Fig 1: PD-L1 directly interacts with PPM1B and inhibits its phosphatase activity, thereby activating p38 MAPK pathway and TGFβ production.a HEK293T cells were transfected with Flag-tagged PD-L1 and Myc-tagged PPM1B for 48 h and then subjected to immunoprecipitation followed by immunoblotting. b, c A549 and H460 cells were transfected with PD-L1 overexpressing vector or PD-L1 siRNA. At 24 h of transfection, cells were submitted to co-immunoprecipitation assay with PD-L1 to evaluate the protein complex formation with PPM1B and p38 MAPK. d Full length (FL), extracellular domain (ECD), and intracellular domain (ICD) of GST-tagged PD-L1 were purified from E. coli. GST pull-down assays were performed using 1 μg of each indicated protein. e In vitro phosphatase assay was performed using recombinant p-p38 as a substrate and recombinant PPM1B as a phosphatase in the presence and absence of recombinant PD-L1 and phosphatase inhibitor (PPase inhibitor). Phosphatase activity was evaluated by measurement of free phosphate (upper) and immunoblotting for p-p38 (lower). Relative intensity of p-p38 was estimated on immunoblot. f H460 cells were transfected with PD-L1 siRNA and/or PPM1B siRNA. At 24 h after transfection, cell were submitted to Western blotting for indicated molecules. g H460 cells were transfected with PD-L1 siRNA and/or PPM1B siRNA for 24 h and then transfected with luciferase-expressing vector with wild-type (wt) promoter sequence of TGFβ containing ATF2- and c-Jun-binding motif. At 12 h after transfection, luciferase activity was measured. As a negative control, the plasmid with truncated mutation (mut) of the TGFβ promoter sequence were used. h A549 and H460 cells were incubated with rhPD-1 (1 μg/ml) for 24 h, and TGFβ production in the culture supernatant was measured using an ELISA. i A549 and H460 cells were treated with rhPD-1 (1 μg/ml) in the presence or absence of anti-TGFβ neutralizing antibody (1 μg/mL). At 24 h after treatment, total protein was extracted and submitted to Western blotting for EMT markers. j, k After 24 h treatment of rhPD-1, protein complex of endogenous PD-L1, PPM1B, and p38-MAPK was analyzed by co-immunoprecipitation assay (j), and phosphorylation of p38, c-Jun, and ATF2 was evaluated using Western blotting (k) in A549 and H460 cells. l, m A549 and H460 cells were treated with rhPD-1 in the presence or absence of p38 inhibitor (SB203580, 10 μM). Cells were then transfected with TGFβ-luc vector and at the 12 h of transfection luciferase activity of promoter sequence of TGFβ containing ATF2- and c-Jun-binding motif were measured. Data are presented as mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 2: CRC organoids are resistant to type I but sensitive to type 1.5 p38α inhibitors.a, Generation of KAP organoids expressing Mapk14 shRNAs. b, Knockdown test of Mapk14 shRNAs. Representative western blot analysis of p38α in KAP2D cells upon 6 days of treatment with doxycycline (Dox; cropped blot images, n = 3 biologically independent experiments). c, Cell viability analysis in KAP-shMapk14 and KAP-shNC organoids upon 12 days of treatment with doxycycline (n = 4 biologically independent experiments; data are presented as the mean ± s.d.). Statistical significance was calculated using an ANOVA and Dunnett’s multiple-comparisons test (P < 0.0001). d, Cell viability analysis in KAP organoids upon 4 days of treatment with SKL, PH-797804, LY2228820 or DMSO (n = 3 biologically independent experiments; data are presented as the mean ± s.d.). e, Representative western blot analysis of KAP2D cells upon 1 day of treatment with 5 µM SKL, PH-797804 or DMSO (cropped blot images; n = 3 biologically independent experiments). f, Schematic picture showing the binding of 1639 to HRI, HRII and R-spine of the p38α kinase. g, Cell viability analysis of KAP organoids upon 4 days of treatment with SKL, 1639 or DMSO (n = 3 biologically independent experiments; data are presented as the mean ± s.d.). Statistical significance was calculated using a two-tailed Student’s t-test (P < 0.0001). Conc., concentration. h, Generation of a CRC mouse model based on subcutaneous injection of KAP organoids into wild-type (WT) mice. i, Representative pictures of hematoxylin and eosin (H&E) and immunohistochemical staining for pan-CK and CDX2 in KAP subcutaneous tumors, 19 days after tumor initiation (n = 4 tumors per group). Scale bars, 100 µm. j, Treatment of subcutaneous KAP CRCs with SKL, 1639 or carrier (n = 10 tumors per group; data are presented as the mean ± s.e.m.). Statistical significance was calculated using an ANOVA and Dunnett’s multiple-comparisons test (P = 0.0192). NS, not significant. Treatment was started 1 week after organoid transplantation. The experiments in b, d, e and g were independently performed three times, the experiment in c was independently performed four times and the stainings in i were independently performed twice, all with similar results.Source data
Fig 3: Chemical structures of nilotinib and ten analogs evaluated for p38/MK2 PPI inhibition using a TR-FRET assay with recombinant purified proteins. IC50 values are shown for compounds exhibiting measurable activity; compounds with less than 50% inhibition at 30 μM are indicated as not determined (N.D.).
