Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: (A and B) Nuclear extracts containing activated (A) Stat3 from NIH3T3/v-Src fibroblasts or (B) Stat1, Stat3 and Stat5 from EGF-stimulated NIH3T3/hEGFR were pre-incubated with the designated hirsutinolides for 30 min at room temperature prior to incubating with the radiolabeled hSIE probe that binds Stat1 and Stat3 or MGFe probe that binds Stat1 and Stat5 and performing EMSA analysis; bands corresponding to STAT:DNA complexes in gel were quantified using ImageQuant and represented as percent of control (B (i), lower panel); (C and D) Stat3 DNA-binding activity with EMSA analysis using the hSIE probe that binds Stat3 of nuclear extracts prepared from U251MG or U373MG cells treated with the designated hirsutinolides at (C) 5 μM for 0–24 h or (D) 0–5 μM for 1.5 h; (E–G) Immunoblotting analysis of whole-cell lysates prepared from (E and F) U251MG cells treated with the designated hirsutinolides at (E) 5 μM for 0–24 h or (F) 0–5 μM for 2 h, or (G) human glioma patients-derived xenograft cells (i) G6-G102, untreated, or (ii) G22, treated with 5 μM for 0–24 h 6, 10, or 22 and probing for pStat3, Stat3 or GAPDH. Positions of proteins or DNA-bound STATs in gel are labeled; control lane (c, 0) represents whole-cell lysates or nuclear extracts prepared from 0.025% DMSO-treated cells or nuclear extracts pre-treated with 0.025% DMSO. Bands corresponding to Stat3:DNA complexes were scanned and quantified using ImageJ, plotted against concentration of agent from which IC50 values were derived. Data are representative of 2–3 independent determinations. *Position of supershifted complex.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Incubation, Binding Assay, Activity Assay, Western Blot, Derivative Assay, Labeling, Concentration Assay

Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: Compounds, and their side chains, inhibitory activities against U251MG, MDA-MB-231, NIH3T3 cell growth, and Stat3-inhibitory activity inside and outside of cells.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Activity Assay, Inhibition, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: (A–C) Immunoblotting analysis of whole-cell lysates of equal total protein prepared from (A) NIH3T3/hEGFR fibroblasts pre-treated with or without 5 μM of 6, 10, 20 or 22 for 3 h prior to stimulation with 1 μg/ml EGF for 12 min, (B) U251MG cells treated with 5 μM 6, 10 or 22 for 0–24 h, or (C) U251MG cells treated with 5 μM 6, 10 or 22 for 3 h in the absence or presence of 10–300 μM Na3VO4 and probing for pStat3, Stat3, pStat1, Stat1, pStat5, Stat5, pErk1/2, Erk1/2, pS-Akt, Akt, pJAK2, JAK2, SOCS3, PTP1B, β-actin or GAPDH. Positions of proteins in gel are labeled; control lanes (0, −) represent whole-cell lysates prepared from 0.025% DMSO-treated cells. Data are representative of 2–4 independent determinations.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Western Blot, Labeling

Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: (A) Molecular modeling and docking of 33, 31, 29, 22, 6 and 7 into the Stat3 DNA-binding domain pocket showing the interactions with key amino acid residues; (B and C) Overlay of the 1H-13C HMQC spectra of (B) wild-type Stat3, free (black) or bound to 100 μM (red) or 200 μM (green) 6 and 10, and (C) wild-type Stat3, free (red) or bound to 100 or 200 μM (blue) 7, 9, 11 or 22 and showing residues with significant changes in either resonance line-widths or NMR chemical shifts that are indicated by arrowheads; and (D) immunoblots of (i) FLAG or GAPDH, or (ii) pStat3 or Stat3 in whole-cell lysates from U251MG cells transiently-transfected with Stat3 FLAG-tagged N-terminal (NTD), Coiled coil (CCD), C-terminal (CTD) or DNA-binding (DBD) domain and treated with 5 μM 6 for 1.5 h. Positions of proteins in gel are labeled; control lane (−) represents whole-cell lysates prepared from cells treated with 0.025% DMSO. Data are representative of 2 independent determinations.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Binding Assay, Western Blot, Transfection, Labeling

Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: (A) Mice bearing U251MG subcutaneous tumor xenografts were administered 6 or 22 via oral gavage, 2 mg/kg or vehicle (1% DMSO) every other day for 33 days. Tumor sizes, measured every 3 days, were converted to tumor volumes and plotted against days of treatment; and (B) immunoblots of pStat3, Stat3 or GAPDH in tumor tissue lysates prepared from control and treated mice. Positions of proteins in gel are labeled; control lanes (c) represent tissue lysates prepared from mice treated with 1% DMSO. Values, mean ± S.D., n=6. * - <0.05.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Western Blot, Labeling

Journal: Journal of medicinal chemistry

Article Title: Hirsutinolide series inhibit Stat3 activity, alter GCN1, MAP1B, Hsp105, G6PD, vimentin, TrxR1, and importin α-2 expression, and induce antitumor effects against human glioma

doi: 10.1021/acs.jmedchem.5b00686

Figure Lengend Snippet: (A) Single-cell cultures of U251MG, SF295, MDA-MB-231, MCF7 and NIH3T3 cells treated once with 0–5 μM 6, 10 or 22 and allowed to grow until large colonies were visible, which were stained with crystal violet, counted and plotted. U251MG colony formation was completely inhibited by 1 μM 6 or 22; (B) 5-bromo-2′-deoxyuridine (BrdU) incorporation analysis for proliferating U251MG cells treated with 0 or 5 μM 6 or 22 for 3–24 h. Images were captured under a fluorescence microscope and analyzed with ImageJ software; (C) cultured U251MG cells were wounded, treated once with 0–5 μM 6, 10 or 22 and allowed to migrate to the denuded area over 19 h and imaged; (D) cell cycle distribution analysis of U251MG cells treated or untreated (DMSO) with 15 μM Cmpd1 for 24 or 72 h, processed by propidium iodide (PI) staining, and analyzed by flow cytometry for DNA content, which is plotted; and (D) immunoblots of pStat3, Stat3, c-Myc, Bcl-2, Bcl-xL, Mcl-1 or GAPDH from whole-cell lysates from U251MG cells treated with 5 μM 6 for 0–24 h. Positions of proteins in gel are shown; control lane (0) represents 0.025% DMSO-treated cells or whole-cell lysate preparation from 0.025% DMSO-treated cells. Values are the mean ± S.D., n=3–6. Data are representative of 2–3 independent determinations.

Article Snippet: Computational modeling predicts active hirsutinolides directly bind to Stat3 An unbiased computational modeling analysis of Stat3:hirsutinolide binding (MOE Site Finder, Molecular Operating Environment version 2013, Chemical Computing Group, Montreal, QC) mapped thirty-six potential binding sites within Stat3 (Stat3β/DNA complex, Protein Data Bank ID: 1BG1).

Techniques: Staining, BrdU Incorporation Assay, Fluorescence, Microscopy, Software, Cell Culture, Flow Cytometry, Western Blot