Journal: Nature chemical biology

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

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( a ) Ion dependency and ( b ) pH preference of the dominant hydrolase activity in MDA-MB231 cells. ( c ) Activity of recombinant ENPP1 alone or ( d ) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca 2+ , 200 μM Zn 2+ . ( e ) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as ( f ) and ( g ). Data are presented as mean and standard error.

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 non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( 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. 4 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: 1% NP-40, 20 mM Tris-HCl, pH 7.5, protease inhibitor cocktail. The assay was conducted in 0.2% NP-40, 20 mM Tris-HCl, 150 mM KCl, 2 mM Ca 2+ , 2 mM Mg 2+ , 200 μM Zn 2+ 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 and 2′3′-cGAMP runs at a lower R f in this buffer; Tris-HCl buffer was used for pH 7.5-9.0, and Borate buffer was used pH 9.5. These buffer conditions were also used in liver and spleen extract studies.

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

Techniques: Knockdown, Activity Assay, 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 non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( 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, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL 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). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( 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 media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

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

Techniques: Incubation, Scintillation Proximity Assay, Binding Assay, Labeling

Journal: Nature chemical biology

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

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Lung fibroblast cells from wild type and Enpp1 -/- female mice were incubated with 2′3′-cGAMP analogs and HSV-60 at the indicated concentrations for 24 h. IFN-β in the media was measured using a B16-SEAP cell line. N=3 samples. Data are presented as mean and standard error.

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

Techniques: Incubation

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