Advertisement

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis*

  • Alison L. Reynolds
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Yolanda Alvarez
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Temitope Sasore
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Nora Waghorne
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Clare T. Butler
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Claire Kilty
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Andrew J. Smith
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Carmel McVicar
    Affiliations
    Centre for Experimental Medicine, Queen's University Belfast, Wellcome-Wolfson Building, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom
    Search for articles by this author
  • Vickie H.Y. Wong
    Affiliations
    Centre for Experimental Medicine, Queen's University Belfast, Wellcome-Wolfson Building, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom
    Search for articles by this author
  • Orla Galvin
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Stephanie Merrigan
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Janina Osman
    Affiliations
    Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, 20502 Malmö, Sweden
    Search for articles by this author
  • Gleb Grebnev
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Anita Sjölander
    Affiliations
    Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, 20502 Malmö, Sweden
    Search for articles by this author
  • Alan W. Stitt
    Affiliations
    Centre for Experimental Medicine, Queen's University Belfast, Wellcome-Wolfson Building, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom
    Search for articles by this author
  • Breandán N. Kennedy
    Correspondence
    To whom correspondence should be addressed: F062 UCD Conway Institute, UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: 353-1-716-6740.
    Affiliations
    University College Dublin School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
    Search for articles by this author
  • Author Footnotes
    * This work was supported by funding from an Enterprise Ireland proof of concept award, an Enterprise Ireland Commercialisation Fund award, a Health Research Board Ireland Health Research Award, and a Science Foundation Ireland Technology innovation development award. B. N. K. and Y. A. are authors on granted patent WO2012095836 A1. A. R., C. K., and B. N. K. are authors on patent filing WO2014012889 A1.
Open AccessPublished:February 04, 2016DOI:https://doi.org/10.1074/jbc.M115.710665
      Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge DiversetTM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μm in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1–2) at micromolar IC50 values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.

      Introduction

      In the eye, developmental angiogenesis is a critical biological process enabling vision (
      • Fruttiger M.
      Development of the retinal vasculature.
      ). Morphogenesis of the retinal vasculature is strictly controlled to balance high metabolic requirements while maintaining visual function. Uncontrolled pathological angiogenesis in the retina, choroid, and iris results in proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinal vein occlusion, and retinopathy of prematurity, which are leading causes of blindness worldwide (
      • Cheung N.
      • Mitchell P.
      • Wong T.Y.
      Diabetic retinopathy.
      ,
      • Jager R.D.
      • Mieler W.F.
      • Miller J.W.
      Age-related macular degeneration.
      ,
      • Hellström A.
      • Smith L.E.
      • Dammann O.
      Retinopathy of prematurity.
      ,
      • Rehak M.
      • Wiedemann P.
      Retinal vein thrombosis: pathogenesis and management.
      ). Our understanding of the endogenous and exogenous factors regulating the overlapping but distinct phenotypes of developmental and pathological ocular angiogenesis is limited (
      • Gariano R.F.
      • Gardner T.W.
      Retinal angiogenesis in development and disease.
      ). Unbiased, phenotype-based chemical screens provide an opportunity to efficiently identify novel pharmacological inhibitors of angiogenesis in the eye (
      • Alvarez Y.
      • Astudillo O.
      • Jensen L.
      • Reynolds A.L.
      • Waghorne N.
      • Brazil D.P.
      • Cao Y.
      • O'Connor J.J.
      • Kennedy B.
      Selective inhibition of retinal angiogenesis by targeting PI3 kinase.
      ,
      • Kitambi S.S.
      • McCulloch K.J.
      • Peterson R.T.
      • Malicki J.J.
      Small molecule screen for compounds that affect vascular development in the zebrafish retina.
      ). These drugs and their molecular targets enhance our fundamental knowledge of the signaling networks regulating ocular angiogenesis and highlight alternative therapeutic interventions for angiogenesis-related disease.
      An intricate balance of growth and inhibitory factors regulates angiogenesis (
      • Folkman J.
      ). Imbalances can result in growth of abnormal, leaky vessels (
      • Gariano R.F.
      • Gardner T.W.
      Retinal angiogenesis in development and disease.
      ). Pathological angiogenesis is a hallmark of blinding ocular neovascular disease (
      • Jager R.D.
      • Mieler W.F.
      • Miller J.W.
      Age-related macular degeneration.
      ,
      • Gariano R.F.
      • Gardner T.W.
      Retinal angiogenesis in development and disease.
      ), including neovascular age-related macular degeneration, which affects 2.6 million European Union and United States patients and whose prevalence is increasing because of an aging population (

      Frost & Sullivan (June 7, 2011) Analysis of the US Retinal Therapeutics Market: Improvements in Administration and Efficacy Drive Growth, Frost & Sullivan Report NC77–52

      ,

      Frost & Sullivan (July 3, 2010) European Ophthalmic Pharmaceuticals Market, Frost & Sullivan Report M4AC-52

      ). Ocular neovascular disorders are regularly treated with biological agents (e.g. ranibizumab, bevacizumab, and aflibercept) targeting VEGF, a key proangiogenic mediator (
      • Martin D.F.
      • Maguire M.G.
      • Ying G.S.
      • Grunwald J.E.
      • Fine S.L.
      • Jaffe G.J.
      CATT Research Group
      Ranibizumab and bevacizumab for neovascular age-related macular degeneration.
      ,
      • Heier J.S.
      • Brown D.M.
      • Chong V.
      • Korobelnik J.-F.
      • Kaiser P.K.
      • Nguyen Q.D.
      • Kirchhof B.
      • Ho A.
      • Ogura Y.
      • Yancopoulos G.D.
      • Stahl N.
      • Vitti R.
      • Berliner A.J.
      • Soo Y.
      • Anderesi M.
      • Groetzbach G.
      • Sommerauer B.
      • Sandbrink R.
      • Simader C.
      • Schmidt-Erfurth U.
      VIEW 1 and VIEW 2 Study Groups
      Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration.
      ,
      • Rosenfeld P.J.
      • Brown D.M.
      • Heier J.S.
      • Boyer D.S.
      • Kaiser P.K.
      • Chung C.Y.
      • Kim R.Y.
      MARINA Study Group
      Ranibizumab for neovascular age-related macular degeneration.
      ). Unfortunately, many patients do not respond clinically or become refractory to treatment (
      • Rofagha S.
      • Bhisitkul R.B.
      • Boyer D.S.
      • Sadda S.R.
      • Zhang K.
      SEVEN-UP Study Group
      Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP).
      ). For example, ∼46% of diabetic macular edema patients require additional laser treatment, and limited improvement in visual acuity is achieved (
      • Elman M.J.
      • Qin H.
      • Aiello L.P.
      • Beck R.W.
      • Bressler N.M.
      • Ferris 3rd, F.L.
      • Glassman A.R.
      • Maturi R.K.
      • Melia M.
      Diabetic Retinopathy Clinical Research Network
      Intravitreal ranibizumab for diabetic macular edema with prompt vs. deferred laser treatment: 3-year randomized trial results.
      ). This, coupled with an undesirable intraocular delivery route, large clinical burden and expensive biological therapies highlight the need for improved ocular neovascular therapeutic agents (

