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,Alison L Reynolds,Miriam Tosetto,Eugene T Dillon,Patrick J Guiry,Gerard Cagney,Jacintha O'Sullivan,Breandán N Kennedy

doi:10.1074/jbc.m116.747766

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.

Highlights

Excess blood vessel growth contributes to the pathology of metastatic cancers and age-related retinopathies
The VEGFindependent 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-vascular endothelial growth factor (VEGF) therapies
Further to what we previously reported regarding the mechanism of action of quininib [22], the structurally distinct Q8 analogue significantly reduces cellular levels of proangiogenic signals NF-␬B and calpain-2 and secreted levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and VEGF compared with quininib

Summary

Results

Analogues Have Enhanced Antiangiogenic Effects in Vivo Compared with Quininib (Q1)—The small molecule Q1 was previously identified to inhibit ocular angiogenesis in the zebrafish hyaloid vascular assay and tumor angiogenesis [22,23,24]. Preliminary analyses of 37 structural quininib analogues identified drugs that robustly inhibited developmental angiogenesis in larval zebrafish eyes [29] In these analogues, the position of the phenyl ring hydroxy group and/or the linkage between the quinoline and phenyl ring was modified (Fig. 1A). We ranked the bioactivity of 24 salt or amine analogue formulations by comparing the ability of a 10 ␮M concentration of the drugs to inhibit developmental angiogenesis in the intersegmental vessel (ISV) assay using Tg(fli: EGFP) zebrafish (Fig. 1, B and C). Twelve analogue formulations produced a statistically significant inhibition of developmental angiogenesis compared with control (Fig. 1C). C, ranking graph of the bioactivity of 24 salt or amine analogue formulations comparing the ability of a 10 ␮M concentration of each analogue to inhibit developmental angiogenesis in the ISV assay using Tg(fli1:EGFP) zebrafish. 2.5 ␮g/␮l bevacizumab in combination with 3 ␮M Q8 significantly reduced migration compared with control (p Ͻ 0.046) and with 2.5 ␮g/␮l bevacizumab (p Ͻ 0.039) but not with 3 ␮M Q8 alone

Total score

Discussion

Experimental Procedures