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. 2017 Mar 3;292(9):3552-3567.
doi: 10.1074/jbc.M116.747766. Epub 2016 Dec 29.

A Quininib Analogue and Cysteinyl Leukotriene Receptor Antagonist Inhibits Vascular Endothelial Growth Factor (VEGF)-independent Angiogenesis and Exerts an Additive Antiangiogenic Response with Bevacizumab

Clare T Butler  1 , Alison L Reynolds  1 , Miriam Tosetto  2 , Eugene T Dillon  1 , Patrick J Guiry  3 , Gerard Cagney  1 , Jacintha O'Sullivan  4 , Breandán N Kennedy  5
Affiliations

A Quininib Analogue and Cysteinyl Leukotriene Receptor Antagonist Inhibits Vascular Endothelial Growth Factor (VEGF)-independent Angiogenesis and Exerts an Additive Antiangiogenic Response with Bevacizumab

Clare T Butler et al. J Biol Chem. .

Abstract

Excess blood vessel growth contributes to the pathology of metastatic cancers and age-related retinopathies. Despite development of improved treatments, these conditions are associated with high economic costs and drug resistance. Bevacizumab (Avastin®), a monoclonal antibody against vascular endothelial growth factor (VEGF), is used clinically to treat certain types of metastatic cancers. Unfortunately, many patients do not respond or inevitably become resistant to bevacizumab, highlighting the need for more effective antiangiogenic drugs with novel mechanisms of action. Previous studies discovered quininib, an antiangiogenic small molecule antagonist of cysteinyl leukotriene receptors 1 and 2 (CysLT1 and CysLT2). Here, we screened a series of quininib analogues and identified a more potent antiangiogenic novel chemical entity (IUPAC name (E)-2-(2-quinolin-2-yl-vinyl)-benzene-1,4-diol HCl) hereafter designated Q8. Q8 inhibits developmental angiogenesis in Tg(fli1:EGFP) zebrafish and inhibits human microvascular endothelial cell (HMEC-1) proliferation, tubule formation, and migration. Q8 elicits antiangiogenic effects in a VEGF-independent in vitro model of angiogenesis and exerts an additive antiangiogenic response with the anti-VEGF biologic bevacizumab. Cell-based receptor binding assays confirm that Q8 is a CysLT1 antagonist and is sufficient to reduce cellular levels of NF-κB and calpain-2 and secreted levels of the proangiogenic proteins intercellular adhesion molecule-1, vascular cell adhesion protein-1, and VEGF. Distinct reductions of VEGF by bevacizumab explain the additive antiangiogenic effects observed in combination with Q8. In summary, Q8 is a more effective antiangiogenic drug compared with quininib. The VEGF-independent activity coupled with the additive antiangiogenic response observed in combination with bevacizumab demonstrates that Q8 offers an alternative therapeutic strategy to combat resistance associated with conventional anti-VEGF therapies.

Keywords: G protein-coupled receptor (GPCR); angiogenesis; calpain; cell migration; colorectal cancer; drug development; endothelial cell; leukotriene; pharmacology; vascular endothelial growth factor (VEGF).

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Conflict of interest statement

J. O. and B. N. K. are inventors on United States Patent 8916586 B2, and A. L. R., J. O., and B. N. K. are inventors on United States Patent 9388138 B2