      Frost & Sullivan (June 7, 2011) Analysis of the US Retinal Therapeutics Market: Improvements in Administration and Efficacy Drive Growth, Frost & Sullivan Report NC77–52

      ).
      The retina is a highly metabolically active tissue needing significant nourishment and oxygen supply (
      • Wangsa-Wirawan N.D.
      • Linsenmeier R.A.
      Retinal oxygen: fundamental and clinical aspects.
      ). The adult retina in most mammals is nourished by two vascular networks. The choroid vessels overlying the retinal pigmented epithelium (RPE) nourish the outer retina. The inner retinal vessels at the ganglion cell layer develop at late embryonic stages and complete their morphogenesis after birth (
      • Fruttiger M.
      Development of the retinal vasculature.
      ,
      • Saint-Geniez M.
      • D'Amore P.
      Development and pathology of the hyaloid, choroidal and retinal vasculature.
      ). During development, the inner mammalian retina is nourished by the hyaloid vasculature, a transient capillary network located between the lens and retina. Later, hyaloid vessels undergo programmed regression, and a retinal vasculature forms by angiogenesis (
      • Fruttiger M.
      Development of the retinal vasculature.
      ,
      • Saint-Geniez M.
      • D'Amore P.
      Development and pathology of the hyaloid, choroidal and retinal vasculature.
      ,
      • Fruttiger M.
      Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis.
      ). Defects in hyaloid vasculature regression, known as persistent fetal vasculature, result in pathological eye conditions (
      • Shastry B.S.
      Persistent hyperplastic primary vitreous: congenital malformation of the eye.
      ). In zebrafish, intraocular vasculature development is initially similar to mammals. However, hyaloid vessels do not regress after embryonic development but progressively lose contact with the lens and, by 30 days after fertilization, adhere to the inner limiting membrane of the juvenile retina (
      • Alvarez Y.
      • Cederlund M.L.
      • Cottell D.C.
      • Bill B.R.
      • Ekker S.C.
      • Torres-Vazquez J.
      • Weinstein B.M.
      • Hyde D.R.
      • Vihtelic T.S.
      • Kennedy B.N.
      Genetic determinants of hyaloid and retinal vasculature in zebrafish.
      ). In adult zebrafish, these vessels are found attached to the ganglion cell layer, exhibiting distinctive hallmarks of mammalian retinal vasculature (
      • Alvarez Y.
      • Cederlund M.L.
      • Cottell D.C.
      • Bill B.R.
      • Ekker S.C.
      • Torres-Vazquez J.
      • Weinstein B.M.
      • Hyde D.R.
      • Vihtelic T.S.
      • Kennedy B.N.
      Genetic determinants of hyaloid and retinal vasculature in zebrafish.
      ,
      • Hartsock A.
      • Lee C.
      • Arnold V.
      • Gross J.M.
      In vivo analysis of hyaloid vasculature morphogenesis in zebrafish: a role for the lens in maturation and maintenance of the hyaloid.
      ). Although the cellular morphogenesis of zebrafish hyaloid vasculature is well characterized, our understanding of the molecular regulators is limited to a small number of genetic and pharmacological studies (
      • Alvarez Y.
      • Astudillo O.
      • Jensen L.
      • Reynolds A.L.
      • Waghorne N.
      • Brazil D.P.
      • Cao Y.
      • O'Connor J.J.
      • Kennedy B.
      Selective inhibition of retinal angiogenesis by targeting PI3 kinase.
      ,
      • Kitambi S.S.
      • McCulloch K.J.
      • Peterson R.T.
      • Malicki J.J.
      Small molecule screen for compounds that affect vascular development in the zebrafish retina.
      ,
      • Kalén M.
      • Wallgard E.
      • Asker N.
      • Nasevicius A.
      • Athley E.
      • Billgren E.
      • Larson J.D.
      • Wadman S.A.
      • Norseng E.
      • Clark K.J.
      • He L.
      • Karlsson-Lindahl L.
      • Häger A.-K.
      • Weber H.
      • Augustin H.
      • Samuelsson T.
      • Kemmet C.K.
      • Utesch C.M.
      • Essner J.J.
      • Hackett P.B.
      • Hellström M.
      Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.
      ).
      Zebrafish are particularly amenable to phenotype-based drug discovery (
      • Peterson R.T.
      • Link B.A.
      • Dowling J.E.
      • Schreiber S.L.
      Small molecule developmental screens reveal the logic and timing of vertebrate development.
      ,
      • Rennekamp A.J.
      • Peterson R.T.
      15 years of zebrafish chemical screening.
      ). This “target-agnostic” approach focuses on a chosen phenotype and does not require prior selection of a molecular target. In this study, we identify unique drugs inhibiting developmental angiogenesis of the eye by performing an unbiased screen of ∼1800 small-molecule drugs in the zebrafish hyaloid vessel assay (
      • Alvarez Y.
      • Astudillo O.
      • Jensen L.
      • Reynolds A.L.
      • Waghorne N.
      • Brazil D.P.
      • Cao Y.
      • O'Connor J.J.
      • Kennedy B.
      Selective inhibition of retinal angiogenesis by targeting PI3 kinase.
      ). The screen uncovered 2-[(E)-2-(quinolin-2-yl)vinyl]phenol (quininib)
      The abbreviations used are: quininib, 2-[(E)-2-(quinolin-2-yl)vinyl]phenol; CysLT, cysteinyl leukotriene; DMSO, dimethyl sulfoxide; OIR, oxygen-induced retinopathy; P12, postnatal day 12; ANOVA, analysis of variance; HV, hyaloid vessel; dpf, day(s) post-fertilization.
      as a potent inhibitor of developmental angiogenesis in the zebrafish eye. Subsequently, quininib demonstrated significant anti-angiogenic activity in human endothelial cell, murine aortic ring, and murine oxygen-induced retinopathy models of angiogenesis. Target profiling identified quininib as a cysteinyl leukotriene 1 and 2 receptor (CysLT1–2) antagonist. The cysteinyl leukotrienes LTC4, LTD4, LTE4, and LTF4 are bioactive lipids synthesized from cell membrane arachidonic acid via a 5-hydroxyeicosatetraenoic acid intermediate and signal via G protein-coupled receptors (CysLT1, CysLT2, GPR17, and GPR99) (
      • Kanaoka Y.
      • Maekawa A.
      • Austen K.F.
      Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.
      ,
      • Singh R.K.
      • Gupta S.
      • Dastidar S.
      • Ray A.
      Cysteinyl leukotrienes and their receptors: molecular and functional characteristics.
      ,
      • Kanaoka Y.
      • Boyce J.A.
      Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses.
      ,
      • Bäck M.
      • Powell W.S.
      • Dahlén S.E.
      • Drazen J.M.
      • Evans J.F.
      • Serhan C.N.
      • Shimizu T.
      • Yokomizo T.
      • Rovati G.E.
      Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
      ,
      • Ciana P.
      • Fumagalli M.
      • Trincavelli M.L.
      • Verderio C.
      • Rosa P.
      • Lecca D.
      • Ferrario S.
      • Parravicini C.
      • Capra V.
      • Gelosa P.
      • Guerrini U.
      • Belcredito S.
      • Cimino M.
      • Sironi L.
      • Tremoli E.
      • Rovati G.E.
      • Martini C.
      • Abbracchio M.P.
      The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor.
      ). CysLT1 antagonists are commonly used to treat asthma and allergic rhinitis (
      • Meltzer E.O.
      • Malmstrom K.
      • Lu S.
      • Prenner B.M.
      • Wei L.X.
      • Weinstein S.F.
      • Wolfe J.D.
      • Reiss T.F.
      Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: a randomized, placebo-controlled clinical trial.
      ,
      • Reiss T.F.
      • Altman L.C.
      • Chervinsky P.
      • Bewtra A.
      • Stricker W.E.
      • Noonan G.P.
      • Kundu S.
      • Zhang J.
      Effects of montelukast (MK-0476), a new potent cysteinyl leukotriene (LTD4) receptor antagonist, in patients with chronic asthma.
      ). Previous studies have reported a role for CysLTs in inflammation, vascular permeability, immune responses, tissue repair, and regeneration (
      • Kanaoka Y.
      • Boyce J.A.
      Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses.
      ,
      • Kyritsis N.
      • Kizil C.
      • Zocher S.
      • Kroehne V.
      • Kaslin J.
      • Freudenreich D.
      • Iltzsche A.
      • Brand M.
      Acute inflammation initiates the regenerative response in the adult zebrafish brain.
      ,
      • Qian X.-D.
      • Wei E.-Q.
      • Zhang L.
      • Sheng W.-W.
      • Wang M.-L.
      • Zhang W.-P.
      • Chen Z.
      Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, protects mice against brain cold injury.
      ,
      • Wang X.Y.
      • Tang S.S.
      • Hu M.
      • Long Y.
      • Li Y.Q.
      • Liao M.X.
      • Ji H.
      • Hong H.
      Leukotriene D4 induces amyloid-β generation via CysLT1R-mediated NF-κB pathways in primary neurons.
      ,
      • Kanaoka Y.
      • Boyce J.A.
      Cysteinyl leukotrienes and their receptors: emerging concepts.
      ). CysLT2 and CysLT1 are expressed in the murine retina, and exogenous cysLTs are sufficient to induce retinal edema (
      • Barajas-Espinosa A.
      • Ni N.C.
      • Yan D.
      • Zarini S.
      • Murphy R.C.
      • Funk C.D.
      The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
      ). Here quininib inhibits known cysteinyl leukotriene receptor signaling pathways, reducing ERK phosphorylation in response to leukotriene D4 agonism (
      • Duah E.
      • Adapala R.K.
      • Al-Azzam N.
      • Kondeti V.
      • Gombedza F.
      • Thodeti C.K.
      • Paruchuri S.
      Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT(2) and CysLT(1) receptors.
      ,
      • Jiang Y.
      • Borrelli L.A.
      • Kanaoka Y.
      • Bacskai B.J.
      • Boyce J.A.
      CysLT(2) receptors interact with CysLT(1) receptors and down-modulate cysteinyl leukotriene-dependent mitogenic responses of mast cells.
      ). In summary, from unbiased chemical screens, we advance prior reports on cysLTs and demonstrate that a CysLT1–2 antagonist significantly attenuates angiogenesis in the eye.

