Research ArticleMOLECULAR BIOLOGY

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB–VCAM-1 axis

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eaaz7815
DOI: 10.1126/sciadv.aaz7815
  • Fig. 1 BT2, T4, and T6 block endothelial FosB/ΔFosB expression, proliferation, migration and wound repair after in vitro injury and network formation.

    (A) HMEC-1 cells were serum-arrested for 20 hours, treated with 30 μM compound in serum-free medium (SFM) (without EGF/hydrocortisone) for 4 hours, and stimulated with 10% fetal bovine serum (FBS) (with EGF/hydrocortisone) and compound (same concentration) for 1 hour. Western blotting was then performed with independent biological duplicates where indicated. Approximate positions of molecular weight markers are shown. Data represent three biologically independent experiments. Veh, vehicle. (B) Serum-deprived HMEC-1 cells were treated with compound in 5% FBS (with EGF/hydrocortisone), and proliferation was monitored using xCELLigence. Top: Representative real-time profiles from one experiment. Cell index is a quantitative measure of cell growth. Bottom: Data represent means ± SEM of five to eight independent experiments after 79 hours. One-way analysis of variance (ANOVA). (C) BAECs in 10% FBS were seeded into wells containing 0.8-μm Transwell inserts. After 48 hours, medium was changed to 0.01% FBS for 48 hours. Compounds (1 μM) were added to the upper chamber in 0.01% FBS, and medium in the lower chamber was changed to 10% FBS and VEGF-A165 (50 ng/ml). Cells were left for 24 hours. Typical 4′,6-diamidino-2-phenylindole–stained nuclei are shown. Nuclei were quantified with National Institutes of Health (NIH) ImageJ. Data represent means ± SEM of four to five independent experiments. Kruskal-Wallis multiple comparisons test. (D) Scraped HMEC-1 monolayers were treated with 0.6 μM compound in 5% FBS. Regrowth in denuded area was measured 48 hours after scraping. Representative regrowth from one experiment is shown. Regrowth area was determined with Image-Pro Plus. Data represent means ± SEM of five independent experiments. One-way ANOVA. (E) HMEC-1 cells in 1% FBS and fibroblast growth factor 2 (FGF-2; 50 ng/ml) were mixed with compound (3 μM) and seeded in wells coated with Matrigel. Network formation over 24 hours was quantified using Image-Pro Plus. Data represent means ± SEM of five to six independent experiments. Kruskal-Wallis multiple comparisons test. Representative network formation on Matrigel from same control well over time is shown. Where indicated, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 BT2 inhibits retinal vascular permeability and inhibits CD31, VEGF-A165, pERK, FosB/ΔFosB, and VCAM-1.

    (A) Compound or vehicle (control) was injected intravitreally in rat eyes twice, Kenacort once, and Aflibercept/Eylea six times following six laser burns of retina. On days 14 and 21, sodium fluorescein was subcutaneously injected. After 10 min, ocular fluorescence was recorded using Heidelberg retinal angiography (HRA) and scored, combining days 14 and 21 data. Data represent means ± SEM. One-way ANOVA (plot at left) or t test (plot at right and BT2 versus Kenacort in plot at left). n = 5 to 29 per group. (B) Compound (600 μg) or vehicle was injected intravitreally into the right (R) eyes of rabbits 5 days before intravitreal injection of 500 ng of rhVEGF-A165 in 50 μl in same eyes. After 2 days, sodium fluorescein was injected intravenously, and after 1 hour, ocular fluorescence measured in R and left (L) eyes and expressed as ratio (R/L) for each rabbit. Ratio data from vehicle and BT3 groups pooled (control) as both conditions were inactive and did not differ statistically, for comparison with BT2. Data represent means ± SEM. t test, n = 6 to 8 per group. Immunohistochemical staining in rat retinal lesions for (C) CD31, (D) VEGF-A165, and (E) VEGF-A165 in 100-μm boxed increments relative to wound. Untreated refers to eyes not lasered or injected. Slides photographed under 10× or 20× objective and magnified views are shown. n = 4 to 6 per group for CD31, n = 3 to 6 per group for VEGF-A165, or n = 3 to 5 per group for VEGF-A165 gradient analysis. Data represent means ± SEM of mean/animal. One-way ANOVA, Mann-Whitney, or t test, as appropriate. Arrows indicate positive staining. (F) Matrigel (500 μl) containing VEGF-A165 (100 ng/ml), heparin (10 U), and compound (2.5 mg per mouse) or vehicle injected subcutaneously into left flanks of mice. Plugs after 7 days stained for CD31. Representative images under 10× objective with inset providing magnified view (40×). Data represent means ± SEM of mean/animal. Kruskal-Wallis multiple comparisons test. n = 10 to 11 per group. ns, not significant; IOD, integrated optical density; OPL, outer plexiform layer; ONL, outer nuclear layer. Where indicated, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 BT2 inhibits ERK phosphorylation, FosB/ΔFosB, and VCAM-1 expression.

