p irak4  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): Schema of IL1β/TLR/IRAK1/2/4 signaling pathway. ( B-D ): Analysis of ovarian cancer patient’s microarray data (TCGA-381-tpm-gencode36) using R2 Genomics Analysis and Visualization Platform tools showed that IRAK1, -2 and -4 mRNA overexpression predict poor survival. ( E ): IRAK4 mRNA was overexpressed in malignant than normal ovaries. The data present in GENT2 ovarian cancer databases were analyzed using the inbuilt tools. ( F ): Analysis of the EOC patient’s microarray data using GENT2 tools showed that IRAK4 mRNA expression was altered in various stages of disease. Two-sample T-test showed statistical differentiations: IA vs I (p=0.004); IA vs II (p=0.007); IA vs III (p<0.001); IC vs IIA (p=0.002); IC vs III (p<0.001); IIA vs I (p<0.001); IIA vs II (p<0.001); IIA vs III (p<0.001); IIA vs IIC (p=0.006); IIA vs IIIC (p=0.001); IIC vs III (p<0.001); III vs IIB (p=0.004); IIIA vs I (p=0.001); IIIA vs II (p=0.004); IIIA vs III (p<0.001); IIB vs I (p=0.008); IIIB vs III (p=<0.001); IV vs I (p=0.004); IV vs IIA (p=0.005); IV vs III (p=<0.001). ( http://gent2.appex.kr/gent2/ , date accessed 10/2/2023). ( G ): Compared to non-invasive EOC, tissues from invaded EOC phenotype showed greater IRAK4 mRNA. EOC patient microarray data (TCGA-541-custom-tcgaovag1) deposited at R2-Genomics Analysis and Visualization Platform were analyzed using their MegaSnitch tools. ( H ): Chemical structures of three representative IRAK4–kinase inhibitors undergoing clinical trials. ( I ): Structure-activity relationship (SAR) guided optimization of JH-I-25 scaffold, a literature described IRAK4 inhibitor leading to 3 potent novel analogs. The chemical structure of UR241-2 (sulfone), PSP-099 (sulfide) and PSP-100 (sulfoxide), from among the many analogs synthesized, are shown. ( J ): Calculated drug-likeness/physicochemical properties (LogP, cLogP and topological polar surface area-tPSA are shown. These properties were calculated using Chemdraw software. ( K ): Comparison of IRAK1-, IRAK2-, and IRAK4-kinase inhibitory IC50s of UR241-2 versus CA4948, PF-06650833, PSP-099 and PSP-100 are shown. Compounds were screened using HotSpot Kinase assay available in Reaction Biology Laboratories using 1μM ATP concentration under a 10-dose singlet screening program. ( L ): Dendrograms of global kinome activity of UR241-2 at 50- and 500nM doses. Red indicates kinase affected. At 50nM 13 of 682 kinases were significantly inhibited, whereas at 500nM 34 kinases, shown as red dots, from among 682 total kinases were inhibited. ( M ). A HEK293 cell-based NanoBret target Engagement screening assay was conducted to determine the selectivity of UR241-2 among 10 most affected kinases revealed by HotSpot kinase assay. The nanoBret assay showed that UR241-2 inhibits IRAK4 kinase activity only and other kinases including IRAK1 are affected at 10-100 folds higher doses, making UR241-2 as one of the most selective and specific IRAK4 kinase inhibitors known currently.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Microarray, Over Expression, Expressing, Clinical Proteomics, Activity Assay, Synthesized, Software, Comparison, Kinase Assay, Concentration Assay, Drug discovery, Screening Assay

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): JH-I-25 and its newer analogs UR241-2, PSP-099 and PSP-100 were docked individually to IRAK4 crystal structure using Gnina docking software, which is built on neural networks (CNNs) as a scoring function. ( B ): The interacting amino acid residues and their nature of interactions are shown. Green=aliphatic, magenta=aromatic, blue=basic, sky blue=polar, and yellow=sulfur. Red indicates unfavorable interactions. ( C ): JH-I-25, UR241-2, PSP-099 and PSP-100 analogs were docked together to IRAK4 protein using Gnina tools. ( D ): Root-mean square density (RMSD) simulations of JH-I-25, UR241-2, PSP-099 and PSP-100 ligands docked to IRAK4 falling within 2Å unit range are shown. RMSD-ligand is the root mean deviation of the bounded ligand compared to the ligand in the crystal structure. ( E ): Root-mean square density (RMSD) simulations of IRAK4 protein docked with JH-I-25, UR241-2, PSP-099 and PSP-100 ligands falling with 2Å unit range are shown. RMSD-Protein is the root mean deviation of the protein for each frame of the simulation compared to the crystal structure. Both D and E indicate that docking quality was acceptable. ( F ): RMSF (root mean square fluctuation) of JH-I-25, UR241-2, PSP-099 and PSP-100 ligands are shown. The ligands, when bound, induce the most notable structural changes in residues 172-181 of IRAK4 which reflected in the spike in the RMSF charts. RMSF is the mean fluctuation of IRAK4 protein residues after binding of ligands. RMSF indicates the conformational flexibility of the complex. The time/frame element is removed from RMSF in order to show an average fluctuation of the residues. ( H ): The affinity score measures affinity of a ligand (in that particular frame) binding to the protein. The frames represent different conformations that a ligand may adopt. The affinity is measured in KCal/mol and the lowest frame (lowest energy) is selected as the most stable binder. UR241-2 showed best affinity score (−11.95), hence was considered the most stable binder ligand of IRAK4 crystal structure.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Software, Binding Assay