      Discussion

      From phenotype-based screens of a randomized small molecule library, quininib was uncovered as a novel, robust inhibitor of angiogenesis in vivo. Quininib is an orthosteric cysteinyl leukotriene receptor 1 antagonist and a weak cysteinyl leukotriene receptor 2 antagonist. Overall, this research enhanced our understanding of the molecular pathways controlling ocular angiogenesis and identified drugs and molecular targets with potential to be developed into therapeutic agents for angiogenesis-related diseases.
      Two studies previously identified inhibitors of zebrafish hyaloid vasculature development using phenotype-based chemical screens. Alvarez et al. (
      • Alvarez Y.
      • Astudillo O.
      • Jensen L.
      • Reynolds A.L.
      • Waghorne N.
      • Brazil D.P.
      • Cao Y.
      • O'Connor J.J.
      • Kennedy B.
      Selective inhibition of retinal angiogenesis by targeting PI3 kinase.
      ) identified LY 294,002, a PI3K inhibitor, in a screen of 11 known angiogenic modulators. In a screen of ∼2000 bioactive drugs from the MicroSource Spectrum collection, Kitambi et al. (
      • Kitambi S.S.
      • McCulloch K.J.
      • Peterson R.T.
      • Malicki J.J.
      Small molecule screen for compounds that affect vascular development in the zebrafish retina.
      ) identified pyrogallin, an ATP-competitive inhibitor of JAK3, plus albendazole and mebendazole, both anti-helminthic medications that inhibit microtubule assembly. Thus, both studies screened existing bioactive compounds. Our study differs in that a randomized library (ChemBridge DiverSETTM) of ∼1800 molecules with predicted drug-like physiochemical properties was screened with the intent of identifying novel regulators of developmental angiogenesis in the eye.
      Analysis in zebrafish, human, and rodent models demonstrates that quininib is an effective and well tolerated inhibitor of angiogenesis. Efficacy in the zebrafish model upon administration into the larval medium confirms desirable in vivo pharmacokinetic properties that support bioavailability beyond effective threshold concentrations in ocular tissue. Quininib inhibits the formation of newly forming but not existing vessels and so fits the properties of a vascular targeting agent and not a vascular disrupting agent (
      • Lorusso P.M.
      • Boerner S.A.
      • Hunsberger S.
      Clinical development of vascular disrupting agents: what lessons can we learn from ASA404?.
      ). The anti-angiogenic effect of quininib is a specific pharmacological response and cannot be attributed to toxic effects or developmental delay in the models investigated. Quininib-treated larvae, however, present with a defective optokinetic response. In agreement, previous genetic studies report that zebrafish lacking ocular vasculature (cloche and silent heart mutants or VEGF-A morphants) exhibit reduced differentiation of retinal neurons and impaired synaptic processes (
      • Dhakal S.
      • Stevens C.
      • Weiss O.
      • Inbal A.
      • Stenkamp D.
      Role of the early ocular vasculature in regulation of retinal neurogenesis.
      ,
      • Dhakal S.
      • Stevens C.B.
      • Sebbagh M.
      • Weiss O.
      • Frey R.A.
      • Adamson S.
      • Shelden E.A.
      • Inbal A.
      • Stenkamp D.L.
      Abnormal retinal development in Cloche mutant zebrafish.
      ). The reduced visual behavior of quininib-treated zebrafish larvae is not an overt concern for further drug development because it likely reflects an indirect developmental defect that would not be encountered in adult eyes.
      In mammalian models, quininib is well tolerated in mice and is effective at inhibiting angiogenesis in the mouse oxygen-induced retinopathy model of ocular angiogenesis via an intravitreal delivery route. Two phases of blood vessel growth occur following the relative hypoxia that occurs when these mice are returned to normoxia on P12. These phases are a revascularization (normal intraretinal vessel regrowth), which is similar to a recapitulation of developmental angiogenesis, and a preretinal pathological neovascularization (
      • Stahl A.
      • Connor K.M.
      • Sapieha P.
      • Chen J.
      • Dennison R.J.
      • Krah N.M.
      • Seaward M.R.
      • Willett K.L.
      • Aderman C.M.
      • Guerin K.I.
      • Hua J.
      • Löfqvist C.
      • Hellström A.
      • Smith L.E.
      The mouse retina as an angiogenesis model.
      ). Both 0.5 and 3 μm quininib are effective at blocking retinal revascularization in the OIR model, with equivalent responses observed with 0.5 and 3 μm quininib, suggesting attainment of a maximum response plateau. Curiously, neovascularization in drug-treated eyes shows that the lower quininib dose slightly reduces neovascularization, whereas the higher dose increases the relative neovascular area. This may be due to the drug targeting one specific CysLT receptor at lower concentrations and more than one CysLT receptor at higher concentrations. In agreement, Barajas-Espinosa et al. (
      • Barajas-Espinosa A.
      • Ni N.C.
      • Yan D.
      • Zarini S.
      • Murphy R.C.
      • Funk C.D.
      The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
      ) reported a similar finding when Cyslt2r knockout mice were exposed to the OIR experimental paradigm. P17 flat-mount retinas, the same time point analyzed in our study, exhibited both reduced revascularization and increased neovascularization. They postulated that the increased neovascularization is due to the compensatory up-regulation of CysLT1 in the knockout.
      Adult Cysltr2 knockout mice show no retinal vascular phenotype compared with wild types (
      • Barajas-Espinosa A.
      • Ni N.C.
      • Yan D.
      • Zarini S.
      • Murphy R.C.
      • Funk C.D.
      The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
      ). In agreement, preliminary analysis of isolectin-stained retinal flat mounts from P8 Cysltr1 or Cysltr2 knockout mice suggest that neither receptor alone is required for retinal developmental angiogenesis (data not shown). This supports the theory that cysteinyl leukotriene receptors exhibit functional redundancy.
