recombinant human pad4  (Millipore)

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Chemical structures of PAD-PF1, GSK147, and PAD-PF2. b PAD-PF1 inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala) 3 in a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca 2+ . Data points and error bars represent the mean and SD of quadruplicate determinations of a representative experiment. IC 50 = 15.9 µM, pIC 50 = 4.80 (0.26), mean (SD), n = 29 biologically independent experiments. c – e Representative surface plasmon resonance (SPR) traces characterizing binding of respective ligands to Bap-tagged PAD4 captured on streptavidine sensor. The red line represents the fitted curve. c PAD-PF1. K D = 2.82 µM, pK D = 5.55 (0.12), mean (SD), n = 4 technical replicates. The highest concentration of PAD-PF1 tested was 10 µM, with five more 3-fold dilutions. d , GSK147. K D = 0.469 µM, pK D = 6.33 (0.016), mean (SD), n = 4 technical replicates. The highest concentration of GSK147 tested was 3.33 µM with five more 3-fold dilutions. e Same concentration series of GSK147 in the presence of 10 µM PAD-PF1. K D = 0.380 µM, pK D = 6.42 (0.071), mean (SD), n = 4 technical replicates. Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Fluorescence, SPR Assay, Binding Assay, Concentration Assay

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Crystal structure of human full-length recombinant PAD4 simultaneously bound to GSK147 (orange carbon sticks and van der Waals surface) and PAD-PF1 (green carbon sticks and van der Waals surface). Protein backbone displayed as a ribbon diagram. b PAD4 binding pocket of GSK147 (orange carbon sticks). c PAD4 binding pocket of PAD-PF1 (green carbon sticks). In ( b , c ), side chains interacting with the small molecules are shown as white carbon sticks, hydrogen bonds are dotted lines, and water molecules are represented as red spheres. Key protein residues with hydrophobic interactions with the ligand are labeled in red.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Recombinant, Binding Assay, Labeling

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a PAD-PF2 (open circles) inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala) 3 employing a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca 2+ . Data points and error bars represent the mean and SD of three independent experiments in duplicate. IC 50 = 42.7 nM, pIC 50 = 7.37 (0.03), mean (SD), n = 3 independent experiments. The enantiomer of PAD-PF2 ( ent -PAD-PF2, open squares) was less potent. IC 50 = 26.0 µM, pIC 50 = 4.59 (0.45), mean (SD), n = 3 independent experiments. b PAD-PF2 induces a thermal stabilization of PAD2 (open circles, EC 50 = 2.04 µM) and PAD4 (open squares, EC 50 = 1.95 µM) protein. Individual data points shown for a duplicate determination. c – f Inhibition of the respective PAD isoform by PAD-PF2 determined using the GDH-enzyme coupled assay in the presence of 0.25 mM Ca 2+ . Individual data points shown for a duplicate determination along with the fitted IC 50 curves (line). c PAD1, IC 50 = 109 nM. d PAD2, IC 50 = 28.5 nM. e PAD3, IC 50 = 106 nM. f PAD4, IC 50 = 24.0 nM. g , h Calcium dependence of PAD2 or PAD4 inhibition by PAD-PF2. Inhibition was determined using the GDH enzyme-coupled assay. Data points from duplicate experiments performed at high (open squares) and low (open circles) Ca 2+ concentrations are plotted together, and the fitted IC 50 curves are shown (line). g The determined IC 50 values for PAD2 inhibition were 27.9 nM and 3.09 µM (0.25 and 1.5 mM Ca 2+ , respectively). h The determined IC 50 values for PAD4 inhibition were 20.1 nM and 694 nM (0.25 and 1.5 mM Ca 2+ , respectively). Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Fluorescence, Inhibition

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: The method of Loewe was used to assess synergy between PAD-PF2 and GSK147 towards inhibition of PAD4. Serial dilutions of the inhibitors (0–3.6 μM) were tested in all combinations (two-by-two matrix) under the PAD4 GDH-coupled assay condition with 1.5 mM CaCl 2 . Plotted are the normalized doses of the two compounds that were observed to produce half-maximal activity. For circles, x = [GSK147]/GSK147 IC 50 in the absence of PAD-PF2, y = PAD-PF2 IC 50 /PAD-PF2 IC 50 in the absence of GSK147. For squares, x = GSK147 IC 50 /GSK147 IC 50 in the absence of PAD-PF2, y = [PAD-PF2]/PAD-PF2 IC 50 in the absence of GSK147. Isobole represented by the dotted line. Source data are provided as a Source Data file.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Inhibition, Activity Assay

Journal: Nature Communications

Article Title: Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca 2+ dependent allosteric binding site

doi: 10.1038/s41467-025-59919-4

Figure Lengend Snippet: a Crystal structure of human full-length recombinant PAD4 bound to PAD-PF2 (green carbon sticks). The disordered side chains of Asp369 and Ser370 were constructed using the program ADDS . b Crystal structure of human full-length recombinant PAD2 bound to PAD-PF2 (green carbon sticks). In a , b , side chains interacting with the small molecules are shown as white carbon sticks, hydrogen bonds are dotted lines, and water molecules are represented as red spheres. Key protein residues with hydrophobic interactions with the ligand are labeled in red.

Article Snippet: Human recombinant PAD4 (Cayman Chemical) in Ammonia assay buffer (50 mM borate, pH 8.0, 5 mM DTT, and 10 mM CaCl 2 ) was preincubated with various concentrations of compound or DMSO (1% final) in a Corning 3820 microplate.

Techniques: Recombinant, Construct, Labeling