    (A) HMEC-1 cells treated with 30 μM compound were stimulated with IL-1β (20 ng/ml) for times indicated. Western blots representative of two to three biologically independent experiments each performed with two biologically independent replicates run in separate lanes (where shown). (B) Flow cytometry performed with HMEC-1 treated with 30 μM compound and IL-1β (20 ng/ml) for 4 hours. Data represent means ± SEM of three independent experiments. One-way ANOVA. n = 3 per group. (C) RNA-seq performed with total RNA prepared from HMEC-1 pretreated with 30 μM BT2 and IL-1β (20 ng/ml) for 4 hours. PCA plot (top left) shows close association between biological replicates within untreated (UT), IL-1β, and IL-1β + BT2 and clear separation across conditions. Heat map (center, 1579 genes) was generated for all up-regulated genes for comparison of IL-1β versus UT. Counts per million (cpm) values used and genes (rows) grouped using hierarchical clustering with cpm for FosB and VCAM-1 are plotted. Heat map (right) shows 325 genes with logFC ≥ 2. FosB, c-Fos, and VCAM-1 are indicated in the figure together with other genes inhibited by BT2. The figure also shows small subset of genes (in red) further induced by BT2. (D) BT2 inhibits IL-1β–inducible VCAM-1 expression and ERK phosphorylation more potently than PD98059. Concentrations of BT2 and PD98059 (1 to 30 μM) indicated. Data represent three biologically independent experiments. SMI, small molecule inhibitor. (E) HMEC-1 treated with 0.6 μM siRNA then stimulated with IL-1β (20 ng/ml) for 2 or 4 hours. Western blotting was performed with indicated antibodies. Data are representative of two biologically independent experiments. Approximate positions of molecular weight markers are shown. Where indicated, ***P < 0.001.

  • Fig. 4 BT2 inhibits ERK phosphorylation, FosB/ΔFosB, and VCAM-1 expression in retinas and Matrigel plugs.

    Immunohistochemical staining in retinal lesions was performed for (A) pERK, (B) FosB, and (C) VCAM-1. IOD of positive staining (red chromogen) was assessed using Image-Pro Plus software. Slides were photographed under 20× or 40× objective, and magnified views are shown. n = 3 to 5 per group for pERK and FosB and n = 4 to 6 per group for VCAM-1. Data represent means ± SEM of the mean/animal. Statistical significance was assessed by one-way ANOVA, Kruskal-Wallis, Mann-Whitney, or t test, as appropriate. Arrows provide examples of positive staining. Alternatively, Matrigel plugs were stained for (D) FosB or (E) VCAM-1. Representative FosB or VCAM-1 staining was photographed under 40× or 20× objective, respectively, with the inset providing a magnified view. Staining was quantified using Image-Pro Plus software. Data represent means ± SEM. Statistical significance was assessed by Kruskal-Wallis multiple comparisons test (FosB, n = 9 to 11 per group) or one-way ANOVA (VCAM-1, n = 10 to 11 per group). Arrows provide examples of positive staining. Where indicated, *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Carbamate moiety in BT2 is critical to its interaction with MEK1 and functional effects.

    (A) Serum-deprived HMEC-1 cells were treated with compound (0.4 or 0.8 μM) in 5% FBS, and proliferation was monitored using xCELLigence. Left: Representative growth profiles from one experiment. Right: xCELLigence data representing means ± SEM of three independent experiments after 79 hours. One-way ANOVA or Mann-Whitney test. (B) HMEC-1 network formation in 1% FBS and FGF-2 (50 ng/ml) with compound (1 μM final) in Matrigel-coated wells. Networks quantified with NIH ImageJ. Data represent means ± SEM of three to four independent experiments. Kruskal-Wallis multiple comparisons test. (C) SPR analysis testing interaction of PD98059, BT2, and BT2 analogs with His-MEK1 (left) and His-MEK2 (right). Also shown are fits to equilibrium data for binding of PD98059 and BT2 to MEK1 (top left) and for binding of PD98059 to MEK2 (top right). Data were fitted using a simple Langmuir 1:1 binding isotherm in Biacore software. Measurements made on a Biacore T200 at 15°C in buffer comprising 20 mM Hepes, 150 mM NaCl, and 5% DMSO (pH 7.4). Data are representative of two independent experiments. (D) HMEC-1 cells were treated with 1 μM compound (BT2 and analogs) in serum-free medium for 4 hours. Medium was changed to IL-1β (20 ng/ml) with compound and incubated for 15 min. Western blotting was performed for pERK or total ERK. Data are representative of two biologically independent experiments. Approximate positions of molecular weight markers are shown. Where indicated, *P < 0.05, **P < 0.01, ****P < 0.0001.