      Quininib acts as an orthosteric antagonist of the G protein-coupled receptors cysteinyl leukotriene receptor 1 and 2. Experimentally, we demonstrated quininib to significantly compete with the endogenous ligand LTD4 for binding to CysLT1 preferentially over CysLT2. Additionally, quininib significantly attenuated reporter activity from CysLT1 more than CysLT2. It may be that a higher concentration of quininib is required to elicit a response through CysLT2. These findings are in agreement with a medicinal chemistry report in 1992 by Zamboni et al. (
      • Zamboni R.
      • Belley M.
      • Champion E.
      • Charette L.
      • DeHaven R.
      • Frenette R.
      • Gauthier J.Y.
      • Jones T.R.
      • Leger S.
      • Masson P.
      Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+−)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.
      ) who generated a series of CysLT1 antagonists by structural modification of 3(-2-quinolinyl-(E)-ethenyl)pyridine. A compound equivalent to the free-base version of quininib generates an IC50 of >50 μm for competition with LTD4 binding to guinea pig lung strips (
      • Zamboni R.
      • Belley M.
      • Champion E.
      • Charette L.
      • DeHaven R.
      • Frenette R.
      • Gauthier J.Y.
      • Jones T.R.
      • Leger S.
      • Masson P.
      Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+−)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.
      ). The significantly lower IC50 of 1.4 μm for quininib reported here likely reflects that the human CysLT1- or CysLT2-overexpressing cells more specifically report on these receptors compared with the more heterogeneous guinea pig lung strips. Alternatively, the quininib hydrochloric salt used here may result in greater activity than the amine form. Finally, the chemical structure of quininib (molecular weight, 283.75 g/mol) extensively overlaps with a portion of the much larger, clinically approved CysLT1 antagonist montelukast (Merck, MW 586.18g/mol) (
      • Reiss T.F.
      • Altman L.C.
      • Chervinsky P.
      • Bewtra A.
      • Stricker W.E.
      • Noonan G.P.
      • Kundu S.
      • Zhang J.
      Effects of montelukast (MK-0476), a new potent cysteinyl leukotriene (LTD4) receptor antagonist, in patients with chronic asthma.
      ). Recently montelukast has been reported to reduce neuroinflammation in an aged rat model, acting via inhibition of a separate cysteinyl leukotriene receptor, GPR17 (
      • Marschallinger J.
      • Schaffner I.
      • Klein B.
      • Gelfert R.
      • Rivera F.J.
      • Illes S.
      • Grassner L.
      • Janssen M.
      • Rotheneichner P.
      • Schmuckermair C.
      • Coras R.
      • Boccazzi M.
      • Chishty M.
      • Lagler F.B.
      • Renic M.
      • Bauer H.-C.
      • Singewald N.
      • Blumcke I.
      • Bogdahn U.
      • Couillard-Despres S.
      • Lie D.C.
      • Abbracchio M.P.
      • Aigner L.
      Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drug.
      ), and a fourth cysteinyl leukotriene receptor, GPR99, has been proposed (
      • Kanaoka Y.
      • Maekawa A.
      • Austen K.F.
      Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.
      ). In genetic loss-of function models, cysteinyl leukotriene receptors demonstrate cognate compensatory up-regulation (
      • Barajas-Espinosa A.
      • Ni N.C.
      • Yan D.
      • Zarini S.
      • Murphy R.C.
      • Funk C.D.
      The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
      ,
      • Jiang Y.
      • Borrelli L.A.
      • Kanaoka Y.
      • Bacskai B.J.
      • Boyce J.A.
      CysLT(2) receptors interact with CysLT(1) receptors and down-modulate cysteinyl leukotriene-dependent mitogenic responses of mast cells.
      ,
      • Maekawa A.
      • Kanaoka Y.
      • Xing W.
      • Austen K.F.
      Functional recognition of a distinct receptor preferential for leukotriene E(4) in mice lacking the cysteinyl leukotriene 1 and 2 receptors.
      ). Thus, there are exciting future opportunities to decipher the cysLT receptors exhibiting co-regulatory expression to understand the underlying control mechanisms and to determine the combinations of cysLT receptors that regulate physiological phenotypes.
      Cysteinyl leukotrienes have been reported previously to up-regulate ERK phosphorylation (
      • Duah E.
      • Adapala R.K.
      • Al-Azzam N.
      • Kondeti V.
      • Gombedza F.
      • Thodeti C.K.
      • Paruchuri S.
      Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT(2) and CysLT(1) receptors.
      ,
      • Jiang Y.
      • Borrelli L.A.
      • Kanaoka Y.
      • Bacskai B.J.
      • Boyce J.A.
      CysLT(2) receptors interact with CysLT(1) receptors and down-modulate cysteinyl leukotriene-dependent mitogenic responses of mast cells.
      ). Here we show that drug treatment with quininib inhibits LTD4-induced phospho-ERK up-regulation, which is completely blocked by the more downstream MEK 1/2 inhibitor TAK-733. TAK-733 and the broad-spectrum PKC inhibitor Gö 6983 phenocopy the effects of quininib on developing hyaloid vessels in zebrafish.
      On the basis of a virtual drug screen, the quininib E-isomer was reported in 2008 to inhibit endothelin-converting enzyme 2 (ECE-2) (
      • Gagnidze K.
      • Sachchidanand, Rozenfeld R.
      • Mezei M.
      • Zhou M.-M.
      • Devi L.A.
      Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2.
      ). This target is unlikely to mediate the anti-angiogenic activity of quininib because alternative endothelin receptor antagonists do not phenocopy quininib (data not shown). In addition, recently Gupta et al. (
      • Gupta A.
      • Gomes I.
      • Wardman J.
      • Devi L.A.
      Opioid receptor function is regulated by post-endocytic peptide processing.
      ) reported that the quininib Z-isomer more potently inhibits ECE-2 than the E-isomer, which does not correlate with our finding of greater anti-angiogenic activity of the E-isomer (
      • Gupta A.
      • Gomes I.
      • Wardman J.
      • Devi L.A.
      Opioid receptor function is regulated by post-endocytic peptide processing.
      ). Also of note is that quininib does not directly inhibit the activity of any VEGF receptor. VEGF, particularly in the eye, is a key target to treat ocular neovascularization using humanized antibodies (e.g. Avastin®, Lucentis®) or soluble decoy receptors (Eylea®), and many small-molecule VEGF receptor inhibitors are available (e.g. the tyrosine kinase inhibitors AL 39324, PTK787, and Tg 100801; reviewed in Ref.
      • Reynolds A.L.