Figures

FIGURE 1.
FIGURE 1.
Analogues more effectively inhibit developmental angiogenesis compared with quininib in vivo. A, structural skeleton of the quininib drug series and table listing the differences in chemical structures of all quininib analogues. A synthesis scheme for each chemical is included in Kennedy et al. (29). B, schematic of the intersegmental vessel assay. Male and female Tg(fli1:EGFP) zebrafish were in-crossed, yielding embryos that were treated with quininib analogues at 6 hpf. Larvae were dechorionated and fixed at 2 dpf, and intersegmental vessels were counted by visualizing under fluorescence microscopy. C, ranking graph of the bioactivity of 24 salt or amine analogue formulations comparing the ability of a 10 μm concentration of each analogue to inhibit developmental angiogenesis in the ISV assay using Tg(fli1:EGFP) zebrafish. D, fluorescence images of Tg(fli1:EGFP) treated with the most effective concentrations of analogues (0.1% DMSO, 10 μm sunitinib, 10 μm Q1, 10 μm Q22, 1 μm Q8, and 5 μm Q18). E, dose-response graph following rescreening of quininib analogues in the ISV assay at concentrations increasing from 0.1 to 10 μm. Individual experiments consisted of treating five embryos per well in duplicate (10 embryos), and individual experiments were conducted three times (n = 3). Statistical analysis was performed by ANOVA and Dunnett's or Bonferroni's post hoc multiple comparison test. Error bars are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
FIGURE 2.
FIGURE 2.
Quininib analogues reduce endothelial cell number after 24 h and inhibit endothelial cell migration. A, effect of 10 μm quininib analogues on HMEC-1 endothelial cell number using the MTT assay following 24-, 72-, and 96-h treatment. 10,000 cells were seeded and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). B, effect of 10 μm quininib analogues on HMEC-1 endothelial cell migration. 50,000 cells were seeded and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). C, effect of Q8 in combination with bevacizumab on HMEC-1 endothelial cell migration. 50,000 cells were seeded and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). Migration was assessed using the xCELLigence system and RTCA software allowing real time monitoring of cell migration over 8 h. The real time traces (B and C, right) represent averaged data of all detected/bound cells from 0 to 8 h. Statistical analysis was performed by ANOVA, Dunnett's post hoc multiple comparison test, and Student's t test. Error bars are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
FIGURE 3.
FIGURE 3.
Quininib analogues inhibit human endothelial cell tubule formation and do not affect HMEC-1 viability. A, total tubule length following tubule formation of HMEC-1 endothelial cells treated with 1, 3, or 10 μm quininib analogues for 16 h. 7,500 cells were seeded in μ-Slide angiogenesis wells and treated in duplicate for each individual experiment, and individual experiments were conducted six times (n = 6). B, representative tubule images following treatment of HMEC-1 endothelial cells with 10 μm Q1 or analogues Q8, Q18, and Q22 for 16 h. C, viability images of Q8 (0.1–20 μm)-treated HMEC-1 endothelial cell tubules stained with calcein AM (green) and propidium iodide (red). 7,500 cells were seeded in μ-Slide angiogenesis wells and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). Statistical analysis was performed by ANOVA and Dunnett's post hoc multiple comparison test. Error bars are mean ± S.E. **, p < 0.01; ***, p < 0.001.
FIGURE 4.
FIGURE 4.
Quininib analogues inhibit VEGF-independent angiogenesis in vitro. A, HMEC-1 endothelial cell tubule formation is not affected following 16-h treatment with bevacizumab alone; however, when cells are treated with a combination of recombinant VEGF and bevacizumab, a significant reduction in tubule formation occurs following 16 h. 7,500 cells were seeded in μ-Slide angiogenesis wells and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). B, representative tubule images of HMEC-1 cells treated with bevacizumab alone or in combination with recombinant VEGF. C, the effects of Q1 and analogue Q8 on HMEC-1 tubule formation are significantly diminished when cells are treated in combination with VEGF. 7,500 cells were seeded in μ-Slide angiogenesis wells and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). D, tubule images of HMEC-1 cells treated with Q1 or analogue Q8 alone or in combination with VEGF. Statistical analysis was performed by ANOVA and Dunnett's or Bonferroni's post hoc multiple comparison test and Student's t test. Error bars are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
FIGURE 5.
FIGURE 5.