Fig 4: Virtual screening and molecular dynamics analysis identify nilotinib as a candidate p38/MK2 PPI inhibitor. (A) Distribution of MM-GBSA binding free energies (ΔG bind) from virtual screening of 1,040 FDA-approved drugs docked to the p38 docking groove. The p38 crystal structure (PDB ID: 6TCA) was used for the modeling studies. Compounds with ΔG bind values more than two standard deviations below the mean (red bars, <−60.9 kcal/mol) were prioritized for further analysis. (B) Representative binding pose of carvedilol highlighting key interactions with the p38 docking groove, including hydrogen bonds with Val158, Glu160, and His126 (yellow lines), hydrophobic interactions with the nonpolar pocket defined by Ile116, Leu122, Leu130, and Val158, and a pi-pi stacking with His126 (cyan line). (C) Carvedilol’s carbazole moiety binds within the hydrophobic cleft of the p38 docking groove, which is shown as a molecular surface representation colored by electrostatic potential (red = negative, blue = positive). (D) Root-mean-square deviation (RMSD) plots from three 200 ns molecular dynamics simulations of the p38–nilotinib complex. The RMSD of protein backbone atoms is shown in aquamarine, and nilotinib in red. The PDB IDs of the p38 structures used for the modeling are indicated in the lower-left corners. (E) The representative binding pose of nilotinib obtained after 200 ns MD simulation (a final snapshot of one of the MDs), highlighting pi-pi and H-bond interactions with His126, the H-bonding with Glu160, and multiple water-bridged contacts that stabilize ligand orientation within the groove. (F) Structural overlay of the nilotinib–p38 complex with the p38/MK2 cocrystal structure, illustrating displacement of key MK2 anchoring residues Ile372 and Ile375 by nilotinib. P38 is shown as green ribbons, the p38 docking groove as gray molecular surface, and MK2 as red ribbons.
Fig 5: α 1 -Adrenergic antagonists disrupt the p38/MK2 interface and suppress cytokine production in microglial cells. (A) Chemical structures of doxazosin, terazosin, and alfuzosin, three α1-adrenergic receptor antagonists identified from the high-throughput screen. (B) Dose–response TR-FRET assays using recombinant purified p38 and MK2 proteins demonstrate that all three compounds inhibit the p38/MK2 protein–protein interaction, with IC50 values of 4.4 μM (doxazosin), 6.2 μM (terazosin), and 6.9 μM (alfuzosin). (C) The compound activity was confirmed in a cell lysate-based TR-FRET format, showing a moderate reduction in potency relative to the recombinant protein assay. (D) In a complementary TR-FRET assay using HEK293T lysates coexpressing VF-tagged p8 and a His-tagged MK2 346–400 docking peptide, all three α1-antagonists and nilotinib dose-dependently disrupted peptide binding to p38, consistent with direct competition at the docking interface. (E) qRT-PCR analysis in HMC3 microglial cells shows that all three compounds significantly (p-values <0.05) suppressed LPS-induced expression of TNF-α, IL-6, and IL-1β, similarly to known p38 inhibitors SR318 and VX745, demonstrating effective functional inhibition of p38/MK2 signaling in a disease-relevant context.
Supplier Page from Abcam for Recombinant human p38 alpha/MAPK14 protein (Active)