      • Kent D.
      • Kennedy B.N.
      Ocular neovascularisation: current and emerging therapies.
      ). Thus, the distinct cysteinyl leukotriene pathway-mediated anti-angiogenic mechanism of action of quininib offers potential for additive effects with anti-VEGFs or alternative therapeutic targets for patients non-responsive to current anti-VEGFs (
      • Rofagha S.
      • Bhisitkul R.B.
      • Boyer D.S.
      • Sadda S.R.
      • Zhang K.
      SEVEN-UP Study Group
      Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP).
      ).
      Although this is the first report demonstrating a significant anti-angiogenic effect of CysLT1 antagonists on ocular angiogenesis, accumulating evidence confirms a key role for cysteinyl leukotrienes in angiogenesis. For example, the cysteinyl leukotrienes LTD4 and LTC4 stimulate angiogenesis in endothelial cells (
      • Tsopanoglou N.E.
      • Pipili-Synetos E.
      • Maragoudakis M.E.
      Leukotrienes C4 and D4 promote angiogenesis via a receptor-mediated interaction.
      ,
      • Kanayasu T.
      • Nakao-Hayashi J.
      • Asuwa N.
      • Morita I.
      • Ishii T.
      • Ito H.
      • Murota S.
      Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro.
      ). Of particular relevance, the CysLT1 antagonist montelukast, but not the CysLT2 antagonist BayCysLT2, blocks the LTD4-induced migratory phenotype of the human endothelial cell line EA.hy926 (
      • Yuan Y.-M.
      • Fang S.-H.
      • Qian X.-D.
      • Liu L.-Y.
      • Xu L.-H.
      • Shi W.-Z.
      • Zhang L.-H.
      • Lu Y.-B.
      • Zhang W.-P.
      • Wei E.-Q.
      Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene CysLT1 receptor via the extracellular signal-regulated kinase pathway.
      ). Similarly, montelukast, but not BayCysLT2, inhibits basal microvessel outgrowth from rat thoracic aortic rings, and both drugs inhibit an LTD4-induced model of aortic ring angiogenesis (
      • Xu L.
      • Zhang L.
      • Liu L.
      • Fang S.
      • Lu Y.
      • Wei E.
      • Zhang W.
      Involvement of cysteinyl leukotriene receptors in angiogenesis in rat thoracic aortic rings.
      ). In vivo, Savari et al. (
      • Savari S.
      • Liu M.
      • Zhang Y.
      • Sime W.
      • Sjölander A.
      CysLT1R antagonists inhibit tumor growth in a xenograft model of colon cancer.
      ) recently reported CysLT1 receptor antagonists to reduce tumor angiogenesis in a mouse xenograft model of colorectal cancer, and this was concomitant with reduced tumor VEGF levels. Interestingly, the recognized anti-asthmatic effect of montelukast is modulated by VEGF polymorphisms, and montelukast has been postulated to attenuate airway inflammation by reducing VEGF expression (
      • Balantic M.
      • Rijavec M.
      • Skerbinjek Kavalar M.
      • Suskovic S.
      • Silar M.
      • Kosnik M.
      • Korosec P.
      Asthma treatment outcome in children is associated with vascular endothelial growth factor A (VEGFA) polymorphisms.
      ,
      • Lee K.S.
      • Kim S.R.
      • Park H.S.
      • Jin G.Y.
      • Lee Y.C.
      Cysteinyl leukotriene receptor antagonist regulates vascular permeability by reducing vascular endothelial growth factor expression.
      ). Focusing on the eye, Barajas-Espinosa et al. (
      • Barajas-Espinosa A.
      • Ni N.C.
      • Yan D.
      • Zarini S.
      • Murphy R.C.
      • Funk C.D.
      The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
      ), in defining a role for CysLT2 in retinal permeability and neovascularization, also demonstrated expression of CysLT1 in the retina, which is significantly increased upon knockout of Cysltr2. Here we demonstrate that the zebrafish cysltr1 and cysltr2 genes are expressed in the eye during hyaloid vasculature development and that the CysLT1 antagonist quininib can attenuate ocular angiogenesis. Intriguingly, quininib is significantly more potent than montelukast at inhibiting angiogenesis in the eye. Pharmacokinetic reasons potentially explain these differences. Quininib is significantly smaller than montelukast, which may facilitate greater absorption into the zebrafish eye and greater penetration across the mouse retinal layers, resulting in enhanced bioavailability and efficacy. Additionally, montelukast is known to undergo light-induced isomerization, which produces structurally related but inactive products (
      • Smith G.A.
      • Rawls C.M.
      • Kunka R.L.
      An automated method for the determination of montelukast in human plasma using dual-column HPLC analysis and peak height summation of the parent compound and its photodegradation product.
      ).
      In summary, we demonstrate a novel role for cysteinyl leukotriene receptor antagonists in attenuating ocular angiogenesis. Cysteinyl leukotrienes are proinflammatory agents that increase vascular permeability (
      • Kanaoka Y.
      • Maekawa A.
      • Austen K.F.
      Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.
      ,
      • Bäck M.
      • Powell W.S.
      • Dahlén S.E.
      • Drazen J.M.
      • Evans J.F.
      • Serhan C.N.
      • Shimizu T.
      • Yokomizo T.
      • Rovati G.E.
      Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
      ) through receptor-mediated activation of phospholipase C, phosphoinositide-3-kinase, and/or extracellular signal-related kinases (
      • Bäck M.
      • Powell W.S.
      • Dahlén S.E.
      • Drazen J.M.
      • Evans J.F.
      • Serhan C.N.
      • Shimizu T.
      • Yokomizo T.
      • Rovati G.E.
      Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
      ,
      • Kim M.H.
      • Lee Y.J.
      • Kim M.O.
      • Kim J.S.
      • Han H.J.
      Effect of leukotriene D4 on mouse embryonic stem cell migration and proliferation: involvement of PI3K/Akt as well as GSK-3β/β-catenin signaling pathways.
      ,
      • Salim T.
      • Sand-Dejmek J.
      • Sjölander A.
      The inflammatory mediator leukotriene D4 induces subcellular β-catenin translocation and migration of colon cancer cells.
      ). This increased permeability can increase extravasation of proangiogenic factors that can remodel the extracellular environment and promote growth of new vessels. CysLTs provide a novel pathway from which to enhance our understanding of developmental angiogenesis and a novel therapeutic target in angiogenesis-driven disease.

