with IR (20 Gy) and monitored. Tumor volumes are shown. d , Established 4T1-luc WT (harboring scrambled sgRNA) ( n = 9) or Enpp1 -/- tumors ( n = 9) (100 ± 20 mm 3 ) were treated with three intratumoral injections of 10 µg cGAMP on day 2, 4, and 7 and monitored. Tumor volumes are shown. e , ENPP1 expression from RNA sequencing data from data generated by the TCGA Research Network: https://www.cancer.gov/tcga .

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Clone Assay, Degradation Assay, Expressing, RNA Sequencing, Generated

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , 4T1-luc WT or Enpp1 -/- cells (1×10 6 ) were orthotopically injected into WT BALB/cJ mice on day 0. Tumors were left untreated ( n = 5) or treated with IR (20 Gy) ( n = 5) on day 2. Tumors were harvested and analyzed by FACS on day 3. * P < 0.05. (Welch’s t test). b , 4T1-luc cells (1×10 6 ) were orthotopically injected into WT BALB/cJ mice on day 0. Tumors were treated with 20 Gy IR and intratumorally injected with PBS ( n = 4) or STF-1084 ( n = 5) on day 2. Tumors were harvested and analyzed by FACS on day 3. * P < 0.05 (Welch’s t test). c , Established 4T1-luc WT (harboring scrambled sgRNA) or Enpp1 -/- cells (100 ± 20 mm 3 ) were treated once with IR (20 Gy) followed by three intratumoral injections of 10 µg cGAMP on day 2, 4, and 7 after IR ( n = 10 for scrambled 4T1-luc, n = 11 for Enpp1 -/- 4T1-luc). Tumor volumes are shown. * P < 0.05. P value determined by pairwise comparisons using post hoc tests with a Tukey adjustment at day 20. In the Enpp1 -/- 4T1-luc + IR (20 Gy) + 10 µg cGAMP treatment group, 3/11 (27%) mice are tumor free verified by bioluminescent imaging (tumor area outline in red). d , Established 4T1-Luc tumors (100 ± 20 mm 3 ) were treated once with IR (20 Gy) followed by three intratumoral injections of 10 µg cGAMP alone or 10 µg cGAMP + 100 µL of 1 mM STF-1084 on day 2, 4, and 7 after IR ( n = 9 per treatment group). Tumor volumes are shown. * P < 0.05. P value determined by pairwise comparisons using post hoc tests with a Tukey adjustment at day 40. e , Chemical structure of ENPP1 inhibitor STF-1623. f , Mice bearing established subcutaneous Panc02 tumors (100 ± 20 mm 3 ) were implanted with a subcutaneous pump containing STF-1623 (50 mg/kg/day) on day 0 and left untreated or treated with IR (20 Gy) on day 1. No IR: n = 10, no IR + STF-1623: n = 10, IR (20 Gy): n = 10, IR (20 Gy) + STF-1623: n = 15. Pumps were removed on day 8. Tumor volumes are shown. P value determined by pairwise comparisons using post hoc tests with a Tukey adjustment. * P < 0.05. *** P < 0.001.

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Injection, Imaging

Journal: bioRxiv

Article Title: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

doi: 10.1101/539312

Figure Lengend Snippet: a , Inhibitory activity of STF-1084 in vitro against purified mouse ENPP1 with 32 P-cGAMP as the substrate at pH 7.5 ( K i,app = 110 ± 10 nM) and in cells against human ENPP1 transiently expressed in 293T cGAS ENPP1 -/- cells (IC 50 = 340 ± 160 nM). Extracellular cGAMP levels were analyzed by LC-MS/MS after 24 hours. Mean ± SEM ( n = 3 independent experiments for in vitro assay, n = 2 for in cells assay), with some error bars too small to visualize. b , Permeability of STF-1623 in intestinal cells Caco-2 assay. PA = peak area, IS = internal standard. Compounds, including STF-1623, atenolol (low passive permeability negative control) and propranolol (high passive permeability positive control), were incubated on the apical side of a Caco-2 monolayer for 2 hours. Compound concentration on the basolateral side was monitored by LC-MS/MS. Apparent permeability rates (P app ) were calculated from the slope. Data are representative of two independent experiments. c , Kinome interaction map (468 kinases tested) for STF-1623 depicting kinase inhibition as a percent of control. d , Cell viability measured by CellTiterGlo. Total PBMCs were incubated with STF-1623 for 16 hours and then assayed for ATP levels using CellTiterGlo. Data was normalized to no STF-1623 to calculate % cell viability. Mean ± SEM ( n = 2) with some error bars too small to visualize. e , Intracellular and extracellular cGAMP concentrations for 293T cGAS ENPP1 -/- cells transfected with empty pcDNA6 vector or vector containing human ENPP1 in the presence or absence of 2 μM STF-1623 after 24 hours. cGAMP levels were analyzed by LC-MS/MS. BQL = below quantification limit. Mean ± SEM ( n = 3). ** P < 0.01. **** P < 0.0001 (one-way ANOVA). Data are representative of two independent experiments. f , Human PBMCs were mock electroporated or electroporated with 200 nM cGAMP and immediately incubated with media ± 2 μM of STF-1623 for 16 h. IFNB1 and CXCL10 mRNA levels were normalized to ACTB and the fold induction was calculated relative to untreated cells. Mean ± SEM ( n = 2). g , Mice were injected subcutaneously with 300 mg/kg STF-1623 at time 0. At indicated times, the mouse was sacrificed, blood was drawn by cardiac puncture, and serum isolated after clotting. STF-1623 concentrations were measure by LC-MS/MS. Individual data points shown ( n = 2).

Article Snippet: Seven- to nine-week-old female C57B6/J WT,STING gt/gt (referred to as Sting -/- ), or Enpp1 -/- mice (Jackson Laboratories) were inoculated with 5 × 10 4 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad.

Techniques: Activity Assay, In Vitro, Purification, Liquid Chromatography with Mass Spectroscopy, Permeability, Negative Control, Positive Control, Incubation, Concentration Assay, Inhibition, Control, Transfection, Plasmid Preparation, Injection, Isolation, Coagulation

Journal: iScience

Article Title: CD38 restrains the activity of extracellular cGAMP in a model of multiple myeloma

doi: 10.1016/j.isci.2024.109814

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for flow cytometry: anti-CD138/FITC (clone MI15, BD Biosciences); anti-CD38/APC (clone HIT2, BD Biosciences); anti-ENPP1/APC (polyclonal sheep IgG, R&D), polyclonal sheep IgG/APC (R&D).

Techniques: Virus, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Labeling, Sequencing, Plasmid Preparation, Software, Staining

Journal: iScience

Article Title: CD38 restrains the activity of extracellular cGAMP in a model of multiple myeloma

doi: 10.1016/j.isci.2024.109814

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for flow cytometry: anti-CD138/FITC (clone MI15, BD Biosciences); anti-CD38/APC (clone HIT2, BD Biosciences); anti-ENPP1/APC (polyclonal sheep IgG, R&D), polyclonal sheep IgG/APC (R&D).

Techniques: Virus, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Labeling, Sequencing, Plasmid Preparation, Software, Staining