Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Illustration of the hypothesis that escapees mount a replication stress response to manage aberrant S phases and tolerate DNA lesions. (B) Western blot analysis of γ-H2AX in the indicated cell lines after 96 h treatments. β-tubulin is used as a loading control. Bar plot displays quantification of γ-H2AX band intensity normalized to respective loading control band, and values are reported as fold change of control. (C) Representative immunofluorescence images of five human MAPK-mutant cancer cell lines after 72 h indicated treatments, stained for DNA content, EdU incorporation, and γ-H2AX. (D) Quantification of γ-H2AX nuclear fluorescence intensity after 72 h indicated treatments in EdU − and EdU + cells, displayed as split violins; p -values determined by Mann-Whitney U tests of MAPK-inhibited EdU + cells compared with DMSO-treated EdU + cells. (E) Top: Representative immunofluorescence images of phospho-CHK1 (S317) in A375 cells escaping BRAF inhibition (cycling status displayed by EdU intensity). Bottom: Histograms of phospho-CHK1 (S317) nuclear intensity in S phase cells (EdU + ) in the indicated cell lines after 96 h MAPK inhibition. (F) Distribution of EdU intensity in escapees (after 100 nM Osimertinib or 1 μM Encorafenib) co-treated with ATR inhibition (100 nM AZ20 or 100 nM AZD6738). 96 h treatments. Line drawn at the mean value; n = 190 cells plotted per condition; p -values determined by Mann-Whitney U test. (G) Quantification of cycling cells (determined by phospho-Rb S807/811 immunofluorescence) after 96 h of indicated treatment combinations with ATR inhibitors. AZ20 doses: 100 nM, 1 μM. AZD6738 doses: 100 nM, 1 μM. Mean ± std of 3 replicates; p -value determined by unpaired t-test.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Western Blot, Control, Immunofluorescence, Mutagenesis, Staining, Fluorescence, MANN-WHITNEY, Inhibition

Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Validation of FANCD2-mCitrine expressing clonal cell lines using hydroxyurea treatment as a positive control. Representative cells in S/G2 phases of the cell cycle (marked by cytoplasmic DHB-mCherry intensity) display many FANCD2-mCitrine nuclear foci after 24 h treatments, a known effect of hydroxyurea. (B) Film strips of FANCD2-mCitrine and DHB-mCherry in A375 cells under indicated times of dabrafenib treatment. Arrowheads follow a cell progressing through an entire cell cycle and experiencing FANCD2-mCitrine nuclear foci while under dabrafenib treatment. Corresponding DHB C/N ratio and FANCD2 foci traces for the cell lineage prior to becoming an escapee and afterward are matched to the film strip time frames, shown in the righthand plot. (C) Validation of siRNA knockdown of FANCD2 by western blotting after 24 h; β-tubulin is used as a loading control. FANCD2 depleted by ~90%. Mean ± std of 3 replicates; p -value determined by unpaired t-test. (D) Single-cell traces of CDK2 activity over time in cells treated with combination treatments. The transfection mix was added to the cells 4 h prior to the time of drug treatment and removed 4 h after the time of drug treatment.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Biomarker Discovery, Expressing, Positive Control, Stripping Membranes, Knockdown, Western Blot, Control, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Ex Vivo Validation of Six FDA-Approved Non-Receptor Tyrosine Kinase Inhibitors (NRTKIs) as Antivirals to Pandemic and Seasonal Influenza A Viruses

doi: 10.1101/2022.01.19.476993

Figure Lengend Snippet: Effect of NRTKIs treatment on STAT3 activation. A549 cells were infected with NL09 (A) or NL11 (B) at MOI=1. Total proteins were isolated from whole cell lysate at 18 and 48 hpi and immunoblot assay was performed for phospho- and total STAT3, IAV-NP and bActin. Chemiluminescence was detected and quantified using the Li-Cor C-DiGit and Image Studio 5.1 CLX software. All measurements were taken from two western blots from two independent experiments (n=2). All values are relative to untreated virus-infected cells. P=phosphor, T=total. Error bars indicate ± standard deviation (SD). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant (P>0.05). P-values determined by students t-test compared to untreated virus-infected cells.

Article Snippet: When necessary, the imaged membrane was subsequently stripped using a mild water-based stripping solution (1.5% glycine; 0.1% SDS; 1% Tween-20; pH 2.2) and restained for total proteins using NFkB p65 (L8F6) mouse mAb (1:1000) (Cell Signaling) or Stat3 (124H6) mouse mAb (1:1000) (Cell Signaling).

Techniques: Activation Assay, Infection, Isolation, Western Blot, Software, Virus, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Cholesterol-rich lipid rafts mediate endocytosis as a common pathway for respiratory syncytial virus entry into different host cells

doi: 10.1128/spectrum.01192-25

Figure Lengend Snippet: RSV entry into cells triggers actin polymerization and endosome formation by cholesterol-rich lipid rafts. ( A ) RSV enters cells by actin-mediated endocytosis. HEp-2 or A549 cells were infected with RSV A2 (MOI = 30) (90 min, 37°C) and fixed (4% PFA, 15 min, room temperature). Cell nuclei were labeled with DAPI (cyan), lipid rafts with Alexa 647-CTB (blue), cholesterol with NBD-cholesterol (green), and F-actin with CellMask orange actin tracking dye (yellow), and images were acquired using confocal microscopy. Scale bar = 10 µm. Intensity profiles show colocalization along the direction of the yellow line. Pearson’s correlation between lipid rafts, cholesterol or F-actin, and RSV. n = 16 cells per group. Error bars represent a 95% confidence interval. ( B ) RSV trafficking from early to late endosomes. HEp-2 or A549 cells were infected with RSV A2 (MOI = 30) (150 min, 37°C) and fixed. Cell nuclei were labeled with DAPI (cyan), lipid rafts with Alexa 647-CTB (blue), and cholesterol with NBD-cholesterol (green). Immunodetection of RSV F (blue), RSV-FITC (green), EEA1, Rab7, and STX 6 (red) was performed using confocal microscopy. Scale bar = 10 µm. Intensity profiles show colocalization along the direction of the yellow line. Pearson’s correlation between lipid rafts, cholesterol or F-actin, and RSV. n = 16 cells per group. Error bars represent a 95% confidence interval.

Article Snippet: The Flotillin-1 (D2V7J) rabbit mAb (#18,934T), caveolin-1 (D46G3) rabbit mAb (#3267), EEA1 (C45B10) rabbit mAb (#3288), Rab7 (D95F2) rabbit mAb (#9367), Rab7 (E9O7E) mouse mAb (#95746), and syntaxin 6 (C34B2) rabbit mAb (#2869) were purchased from Cell Signaling Technology.

Techniques: Infection, Labeling, Confocal Microscopy, Immunodetection

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