      Author Contributions

      A. L. R. conducted validation, visual behavior, analogue, and time-course experiments in zebrafish; safety and efficacy experiments in mice; and target profiling analyses, analyzed and interpreted the results, and wrote the paper with Y. A. and B. N. K. Y. A. and N. W. conducted the initial drug discovery and safety experiments in zebrafish and analyzed the results. T. S. performed zebrafish behavioral assays, expression analyses, and downstream cysLT signaling experiments and analyzed the results. C. B. performed HMEC-1 cell viability studies and the in vitro tubule formation assay. C. K. performed the target profiling analysis and helped with mouse OIR experiments. A. J. S. performed screening of downstream cysteinyl leukotriene inhibitors in zebrafish. C. M. and V. H. Y. W. performed a subset of the mouse OIR experiments, analyzed the results, and assisted with the interpretation of results. O. G. performed the mouse aortic ring experiments. S. M. performed the ARPE-19 cell viability studies. J. O. and A. S. provided animal models. G. G. performed analysis of OIR mouse retinas. A. W. S. provided animal models and assisted with analysis and interpretation of the results. B. N. K. conceived the idea for the project, assisted with analysis and interpretation of the results, and wrote the paper with A. L. R. and Y. A.

      Acknowledgments

      We thank the Conway Institute Imaging Core Technology, especially Dimitri Scholz, for assistance with montaging and Tiina O'Neill and Kasia Welzel for assistance with embedding and sectioning of samples; Pat Guiry for help with interpreting chemical structures; Ken O'Halloran for access to hyperoxia equipment; and Adrian Murphy, Jacintha O'Sullivan, Nils Ohnesorge, and Oliver Blacque for discussions and comments on the manuscript.

      References

        • Fruttiger M.
        Development of the retinal vasculature.
        Angiogenesis. 2007; 10: 77-88
        • Cheung N.
        • Mitchell P.
        • Wong T.Y.
        Diabetic retinopathy.
        Lancet. 2010; 376: 124-136
        • Jager R.D.
        • Mieler W.F.
        • Miller J.W.
        Age-related macular degeneration.
        N. Engl. J. Med. 2008; 358: 2606-2617
        • Hellström A.
        • Smith L.E.
        • Dammann O.
        Retinopathy of prematurity.
        Lancet. 2013; 382: 1445-1457
        • Rehak M.
        • Wiedemann P.
        Retinal vein thrombosis: pathogenesis and management.
        J. Thromb. Haemost. 2010; 8: 1886-1894
        • Gariano R.F.
        • Gardner T.W.
        Retinal angiogenesis in development and disease.
        Nature. 2005; 438: 960-966
        • Alvarez Y.
        • Astudillo O.
        • Jensen L.
        • Reynolds A.L.
        • Waghorne N.
        • Brazil D.P.
        • Cao Y.
        • O'Connor J.J.
        • Kennedy B.
        Selective inhibition of retinal angiogenesis by targeting PI3 kinase.
        PLoS ONE. 2009; 4: e7867
        • Kitambi S.S.
        • McCulloch K.J.
        • Peterson R.T.
        • Malicki J.J.
        Small molecule screen for compounds that affect vascular development in the zebrafish retina.
        Mech. Dev. 2009; 126: 464-477
        • Folkman J.
        Goldberg I. Rosen E. Regulation of Angiogenesis. Birkhäuser, Basel, Switzerland1997: 1-8
      1. Frost & Sullivan (June 7, 2011) Analysis of the US Retinal Therapeutics Market: Improvements in Administration and Efficacy Drive Growth, Frost & Sullivan Report NC77–52