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): UR241-2 (5-150nM) treatment for 4hrs blocks human and murine IL1β (5nM, 30 minutes) induced IRAK4-phosphorylation in both human (HCH-1) and murine high-grade serous EOC cells (HGS-1 and -3) cells. ( B ): Analysis of ovarian cancer patient’s microarray database (Kim_161-DESeq2_vst-ensh38e100) using R2 Genomics Analysis and Visualization Platform tools showed strong correlation of IRAK4 mRNA expression with NF-κβ mRNA expression in ovarian tumors (R=0.581, p=6.29e -016 ). Another database (TCGA-381-tpm-gencode36) showed similar correlation (R=0.523, p=5.28e -018 ) between IRAK4 and NF-κβ mRNA expression levels in ovarian tumors. ( C ): Analysis of ovarian cancer patient’s microarray showed using the GENT2 software showed that NF-κβ mRNA is overexpressed (Log2-Fold-change Log2FC=1.1) in malignant ovaries than normal (p<0.001). ( D ): NF-κβ mRNA overexpression showed significant risk of mortality among EOC patients (p=0.0012). The EOC patient’s microarray data was analyzed using the R2-Genomics Analysis and Visualization Platform tools. ( E-upper ): UR241-2 (2.5μM) treatment reduced NF-κβ-luciferase reporter activity in stably transfected OVCAR-3-NF-κβ-Luc cell-lines. (E-lower ): UR241-2 (2.5μM) treatment reduced NF-κB luciferase reporter activity induced by human TNF-α (3, 10 and 30nM) in stably transfected OVCAR-3-NF-κβ-Luc cell-lines. ( F ): UR241-2 (2.5μM) treatment, IL-1β (5ng), TNF-α (10ng), LPS (100nM) and R848 (TLR-7/8 agonist, 10μM). Ratio of positive/negative NF-κB nuclei are shown. NF-κβ (Green) nuclear migration was inhibited significantly in IL-1β and LPS stimulated cells. The number of NF-κβ nuclear positive cells were counted using ImageJ software. * indicates <0.05 (Student T-test).

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Phospho-proteomics, Microarray, Expressing, Software, Over Expression, Luciferase, Activity Assay, Stable Transfection, Transfection, Migration

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: IRAK4 inhibition via UR241-2 decreases tumor burden at the needle injury site and increases the number of MHCII expressing myeloid cells in the peritoneal cavity and tumor. ( A ): tumor weights on the needle injury site were significantly lower in the UR241-2 treatment group than vehicle treated animals. Statistical significance was determined using Mann-Whitney test, *p < 0.033, **p < 0.002, ***p < 0.001. ( B ): Tumor weights on omentum did not differ between UR241-2 treatment group than vehicle treated animals (NS). Two of the three replicates are combined and shown. IRAK4 inhibition via UR241-2 increases the number of MHCII expressing myeloid cells and decreases MHCII low and CD206+ macrophages in the peritoneal cavity . Leukocytes were isolated from the peritoneal lavage or tumor of HGS-3-tumor bearing mice and stained for flow cytometry. ( C ) Representative flow cytometry plots of F4/80 lo MHCII hi M1 macrophages and F4/80 hi MHCII Low macrophages from the peritoneal lavage of HGS-3 tumor bearing mice treated with Vehicle or UR241-2. ( D ) Percentage and cell number of F4/80 lo MHCII hi M1 macrophages and F4/80 hi MHCII Low macrophages from C. (E) Representative flow cytometry plots of F4/80 hi CD206 + macrophages from the peritoneal lavage of HGS-3 tumor bearing mice treated with Vehicle (Vx) or UR241-2. ( E-right ) Percentage and cell number of F4/80 hi CD206 + macrophages. IRAK4 inhibition reduces neutrophil numbers in the peritoneal cavity and tumors of HGS3 bearing mic e: (F ): Flow cytometry plots showing CD11b + Ly6G + neutrophils and MHCII + neutrophils in the HGS-3 tumor. Percentage and cell number per gram are quantified to the right. ( G ): Flow cytometry plots showing CD11b + Ly6G + and MHCII + neutrophils in the peritoneal lavage of HGS-3 tumor bearing mice. Percentage and cell number are quantified to the right. N= 9 mice per group from two independent experiments.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Inhibition, Expressing, MANN-WHITNEY, Isolation, Staining, Flow Cytometry