Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) HUVEC were repeatedly transfected with non-targeting siRNA (siControl) or co-transfected with GFAT1- and OGT-specific (siGFAT1+OGT) siRNAs (96h, twice) and seeded in full growth medium for BrdU assay; after 24 h, medium was changed for low serum (2% FCS) medium and after additional 4 h, cells were either treated with bFGF (50 ng/ml, 24h) or left unstimulated. (B, G) Spheroids generated from HUVEC depleted for O-GlcNAc using either genetic (B) or pharmacological (G) approach were treated with 10 or 50 ng/ml VEGF, respectively, or left unstimulated. Representative images of spheroids (B, G) and analysis of the number of sprouts per spheroid (C, D, H), mean sprout lengths (E) and sum of sprout lengths (F) are shown. Data are means ± SEM of 4 independent experiments using cells from different donors. *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective unstimulated control (-); # p < 0.05 vs. control siRNA or diluent control ( H ). Two-way ANOVA with Bonferroni’s multiple comparison test (A, C, H) , or paired t-test (D-F) were used.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Transfection, BrdU Staining, Generated, Control, Comparison

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) Human umbilical vein endothelial cell (HUVEC) lysates (Thiamet G-treated) were subjected to O-GlcNAc peptide-level enrichment using an antibody mix. (B) Overlap of unique HexNAc peptide and corresponding protein identifications from biological triplicates (Glycan FDR < 0.05). (C) HexNAc-modified proteins detected in at least two of three replicates (N = 187), overlapping with senescence-associated proteins from the SeneQuest database (filtered for proteins up- or downregulated in ≥3 studies, N = 893). The protein-protein interaction network was visualized using STRING with MCL clustering. (D) Gene Ontology (GO) enrichment for Molecular Function of HexNAc-modified proteins identified in at least two of three replicates (N = 187), shown in a simplified form. (E) Lysates from basal or O-GlcNAc-enriched HUVEC (Thiamet G-treated vs. untreated) were immunoprecipitated using the RL2 antibody or a control IgG and analyzed by LC-MS. (F) Venn diagram showing proteins identified in all three biological replicates, excluding identifications from the IgG control IP. (G) Gene Ontology (GO) enrichment for Molecular Function of HexNAc- modified proteins identified in at least two of three replicates (N = 187), shown in a simplified form. (H) Mean intensities (max-LFQ, n=3) of immunoprecipitated proteins comparing Thiamet G treatment vs. vehicle control (untreated). Only proteins quantified in all three replicates in both groups are shown.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Glycoproteomics, Modification, Immunoprecipitation, Control, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: O-GlcNAc-enriched tryptic peptides (N=3) were measured by LC-TIMS/MS with HCD fragmentation. The analysis was performed on the PSM level. (A) Distribution of unmodified and HexNAc-modified PSMs in the ion mobility (1/K0) vs. m/z dimension. Shown are unique peptide sequences for each charge state (Unmodified: N = 23,157 HexNAc: N = 505). (B) Relative abundance of peptide misscleavages for unmodified and HexNAc-modified PSMs. (C-E) Unmodified and HexNAc-modified PSMs were compared. Shown are unique peptide sequences, with the highest observed intensity (Unmodified: N = 18,73867; O-GlcNAc: N = 416). (C) Peptide length distribution. (D) Retention time distribution. (E) Distribution of the GRAVY score as a metric of hydrophobicity, with a higher score indicating higher hydrophobicity. Asymptotic two-sample Kolmogorov-Smirnov-test, **** p<1e-04.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Modification