      2. Frost & Sullivan (July 3, 2010) European Ophthalmic Pharmaceuticals Market, Frost & Sullivan Report M4AC-52

        • Martin D.F.
        • Maguire M.G.
        • Ying G.S.
        • Grunwald J.E.
        • Fine S.L.
        • Jaffe G.J.
        • CATT Research Group
        Ranibizumab and bevacizumab for neovascular age-related macular degeneration.
        N. Engl. J. Med. 2011; 364: 1897-1908
        • Heier J.S.
        • Brown D.M.
        • Chong V.
        • Korobelnik J.-F.
        • Kaiser P.K.
        • Nguyen Q.D.
        • Kirchhof B.
        • Ho A.
        • Ogura Y.
        • Yancopoulos G.D.
        • Stahl N.
        • Vitti R.
        • Berliner A.J.
        • Soo Y.
        • Anderesi M.
        • Groetzbach G.
        • Sommerauer B.
        • Sandbrink R.
        • Simader C.
        • Schmidt-Erfurth U.
        • VIEW 1 and VIEW 2 Study Groups
        Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration.
        Ophthalmology. 2012; 119: 2537-2548
        • Rosenfeld P.J.
        • Brown D.M.
        • Heier J.S.
        • Boyer D.S.
        • Kaiser P.K.
        • Chung C.Y.
        • Kim R.Y.
        • MARINA Study Group
        Ranibizumab for neovascular age-related macular degeneration.
        N. Engl. J. Med. 2006; 355: 1419-1431
        • Rofagha S.
        • Bhisitkul R.B.
        • Boyer D.S.
        • Sadda S.R.
        • Zhang K.
        • SEVEN-UP Study Group
        Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP).
        Ophthalmology. 2013; 120: 2292-2299
        • Elman M.J.
        • Qin H.
        • Aiello L.P.
        • Beck R.W.
        • Bressler N.M.
        • Ferris 3rd, F.L.
        • Glassman A.R.
        • Maturi R.K.
        • Melia M.
        • Diabetic Retinopathy Clinical Research Network
        Intravitreal ranibizumab for diabetic macular edema with prompt vs. deferred laser treatment: 3-year randomized trial results.
        Ophthalmology. 2012; 119: 2312-2318
        • Wangsa-Wirawan N.D.
        • Linsenmeier R.A.
        Retinal oxygen: fundamental and clinical aspects.
        Arch. Ophthalmol. 2003; 121: 547-557
        • Saint-Geniez M.
        • D'Amore P.
        Development and pathology of the hyaloid, choroidal and retinal vasculature.
        Int. J. Dev. Biol. 2004; 48: 1045-1058
        • Fruttiger M.
        Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis.
        Invest. Ophthalmol. Vis. Sci. 2002; 43: 522-527
        • Shastry B.S.
        Persistent hyperplastic primary vitreous: congenital malformation of the eye.
        Clin. Exp. Ophthalmol. 2009; 37: 884-890
        • Alvarez Y.
        • Cederlund M.L.
        • Cottell D.C.
        • Bill B.R.
        • Ekker S.C.
        • Torres-Vazquez J.
        • Weinstein B.M.
        • Hyde D.R.
        • Vihtelic T.S.
        • Kennedy B.N.
        Genetic determinants of hyaloid and retinal vasculature in zebrafish.
        BMC Dev. Biol. 2007; 7: 114
        • Hartsock A.
        • Lee C.
        • Arnold V.
        • Gross J.M.
        In vivo analysis of hyaloid vasculature morphogenesis in zebrafish: a role for the lens in maturation and maintenance of the hyaloid.
        Dev. Biol. 2014; 394: 327-339
        • Kalén M.
        • Wallgard E.
        • Asker N.
        • Nasevicius A.
        • Athley E.
        • Billgren E.
        • Larson J.D.
        • Wadman S.A.
        • Norseng E.
        • Clark K.J.
        • He L.
        • Karlsson-Lindahl L.
        • Häger A.-K.
        • Weber H.
        • Augustin H.
        • Samuelsson T.
        • Kemmet C.K.
        • Utesch C.M.
        • Essner J.J.
        • Hackett P.B.
        • Hellström M.
        Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.
        Chem. Biol. 2009; 16: 432-441
        • Peterson R.T.
        • Link B.A.
        • Dowling J.E.
        • Schreiber S.L.
        Small molecule developmental screens reveal the logic and timing of vertebrate development.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 12965-12969
        • Rennekamp A.J.
        • Peterson R.T.
        15 years of zebrafish chemical screening.
        Curr. Opin. Chem. Biol. 2015; 24: 58-70
        • Kanaoka Y.
        • Maekawa A.
        • Austen K.F.
        Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.
        J. Biol. Chem. 2013; 288: 10967-10972
        • Singh R.K.
        • Gupta S.
        • Dastidar S.
        • Ray A.
        Cysteinyl leukotrienes and their receptors: molecular and functional characteristics.
        Pharmacology. 2010; 85: 336-349
        • Kanaoka Y.
        • Boyce J.A.
        Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses.
        J. Immunol. 2004; 173: 1503-1510
        • Bäck M.
        • Powell W.S.
        • Dahlén S.E.
        • Drazen J.M.
        • Evans J.F.
        • Serhan C.N.
        • Shimizu T.
        • Yokomizo T.
        • Rovati G.E.
        Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
        Br. J. Pharmacol. 2014; 171: 3551-3574
        • Ciana P.
        • Fumagalli M.
        • Trincavelli M.L.
        • Verderio C.
        • Rosa P.
        • Lecca D.
        • Ferrario S.
        • Parravicini C.
        • Capra V.
        • Gelosa P.
        • Guerrini U.
        • Belcredito S.
        • Cimino M.
        • Sironi L.
        • Tremoli E.
        • Rovati G.E.
        • Martini C.
        • Abbracchio M.P.
        The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor.
        EMBO J. 2006; 25: 4615-4627
        • Meltzer E.O.
        • Malmstrom K.
        • Lu S.
        • Prenner B.M.
        • Wei L.X.
        • Weinstein S.F.
        • Wolfe J.D.
        • Reiss T.F.
        Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: a randomized, placebo-controlled clinical trial.
        J. Allergy Clin. Immunol. 2000; 105: 917-922
        • Reiss T.F.
        • Altman L.C.
        • Chervinsky P.
        • Bewtra A.
        • Stricker W.E.
        • Noonan G.P.
        • Kundu S.
        • Zhang J.
        Effects of montelukast (MK-0476), a new potent cysteinyl leukotriene (LTD4) receptor antagonist, in patients with chronic asthma.
        J. Allergy Clin. Immunol. 1996; 98: 528-534
        • Kyritsis N.
        • Kizil C.
        • Zocher S.
        • Kroehne V.
        • Kaslin J.
        • Freudenreich D.
        • Iltzsche A.
        • Brand M.
        Acute inflammation initiates the regenerative response in the adult zebrafish brain.
        Science. 2012; 338: 1353-1356
        • Qian X.-D.
        • Wei E.-Q.
        • Zhang L.
        • Sheng W.-W.
        • Wang M.-L.
        • Zhang W.-P.
        • Chen Z.
        Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, protects mice against brain cold injury.
        Eur. J. Pharmacol. 2006; 549: 35-40
        • Wang X.Y.
        • Tang S.S.
        • Hu M.
        • Long Y.
        • Li Y.Q.
        • Liao M.X.
        • Ji H.
        • Hong H.
        Leukotriene D4 induces amyloid-β generation via CysLT1R-mediated NF-κB pathways in primary neurons.
        Neurochem. Int. 2013; 62: 340-347
        • Kanaoka Y.
        • Boyce J.A.
        Cysteinyl leukotrienes and their receptors: emerging concepts.
        Allergy Asthma Immunol. Res. 2014; 6: 288-295
        • Barajas-Espinosa A.
        • Ni N.C.
        • Yan D.
        • Zarini S.
        • Murphy R.C.
        • Funk C.D.
        The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.
        FASEB J. 2012; 26: 1100-1109
        • Duah E.
        • Adapala R.K.
        • Al-Azzam N.
        • Kondeti V.
        • Gombedza F.
        • Thodeti C.K.
        • Paruchuri S.
        Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT(2) and CysLT(1) receptors.
        Sci. Rep. 2013; 3: 3274
        • Jiang Y.
        • Borrelli L.A.
        • Kanaoka Y.
        • Bacskai B.J.
        • Boyce J.A.
        CysLT(2) receptors interact with CysLT(1) receptors and down-modulate cysteinyl leukotriene-dependent mitogenic responses of mast cells.
        Blood. 2007; 110: 3263-3270
        • Lipinski C.
        • Lombardo F.
        • Dominy B.
        • Feeney P.
        Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.