  • Table 1 Characteristics of BT2 and Cpd X/B/LK001 and their analogs.

    N/A, not applicable.

    CompoundChemical nameChemical
    formula
    Molecular
    weight
    (Da)
    ClassCAS no.Chemical structure
    BT2(10-Ethyl-11-oxo-10,11-
    dihydro-dibenzo[b,f][1,4]
    oxazepin-2-yl)-carbamic
    acid ethyl ester
    C18H18N2O4326.35DibenzoxazepinoneCAS 922029-50-3Embedded Image
    BT32-Amino-10-
    ethyldibenzo[b,f] [1,4]
    oxazepin-11 (10H)-one
    C15H14N2O2254.28DibenzoxazepinoneCAS 23474-61-5Embedded Image
    CpdX
    CpdB
    LK001
    2-Methoxyethyl[[[4-(4-
    chlorobenzoyl)phenyl]
    amino]carbonyl]
    carbamate
    C18H17ClN2O5376.79Benzophenone351068-74-1Embedded Image
    T42-Methoxyethyl[[[4-(4-
    chlorophenyl)
    (hydroxyimino)methyl)
    phenyl]amino]carbonyl]
    carbamate
    C18H18ClN3O5391.81BenzophenoneN/AEmbedded Image
    T6FlubendazoleC16H12FN3O3313.28BenzophenoneCAS 31430-15-6Embedded Image
    T7(4-Aminophenyl)
    (4-fluorophenyl)
    methanone
    C13H10FNO215.22BenzophenoneCAS 10055-40-0Embedded Image
    BT2-MeOAN-(10-ethyl-11-oxo-10,11-
    dihydrodibenzo[b,f][1,4]
    oxazepin-2-yl)-2-
    methoxyacetamide
    C18H18N2O4326.35DibenzoxazepinoneCAS 922029-59-2Embedded Image
    BT2-EOMeEthyl
    (10-(2-methoxyethyl)-11-
    oxo-10,11-
    dihydrodibenzo[b,f][1,4]
    oxazepin-2-yl)carbamate
    C19H20N2O5356.37DibenzoxazepinoneN/AEmbedded Image
    BT2-PrEthyl (11-oxo-10-propyl-
    10,11-dihydrodibenzo[b,f]
    [1,4]oxazepin-2-yl)
    carbamate
    C19H20N2O4340.37DibenzoxazepinoneCAS 922029-50-3Embedded Image
    BT2-ICIsobutyl (10-ethyl-11-oxo-
    10,11-dihydrodibenzo[b,f]
    [1,4]oxazepin-2-yl)
    carbamate
    C19H20N2O4340.37DibenzoxazepinoneN/AEmbedded Image
    BT2-IMOEthyl (11-(oxetan-3-
    ylmethoxy)dibenzo[b,f]
    [1,4]oxazepin-2-yl)
    carbamate
    C20H20N2O5368.38DibenzoxazepinoneN/AEmbedded Image
    BT2-MOEthyl (10-(oxetan-3-
    ylmethyl)-11-oxo-10,11-
    dihydrodibenzo[b,f][1,4]
    oxazepin-2-yl)carbamate
    C20H20N2O5368.38DibenzoxazepinoneN/AEmbedded Image
    BT2-deutEthyl (10-ethyl(2′,2′,2′-d3)-
    11-oxo-10,11-
    dihydrodibenzo[b,f][1,4]
    oxazepin-2-yl)carbamate
    C18H15D3N2O4329.37DibenzoxazepinoneN/AEmbedded Image

Supplementary Materials

  • Supplementary Materials

    Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB–VCAM-1 axis

    Yue Li, Ahmad M. N. Alhendi, Mei-Chun Yeh, Mina Elahy, Fernando S. Santiago, Nandan P. Deshpande, Ben Wu, Enoch Chan, Shafqat Inam, Leonel Prado-Lourenco, Jessica Marchand, Rohan D. Joyce, Lorna E. Wilkinson-White, Mark J. Raftery, Meidong Zhu, Samuel J. Adamson, François Barnat, Karen Viaud-Quentric, Jim Sockler, Joel P. Mackay, Andrew Chang, Paul Mitchell, Sebastian M. Marcuccio, Levon M. Khachigian

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