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) Lysates of control or Thiamet G-treated HUVEC were immunoprecipitated for CCAR1 and stained by immunoblot for O-GlcNAc (upper panel) and CCAR1 (lower panel). Images are representative of 3 independent experiments using cells from different donors. (B) MS-based analysis of enriched proteins after CCAR1 co-immunoprecipitation. Coverage of the N-terminal region of CCAR1 by LC-MS using chymotrypsin as protease. HexNAc modified peptides are indicated in red. (C) MS-intensity fold change between Thiamet G-treatment and control, normalized to protein fold change, for the two most abundant HexNAc peptides and their unmodified counterpart. Data are means ± SD of 3 independent experiments using cells from different donors. (D, E) Representative MS/MS spectrum of the HexNAc modified peptide “TATAVSQPAALGVQQPSLL” (D) and “TQPAVALPTSL” (E) . Peak colors: orange, green and blue refer to y, b and diagnostic oxonium ions, respectively. The enlarged view in the m/z range of 100 to 225 (D) shows the [HexNAc]+ ion (C8H14NO5) and corresponding fragmentation induced oxonium ions.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Control, Immunoprecipitation, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Modification, Tandem Mass Spectroscopy, Diagnostic Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Structure-guided mapping and peptide validation of the p38/MK2 interaction interface. (A) The cocrystal structure of the p38/MK2 complex (PDB ID: 6TCA ). The molecular surface of p38 is shown in gray. The p38 docking groove is highlighted in yellow. MK2 is shown as green ribbons. (B) The MK2 D345-H400 docking motif bound to the p38 docking groove is colored based on its fragments tested in this study: D345-H400 is colored in green, I370–L393 in blue, and I370-L382 in red. (C) The binding curve from a fluorescence polarization assay showing high-affinity binding of FITC-labeled MK2 370–393 peptide to His-tagged p38 (EC 50 = 26.9 nM). (D) Dose–response curves from TR-FRET inhibition assays demonstrating that both MK2 370–393 and 369–382 peptides disrupt the p38/MK2 complex (IC 50 = 0.42 μM and 4.26 μM, respectively).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Binding Assay, Fluorescence, Labeling, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Virtual screening and molecular dynamics analysis identify nilotinib as a candidate p38/MK2 PPI inhibitor. (A) Distribution of MM-GBSA binding free energies (Δ G 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 Δ G 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.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Validation of nilotinib as a p38/MK2 PPI Inhibitor. (A) Thermal shift assay (TSA) showing dose-dependent stabilization of recombinant His-tagged p38 by nilotinib (Δ T max = 8.22 °C), consistent with direct binding. (B) TSA profile for SR318, a type II ATP-competitive p38 inhibitor, used as a positive control (Δ T max = 13.47 °C). (C) Nilotinib competes with His-MK2 346–400 fragment for VF-p38 in a cell lysate-based TR-FRET assay. (D) Quantitative qRT-PCR analysis showing that nilotinib significantly ( p -value <0.05) suppresses LPS-induced TNF-α, IL-6, and IL-1β expression in HMC3 microglial cells. P38 inhibitors SR318 and VX-745 were used as positive controls. (E) Nilotinib disrupts the endogenous p38/MK2 complex in HMC3 cells, as shown by coimmunoprecipitation, correlating with cytokine suppression. (F) qRT-PCR analysis showing that nilotinib suppresses LPS/IFNγ-induced TNF-α expression in the human iPSC-derived microglia (iMGL). (G) TR-FRET assay with recombinant p38 and MK2 proteins purified from E. coli demonstrated direct inhibition of the complex by nilotinib (IC 50 = 2.2 μM). In contrast, ATP-site inhibitors VX-745 and SR318 failed to disrupt the interaction, supporting a non-ATP-competitive mechanism for nilotinib activity. (H) Nilotinib demonstrates a weak inhibition of p38/ATF2 PPI (IC 50 > 30 μM, maximal inhibition ∼ 37%) in a TR-FRET assay with recombinant purified His-p38 and GST-ATF2. The inhibition of His-p38/GST-MK2 PPI by nilotinib was monitored in parallel.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Thermal Shift Assay, Recombinant, Binding Assay, Positive Control, Quantitative RT-PCR, Expressing, Derivative Assay, Purification, Inhibition, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Chemical structures of nilotinib and ten analogs evaluated for p38/MK2 PPI inhibition using a TR-FRET assay with recombinant purified proteins. IC 50 values are shown for compounds exhibiting measurable activity; compounds with less than 50% inhibition at 30 μM are indicated as not determined (N.D.).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Recombinant, Purification, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Field-based QSAR maps illustrating physicochemical features of nilotinib analogs associated with p38/MK2 PPI inhibition. (A) Compounds 1–6 (white) and 7–10 (orange) docked into the p38 binding groove. The molecular surface of the binding groove is colored based on the electrostatic potential, ranging from the most positive (blue) to the most negative (red) charge. (B) Steric field map showing regions where steric bulk is favorable (green). The pyridine–pyrimidine system is positioned within favorable steric zones, supporting its critical role in activity. (C) Hydrophobic field map with yellow-green and gray surfaces representing positive and negative hydrophobic contributions, respectively. (D) Electrostatic field map colored by potential (red - negative, blue - positive). (E) Hydrogen bond acceptor field map. Red contours indicate favorable contributions of H-bond acceptors, while the magenta contour indicates unfavorable contributions of H-bond acceptors. (F) Hydrogen bond donor field map. The blue-violet contour indicates the region favorable for H-bond donors. The cyan field map indicates the area unfavorable for the H-bond donors.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Binding Assay, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Development of a lysate-based TR-FRET platform for high-throughput screening of p38/MK2 PPI inhibitors. (A) The preferential binding of MK2 to p38α and p38β isoforms was determined by Flag-immunoprecipitation in HEK293T cells. (B) Isoform selectivity of MK2 binding was validated by TR-FRET using lysates coexpressing GST-tagged MK2 and Venus-Flag (VF)-tagged p38 isoforms. Robust signal was observed for p38α and p38β, with negligible interaction detected for p38γ and p38δ. (C) TR-FRET assay shows stable signal over 48 h postantibody addition, indicating excellent temporal stability. (D) The platform tolerates up to 10% DMSO without signal degradation, supporting its suitability for screening applications. (E) Pilot screen of 2036 compounds from the Emory Enriched Library (EEL) in 1536-well format identified 48 compounds that inhibited the p38/MK2 interaction by ≥ 50% relative to vehicle control. Gray dots indicate fluorescence assay-interfering compounds.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Binding Assay, Immunoprecipitation, Control, Fluorescence

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: 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 α 1 -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 IC 50 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 α 1 -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.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Recombinant, Purification, Activity Assay, Binding Assay, Quantitative RT-PCR, Expressing, Functional Assay, Inhibition