Q8 has additive antiangiogenic effects with an anti-VEGF biologic, bevacizumab. A, HMEC-1 endothelial cell tubule formation was significantly reduced following 16-h treatment with analogue Q8 in combination with bevacizumab compared with treatment with Q8 or bevacizumab alone. Q1 did not significantly reduce tubule formation in combination with bevacizumab compared with treatment with quininib or bevacizumab alone. 7,500 cells were seeded in μ-Slide angiogenesis wells and treated in duplicate for each individual experiment, and individual experiments were conducted three times (n = 3). B, representative tubule images of HMEC-1 endothelial cells treated with analogue Q8 or Q1 alone or in combination with bevacizumab for 16 h. Statistical analysis was performed by ANOVA and Dunnett's or Bonferroni's post hoc multiple comparison test and Student's t test. Error bars are mean ± S.E. *, p < 0.05; **, p < 0.01; ns, not significant.
FIGURE 6.
FIGURE 6.
Quininib analogue Q8 is a cysteinyl leukotriene receptor-1 antagonist that affects inflammatory and angiogenic signaling pathways. A, the CysLT1 receptor IC50 of analogue Q8 was determined to be 4.9 μm following testing in a cell-based CysLT1 receptor antagonist assay in CHO cells (Eurofins Cerep SA). The assay measured calcium mobilization using fluor-3-loaded CHO cells overexpressing CysLT1 and stimulated with the CysLT1 receptor antagonist LTD4. 20 μm Q8 inhibited CysLT1 activation by 105.4%. In contrast, 30 μm Q8 produced only a 22.9% antagonism of CysLT2 receptor activation in HEK-293 cells. Thus, Q8 was excluded as a tangible CysLT2 antagonist as results showing an inhibition lower than 50% were not considered significant. Treatment of cells for individual experiments was carried out in duplicate, and each experiment was conducted 3 times (n = 3). B, CysLT1 is present in our angiogenesis models shown by the presence of the CysLT1 transcript in zebrafish larvae at 6 hpf, 1 dpf, 2 dpf, and 3 dpf following PCR and the presence of the nuclear form of the receptor in HMEC-1 endothelial cells by Western blotting. PCR was carried out using RNA isolated from larvae during three separate experiments (n = 3), and Western blotting was conducted three separate times using protein lysates from cells treated in duplicate during three separate experiments (n = 3). C, Western blot of HMEC-1 cells treated for 5 h with 0.1% DMSO or 10 μm Q1, Q8, and montelukast. Expression of calpain-2, a putative calcium-sensitive downstream target of CysLT1 signaling and proangiogenic mediator, is reduced following treatment of endothelial cells with 10 μm Q8. Calpain-2 Western blotting was conducted on protein lysates from cells treated during three separate experiments (n = 3). D, the levels of activated NF-κB p65 were significantly reduced following treatment of HMEC-1 cells with 10 μm Q8 for 5 h, whereas treatment of cells with 10 μm Q1 or the clinically used CysLT1 antagonist montelukast for 5 h had no effect on levels of activated NF-κB p65. NF-κB p65 ELISA was conducted using protein isolated from cells treated on three separate occasions in duplicate (n = 3). E, ELISA of HMEC-1 endothelial cell-conditioned medium following 16-h treatment of cells with 10 μm Q1, Q8, Q22, and Q18 revealing significant reductions in important proangiogenic mediators angiopoietin-2, VEGF, ICAM-1, and VCAM-1. Compared with other analogues of Q1, Q8, our highest ranking analogue, was the only compound to significantly reduce soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1), and VEGF. Conditioned medium collected from three separate experiments was analyzed in duplicate by ELISA (n = 3). Statistical analysis was performed by ANOVA, Dunnett's post hoc multiple comparison test, and Student's t test. Error bars are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 7.
FIGURE 7.
Proposed effects of combined targeting of CysLT1 and VEGFR2 during angiogenesis. Signaling schematic of CysLT1 and VEGFR2. Following antagonism of CysLT1, Q8 inhibits many of the instrumental processes that regulate angiogenesis such as cell motility, cytoskeletal reorganization, proliferation, and survival. Ca2+ influxes following Q8 antagonism are prevented, and the calcium-activated cysteine endopeptidase calpain-2 can no longer facilitate IκBα degradation. Hence, NF-κB is stabilized and will not translocate to the nucleus to induce expression of the angiogenic mediator VEGF. Additionally, antagonism of CysLT1 by Q8, as demonstrated by ELISA, decreases the expression of ICAM-1 and VCAM-1, preventing endothelial cell motility and endothelial cell precursor recruitment. The enhanced additive antiangiogenic activity of Q8 is likely due to dual down-regulation of VEGF by Q8 and bevacizumab and the exclusive effects of Q8 on secreted levels of ICAM-1 and VCAM-1. IP3, inositol trisphosphate; PLC, phospholipase C; APC, adenomatous polyposis coli; TCF, T cell factor; LEF, lymphoid enhancer factor; β-cat, β-catenin. The table at the bottom of the figure denotes the percentage of decrease in secretion of ICAM-1, VCAM-1, and VEGF following treatment of human microvascular endothelial cells with either Q8 or bevacizumab, as determined by ELISA.
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