Journal: RSC Chemical Biology

Article Title: A stress-responsive p38 signaling axis in choanoflagellates †

doi: 10.1039/d4cb00122b

Figure Lengend Snippet: High-throughput screening of a small molecule library revealed inhibitors of S. rosetta cell proliferation. (A) Treatment of S. rosetta cultures with 1255 different small molecules (see Table S1, ESI ) resulted in a distribution of cell counts, assessed by flow cytometry, at the 24-hour endpoint. S. rosetta cell counts were normalized to the average of DMSO controls within the same plate (dark grey). Compounds determined to significantly inhibit S. rosetta cell proliferation by flow cytometry (based on two-tailed p -value <0.05 calculated from z -score), fall below the dotted line and are indicated in red. Compounds that were not detected as significant inhibitors by flow cytometry but were identified by imaging (based on two-tailed p -value <0.05 calculated from z -score) are in blue. Compounds that were not significant inhibitors for either screen are indicated in light grey. Sorafenib (SO), a focus of this study, is labeled. (B) The range of normalized cell counts measured by flow cytometry for compounds that significantly inhibited S. rosetta cell proliferation. Compounds that were the focus of further study – genistein (GE), glesatinib (GL), PP121, masitinib (MA), sotrastaurin (SOT) – are labeled. (C) Comparison of normalized values of compounds that inhibited S. rosetta cell proliferation, assessed by flow cytometry and the corresponding normalized values determined by imaging. Compounds determined to significantly inhibit S. rosetta cell proliferation (based on two-tailed p -value <0.05 calculated from z -score) by flow cytometry fall below the dotted line on the y -axis and by imaging, to the left of the dotted line on the x -axis.

Article Snippet: Glesatinib (#HY-19642A), masitinib (#HY-10209), lapatinib (#HY-50898), PP121 (#HY-10372), gliteritinib (#HY-12432), brigatinib (#HY-12857), RAF265 (#HY-10248), vemurafenib (#HY12057), skepinone-L (#HY-15300), BIRB 796 (#HY-10320), were purchased from MedChem Express.

Techniques: High Throughput Screening Assay, Flow Cytometry, Two Tailed Test, Imaging, Labeling, Comparison

Journal: RSC Chemical Biology

Article Title: A stress-responsive p38 signaling axis in choanoflagellates †

doi: 10.1039/d4cb00122b

Figure Lengend Snippet: Glesatinib and sorafenib, two multi-target tyrosine kinase inhibitors, disrupt S. rosetta cell proliferation and tyrosine phosphosignaling. (A) Treatment of S. rosetta cultures with 1 μM sorafenib and glesatinib led to a complete block of cell proliferation, while treatment with 1 μM masitinib or PP121 led to a partial reduction in cell proliferation relative to DMSO-treated cultures. Two biological replicates were conducted per treatment, and each point represents the mean of three measurements from each biological replicate. For timepoints at 40, 60, and 85 hours, cell densities of inhibitor-treated cultures were significantly different from vehicle (DMSO) ( p -value <0.01). Significance was determined by a two-way ANOVA multiple comparisons test. (B) S. rosetta cultures treated with 1 μM or 10 μM sorafenib, glesatinib, or PP121 for 24 hours had reduced normalized cell density, whereas masitinib only had reduced normalized cell density at 10 μM. Normalized cell densities were determined to be reduced if differences between treatments and vehicle (DMSO) were significant ( p -value <0.01) Significance was determined by determined by a two-way ANOVA multiple comparisons test. Movies show S. rosetta cells treated with 10 μM glesatinib that undergo cell lysis (Movie S1, ESI ) and sorafenib, that have cell body deformation (Movies S2 and S3, ESI ), in comparison to DMSO control (Movie S4, ESI ). (C) Western blot analysis of S. rosetta cultures treated with 1 μM sorafenib and glesatinib for 1 hour showed a decrease in tyrosine phosphorylation of proteins at ∼60 kDa, ∼45 kDa, and ∼35 kDa (indicated by arrows and detected with pY1000 anti-phosphotyrosine antibody) compared to vehicle (DMSO) control. Masitinib and PP121 did not reduce the phosphotyrosine signal.

Article Snippet: Glesatinib (#HY-19642A), masitinib (#HY-10209), lapatinib (#HY-50898), PP121 (#HY-10372), gliteritinib (#HY-12432), brigatinib (#HY-12857), RAF265 (#HY-10248), vemurafenib (#HY12057), skepinone-L (#HY-15300), BIRB 796 (#HY-10320), were purchased from MedChem Express.