        Adv. Drug Deliv. Rev. 2001; 23: 3-26
        • Lawson N.D.
        • Weinstein B.M.
        In vivo imaging of embryonic vascular development using transgenic zebrafish.
        Dev. Biol. 2002; 248: 307-318
        • Brockerhoff S.
        Measuring the optokinetic response of zebrafish larvae.
        Nat. Protoc. 2006; 1: 2448-2451
        • Sasore T.
        • Kennedy B.
        Deciphering combinations of PI3K/AKT/mTOR pathway drugs augmenting anti-angiogenic efficacy in vivo.
        PLoS ONE. 2014; 9: e105280
        • Baker M.
        • Robinson S.D.
        • Lechertier T.
        • Barber P.R.
        • Tavora B.
        • D'Amico G.
        • Jones D.T.
        • Vojnovic B.
        • Hodivala-Dilke K.
        Use of the mouse aortic ring assay to study angiogenesis.
        Nat. Protoc. 2012; 7: 89-104
        • Connor K.M.
        • Krah N.M.
        • Dennison R.J.
        • Aderman C.M.
        • Chen J.
        • Guerin K.I.
        • Sapieha P.
        • Stahl A.
        • Willett K.L.
        • Smith L.E.
        Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.
        Nat. Protoc. 2009; 4: 1565-1573
        • Smith L.E.
        • Wesolowski E.
        • McLellan A.
        • Kostyk S.K.
        • D'Amato R.
        • Sullivan R.
        • D'Amore P.A.
        Oxygen-induced retinopathy in the mouse.
        Invest. Ophthalmol. Vis. Sci. 1994; 35: 101-111
        • Gardiner T.A.
        • Gibson D.S.
        • de Gooyer T.E.
        • de la Cruz V.F.
        • McDonald D.M.
        • Stitt A.W.
        Inhibition of tumor necrosis factor-α improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy.
        Am. J. Pathol. 2005; 166: 637-644
        • Sidman R.L.
        • Li J.
        • Lawrence M.
        • Hu W.
        • Musso G.F.
        • Giordano R.J.
        • Cardó-Vila M.
        • Pasqualini R.
        • Arap W.
        The peptidomimetic Vasotide targets two retinal VEGF receptors and reduces pathological angiogenesis in murine and nonhuman primate models of retinal disease.
        Sci. Transl. Med. 2015; 7: 309ra165
        • Gagnidze K.
        • Sachchidanand, Rozenfeld R.
        • Mezei M.
        • Zhou M.-M.
        • Devi L.A.
        Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2.
        J. Med. Chem. 2008; 51: 3378-3387
        • Gupta A.
        • Gomes I.
        • Wardman J.
        • Devi L.A.
        Opioid receptor function is regulated by post-endocytic peptide processing.
        J. Biol. Chem. 2014; 289: 19613-19626
        • Zamboni R.
        • Belley M.
        • Champion E.
        • Charette L.
        • DeHaven R.
        • Frenette R.
        • Gauthier J.Y.
        • Jones T.R.
        • Leger S.
        • Masson P.
        Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+−)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.
        J. Med. Chem. 1992; 35: 3832-3844
        • Lorusso P.M.
        • Boerner S.A.
        • Hunsberger S.
        Clinical development of vascular disrupting agents: what lessons can we learn from ASA404?.
        J. Clin. Oncol. 2011; 29: 2952-2955
        • Dhakal S.
        • Stevens C.
        • Weiss O.
        • Inbal A.
        • Stenkamp D.
        Role of the early ocular vasculature in regulation of retinal neurogenesis.
        Invest. Ophthalmol. Vis. Sci. 2013; 54 (Abstr. 5145)
        • Dhakal S.
        • Stevens C.B.
        • Sebbagh M.
        • Weiss O.
        • Frey R.A.
        • Adamson S.
        • Shelden E.A.
        • Inbal A.
        • Stenkamp D.L.
        Abnormal retinal development in Cloche mutant zebrafish.
        Dev. Dyn. 2015; 244: 1439-1455
        • Stahl A.
        • Connor K.M.
        • Sapieha P.
        • Chen J.
        • Dennison R.J.
        • Krah N.M.
        • Seaward M.R.
        • Willett K.L.
        • Aderman C.M.
        • Guerin K.I.
        • Hua J.
        • Löfqvist C.
        • Hellström A.
        • Smith L.E.
        The mouse retina as an angiogenesis model.
        Invest. Ophthalmol. Vis. Sci. 2010; 51: 2813-2826
        • Marschallinger J.
        • Schaffner I.
        • Klein B.
        • Gelfert R.
        • Rivera F.J.
        • Illes S.
        • Grassner L.
        • Janssen M.
        • Rotheneichner P.
        • Schmuckermair C.
        • Coras R.
        • Boccazzi M.
        • Chishty M.
        • Lagler F.B.
        • Renic M.
        • Bauer H.-C.
        • Singewald N.
        • Blumcke I.
        • Bogdahn U.
        • Couillard-Despres S.
        • Lie D.C.
        • Abbracchio M.P.
        • Aigner L.
        Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drug.
        Nat. Commun. 2015; 6: 8466
        • Maekawa A.
        • Kanaoka Y.
        • Xing W.
        • Austen K.F.
        Functional recognition of a distinct receptor preferential for leukotriene E(4) in mice lacking the cysteinyl leukotriene 1 and 2 receptors.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 16695-16700
        • Reynolds A.L.
        • Kent D.
        • Kennedy B.N.
        Ocular neovascularisation: current and emerging therapies.
        Adv. Exp. Med. Biol. 2014; 801: 797-804
        • Tsopanoglou N.E.
        • Pipili-Synetos E.
        • Maragoudakis M.E.
        Leukotrienes C4 and D4 promote angiogenesis via a receptor-mediated interaction.
        Eur. J. Pharmacol. 1994; 258: 151-154
        • Kanayasu T.
        • Nakao-Hayashi J.
        • Asuwa N.
        • Morita I.
        • Ishii T.
        • Ito H.
        • Murota S.
        Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro.
        Biochem. Biophys. Res. Commun. 1989; 159: 572-578
        • Yuan Y.-M.
        • Fang S.-H.
        • Qian X.-D.
        • Liu L.-Y.
        • Xu L.-H.
        • Shi W.-Z.
        • Zhang L.-H.
        • Lu Y.-B.
        • Zhang W.-P.
        • Wei E.-Q.
        Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene CysLT1 receptor via the extracellular signal-regulated kinase pathway.
        J. Pharmacol. Sci. 2009; 109: 285-292
        • Xu L.
        • Zhang L.
        • Liu L.
        • Fang S.
        • Lu Y.
        • Wei E.
        • Zhang W.
        Involvement of cysteinyl leukotriene receptors in angiogenesis in rat thoracic aortic rings.
        Pharmazie. 2010; 65: 750-754
        • Savari S.
        • Liu M.
        • Zhang Y.
        • Sime W.
        • Sjölander A.
        CysLT1R antagonists inhibit tumor growth in a xenograft model of colon cancer.
        PLoS ONE. 2013; 8: e73466
        • Balantic M.
        • Rijavec M.
        • Skerbinjek Kavalar M.
        • Suskovic S.
        • Silar M.
        • Kosnik M.
        • Korosec P.
        Asthma treatment outcome in children is associated with vascular endothelial growth factor A (VEGFA) polymorphisms.
        Mol. Diagn. Ther. 2012; 16: 173-180
        • Lee K.S.
        • Kim S.R.
        • Park H.S.
        • Jin G.Y.
        • Lee Y.C.
        Cysteinyl leukotriene receptor antagonist regulates vascular permeability by reducing vascular endothelial growth factor expression.
        J. Allergy Clin. Immunol. 2004; 114: 1093-1099
        • Smith G.A.
        • Rawls C.M.
        • Kunka R.L.
        An automated method for the determination of montelukast in human plasma using dual-column HPLC analysis and peak height summation of the parent compound and its photodegradation product.
        Pharm. Res. 2004; 21: 1539-1544
        • Kim M.H.
        • Lee Y.J.
        • Kim M.O.
        • Kim J.S.
        • Han H.J.
        Effect of leukotriene D4 on mouse embryonic stem cell migration and proliferation: involvement of PI3K/Akt as well as GSK-3β/β-catenin signaling pathways.
        J. Cell Biochem. 2010; 111: 686-698
        • Salim T.
        • Sand-Dejmek J.
        • Sjölander A.
        The inflammatory mediator leukotriene D4 induces subcellular β-catenin translocation and migration of colon cancer cells.
        Exp. Cell Res. 2014; 321: 255-266
        • Savari S.
        • Vinnakota K.
        • Zhang Y.
        • Sjölander A.
        Cysteinyl leukotrienes and their receptors: bridging inflammation and colorectal cancer.
        World J. Gastroenterol. 2014; 20: 968-977