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: Inhibition of CD38 on Dox-treated MM cells causes accumulation of extracellular cGAMP (A) Cells were treated with Dox for 24 h as already described; the medium was then replaced with serum-free medium containing DMSO or CD38i (10 μM for SKO-007(J3) cells, 20 μM for ARK and ARH-77 cells); after additional 24h, cGAMP in the CM (top panel) and in the cellular lysate (50 μL of lysate for 0.5 · 10 6 cells, bottom panel) was quantified through competitive ELISA. Statistical significance was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. (B) Primary PCs from four MM patients at disease onset (1–4 of Table 1 ) were treated for 48 h with CD38i 10 μM or DMSO in serum-free medium enriched with 20 μM MnCl 2, 20 ng/mL IL-3 and 2 ng/mL IL-6; the concentration of cGAMP in the CM was then quantified through ELISA. Statistical significance was calculated using two-tailed paired Student’s T test. (C) ARK cells were transduced with a lentiviral vector carrying short hairpin RNA targeting CD38 (shCD38) or non-targeting (scramble). After selection in G418, surface expression of CD38 was measured by flow cytometry. (D and E) ARK and A549 shCD38 and scramble control were stimulated with Dox and subsequently cGAMP was measured in CM as described previously. Statistical significance was calculated using two-way ANOVA with Bonferroni correction for multiple comparisons. (F) BMSCs from MM patients (12–15 of Table 1 ) were incubated for 16 h with the CM from SKO-007(J3); the transcription of IFN-β and CCL5 was then quantified through real-time PCR (normalized with GUSB ). Statistical significance was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. (G) THP-1 cells were incubated for 6 h with serum-free medium containing cGAMP 100 nM, pre-incubated with rhCD38 10 nM or nuclease-free water (vehicle) for 1 h at 37°C. The induction of IFN-β was then measured by real-time PCR. Statistical significance was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. Error bars represent SEM.

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: Inhibition, Competitive ELISA, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transduction, Plasmid Preparation, shRNA, Selection, Expressing, Flow Cytometry, Incubation, Real-time Polymerase Chain Reaction

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: Molecular docking and multi-copy μs-long MD simulations reveal stable interactions of cGAMP with CD38 and demonstrate that cGAMP binding leads to significant CD38 global rigidification (A) CD38-cGAMP complex total energy score was plotted against cGAMP unsuperimposed root-mean-square deviation (ligand RMSD), from which the top 10 binding models (red dots) were selected. The model with lowest binding energy was set as the reference. (B) Predicted binding mode of cGAMP to CD38. (C) Trajectory median backbone RMSD of unbound (red) and bound (blue) CD38. The increased conformational rigidity of the CD38-cGAMP complex compared to the unbound CD38 is demonstrated by a median backbone RMSD of ∼3 Å and ∼2 Å along the simulation time, respectively (left boxplot). The overall rigidification of the protein in the bound state is reflected in the active site as well (right boxplot). Error bars denote SD. (D) Principal Component Analysis (PCA) of unbound (red) and bound (blue) CD38. The projection to the first two PCA-eigenvectors is based on the Cα of all simulated ensembles. Distributions for the projection on the first and second eigenvectors are plotted previously and on the right of the corresponding plots. (E) Collective motions corresponding to the first PC of unbound and bound CD38. Comparison of the motion described by eigenvector 1 from the unbound (red) and bound (blue) MD ensembles. Motions are illustrated as linear interpolations between the extreme projections of the structures onto the eigenvector 1 are indicated by black arrows. The direction of the arrow in each Cα atom represents the direction of motion, while the length of the arrow characterizes the movement strength. A schematic of the described motions is presented (see also and ). (F) Catalytic pocket volume distributions for unbound (red) and bound (blue) simulations.

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: Binding Assay, Comparison

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: Molecular interactions between CD38 catalytic pocket and cGAMP (A–D) Statistical distribution (% of the total simulation time) of the interactions between CD38 residues and cGAMP ligand. For the hydrogen bonds and salt bridges analyses, given the high number of interactions observed, only the interactions with a persistence higher than 25% of the simulation time have been reported to limit the description to the most relevant ones. For the π—stacking interactions, the contacts observed for more than 20% of the simulation time are reported. Values are reported by averaging the results from the three replicate simulations for both unbound and bound systems. (E) Per-residue Molecular mechanics generalized Born and surface area (MM/GBSA) binding affinity estimate (right) and corresponding hot spot residues (left) are illustrated. The binding free energy from different residues located within a 5 Å radius from the ligand is shown. Only residues contributing with more than k T (∼0.6 kcal/mol at 300 K) are reported. Values are reported by averaging the results from the three replicate simulations for both unbound and bound systems.

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: Residue, Binding Assay

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: Energetic components of the relative binding free energy for cGAMP-CD38 complex

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: Binding Assay

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: Tryptophan intrinsic fluorescence measurements and MST analysis show the interaction between CD38 and cGAMP (A) Fluorescence quenching titration of rhCD38 (1 μM) with increasing cGAMP (0–205 μM) in 50 mM Phosphate buffer pH 7.4 and 50mM NaCl at 25°C upon excitation at 295 nm. The black curves represent the emission spectrum of rhCD38 in the absence of ligands. The colored curves show the progressive decrease in fluorescence intensity of rhCD38 at the emission maximum (345 nm) upon the addition of cGAMP. (B) Plot of normalized fluorescence signal at 345 nm expressed as percentage of quenching versus the concentrations of dinucleotides. By applying a nonlinear regression fitting, dissociation constants values (K D ) of 33.2 ± 5.4 μM and 46.3 ± 9.7 μM were calculated for cGAMP (red) and 2′3′-cG s A s MP (black), respectively. (C) Capillary scan of MST analysis. (D and E) MST traces and dose–response curves reported as ΔF norm = normalized fluorescence. Error bars indicate the standard deviation of two independent replicates ( N = 2). (F) MST dose-response curves reported as Fraction Bound. A dissociation constant (K D ± SD) of 8.1 ± 2.5 μM and signal-to-noise ratios (S/N) of 8.7185 were estimated using the software MO Affinity Analysis, v 2.27.

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: Fluorescence, Titration, Standard Deviation, Software

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