Techniques: Blocking Assay, Lysis, Comparison, Control, Western Blot, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: The structure of glesatinib and its cytotoxic effects to three P-gp overexpressing cancer cells. (A) Chemical structure of glesatinib. (B) Concentration-dependent viability curves for KB-3-1 and KB-C2 cell lines incubated with different concentration of glesatinib for 72 h. (C) Concentration-dependent viability curves for SW620 and SW620/Ad300 cell lines incubated with different concentration of glesatinib for 72 h. (D) Concentration-dependent viability curves for HEK293/pcDNA3.1 and HEK293/ABCB1 cells incubated with different concentration of glesatinib for 72 h. The cell viability was determined by MTT assay. Data are expressed as mean ± SD, and representative of three independent experiments in triplicate are shown.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Concentration Assay, Incubation, MTT Assay

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib sensitized paclitaxel, colchicine, and doxorubicin to P-gp-overexpressing cell lines (KB-C2 and HEK293/ABCB1 cells).

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques:

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib sensitized paclitaxel, colchicine, and doxorubicin to P-gp-overexpressing cell line (SW620/Ad300 cells), but not topotecan to ABCG2-overexpressing cells (NCI-H460/MX20 cells).

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques:

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib did not affect the protein expression of P-gp transporters in ABCB1 overexpressing cell lines. Detection and relative intensity of ABCB1 expression in KB-C2 cells incubated with 0.3, 1, 3 μM for 72 h and 3 μM for 0, 24, 48, 72 h. Data are mean ± SD, representative of three independent experiments. * p < 0.05, compared with control group.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Expressing, Incubation, Control

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib did not affect the localization of ABCB1 transporters in ABCB1 overexpressing cell lines. Sub-cellular localization of ABCB1 expression in SW620/Ad300 cells incubated with 3 μM of glesatinib for 0, 24, 48, and 72 h. ABCB1, green and DAPI (blue) counterstains the nuclei. SW620 cells represented the control group.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Expressing, Incubation, Control

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib increased the accumulation and inhibited the efflux of [ 3 H]-paclitaxel in P-gp overexpressing KB-C2 cells. (A) The effect of glesatinib on the accumulation of [ 3 H]-paclitaxel in KB-3-1 and KB-C2 cell lines. (B) The effect of glesatinib on efflux of [ 3 H]-paclitaxel in KB-3-1 and (C) KB-C2. Verapamil (3 μM) was used as positive controls. Data are mean ± SD, representative of three independent experiments. * p < 0.05, compared with control group. Gle, Glesatinib; Vera, verapamil.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Control

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: Glesatinib stimulated the ATPase activity of P-gp. Effect of various concentrations of glesatinib on the ATPase activity of P-gp. The inset graphs illustrate the effect of 0–10 μM glesatinib on the ATPase activity of P-gp. Data are mean ± SD, representative of three independent experiments.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Activity Assay

Journal: Frontiers in Oncology

Article Title: Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

doi: 10.3389/fonc.2019.00313

Figure Lengend Snippet: The molecular modeling study of glesatinib with human homology ABCB1. (A) Overall view of glesatinib-P-gp complex. (B) 3D figure of Docked position of glesatinib within the drug-binding site of human P-gp homology model. Glesatinib was showed as ball and stick mode with the atoms colored: carbon-cyan, nitrogen-blue, oxygen-red, fluorine-green, sulfur-yellow, hydrogen-purple. Important residues were showed as sticks, with the color pattern: carbon-gray, nitrogen-blue, oxygen-red, hydrogen-purple. π-π stacking interactions are indicated with cyan dotted line. π-cation bond is indicated with green dotted line. Hydrogen bonds were showed by the yellow dotted line. (C) 2D figure of Docked position of glesatinib within the drug-binding site of human P-gp homology model. The cyan bubbles indicate polar residues and the green bubbles indicate hydrophobic residues. Hydrogen bonds are shown by the purple dotted arrow. π-π stacking interactions are shown by the green lines and π-cation bond is indicated with red line.

Article Snippet: Glesatinib (99% purity as measured by high performance liquid chromatography) was purchased from ChemieTek (Indianapolis, IN).

Techniques: Binding Assay