Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations
2018; Springer Nature; Volume: 10; Issue: 10 Linguagem: Inglês
10.15252/emmm.201809155
ISSN1757-4684
AutoresCécile Otten, Jessica Knox, Gwénola Boulday, Mathias Eymery, Marta Haniszewski, Martin Neuenschwander, Silke Radetzki, Ingo Vogt, Kristina Hähn, Coralie De Luca, Cécile Cardoso, Sabri Hamad, Carla Igual Gil, Peter J. Roy, Corinne Albigès‐Rizo, Eva Faurobert, Jens Peter von Kries, Mónica Campillos, Elisabeth Tournier‐Lasserve, W. Brent Derry, Salim Abdelilah‐Seyfried,
Tópico(s)Intracerebral and Subarachnoid Hemorrhage Research
ResumoResearch Article4 September 2018Open Access Source DataTransparent process Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations Cécile Otten Cécile Otten Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Jessica Knox Jessica Knox Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Gwénola Boulday Gwénola Boulday INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Mathias Eymery Mathias Eymery INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Marta Haniszewski Marta Haniszewski Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Developmental and Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Martin Neuenschwander Martin Neuenschwander Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Silke Radetzki Silke Radetzki Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Ingo Vogt Ingo Vogt German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Kristina Hähn Kristina Hähn Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Coralie De Luca Coralie De Luca INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Cécile Cardoso Cécile Cardoso INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Sabri Hamad Sabri Hamad German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Carla Igual Gil Carla Igual Gil Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Peter Roy Peter Roy Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Corinne Albiges-Rizo Corinne Albiges-Rizo INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Eva Faurobert Eva Faurobert INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Jens P von Kries Jens P von Kries Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Mónica Campillos Mónica Campillos German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Elisabeth Tournier-Lasserve Elisabeth Tournier-Lasserve INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France AP-HP, Groupe hospitalier Saint-Louis, Lariboisière, Fernand-Widal, Service de génétique moléculaire neuro-vasculaire, Paris, France Search for more papers by this author W Brent Derry W Brent Derry Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Developmental and Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Salim Abdelilah-Seyfried Corresponding Author Salim Abdelilah-Seyfried [email protected] orcid.org/0000-0003-3183-3841 Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Institute of Molecular Biology, Hannover Medical School, Hannover, Germany Search for more papers by this author Cécile Otten Cécile Otten Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Jessica Knox Jessica Knox Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Gwénola Boulday Gwénola Boulday INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Mathias Eymery Mathias Eymery INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Marta Haniszewski Marta Haniszewski Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Developmental and Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Martin Neuenschwander Martin Neuenschwander Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Silke Radetzki Silke Radetzki Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Ingo Vogt Ingo Vogt German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Kristina Hähn Kristina Hähn Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Coralie De Luca Coralie De Luca INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Cécile Cardoso Cécile Cardoso INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France Search for more papers by this author Sabri Hamad Sabri Hamad German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Carla Igual Gil Carla Igual Gil Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Search for more papers by this author Peter Roy Peter Roy Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Corinne Albiges-Rizo Corinne Albiges-Rizo INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Eva Faurobert Eva Faurobert INSERM U1209, Grenoble, France Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France CNRS UMR 5309, Grenoble, France Search for more papers by this author Jens P von Kries Jens P von Kries Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Mónica Campillos Mónica Campillos German Center for Diabetes Research, Neuherberg, Germany Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Elisabeth Tournier-Lasserve Elisabeth Tournier-Lasserve INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France AP-HP, Groupe hospitalier Saint-Louis, Lariboisière, Fernand-Widal, Service de génétique moléculaire neuro-vasculaire, Paris, France Search for more papers by this author W Brent Derry W Brent Derry Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Developmental and Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Salim Abdelilah-Seyfried Corresponding Author Salim Abdelilah-Seyfried [email protected] orcid.org/0000-0003-3183-3841 Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany Institute of Molecular Biology, Hannover Medical School, Hannover, Germany Search for more papers by this author Author Information Cécile Otten1, Jessica Knox2,3,4,‡, Gwénola Boulday5,‡, Mathias Eymery6,7,8, Marta Haniszewski2,9, Martin Neuenschwander10, Silke Radetzki10, Ingo Vogt11,12, Kristina Hähn1, Coralie De Luca5, Cécile Cardoso5, Sabri Hamad11,12, Carla Igual Gil1, Peter Roy2,3,4, Corinne Albiges-Rizo6,7,8, Eva Faurobert6,7,8, Jens P Kries10, Mónica Campillos11,12, Elisabeth Tournier-Lasserve5,13, W Brent Derry2,9 and Salim Abdelilah-Seyfried *,1,14 1Institute of Biochemistry and Biology, Potsdam University, Potsdam, Germany 2Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 3The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada 4Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada 5INSERM UMR-1161, Génétique et physiopathologie des maladies cérébro-vasculaires, Université Paris Diderot, Paris, France 6INSERM U1209, Grenoble, France 7Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France 8CNRS UMR 5309, Grenoble, France 9Developmental and Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada 10Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany 11German Center for Diabetes Research, Neuherberg, Germany 12Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany 13AP-HP, Groupe hospitalier Saint-Louis, Lariboisière, Fernand-Widal, Service de génétique moléculaire neuro-vasculaire, Paris, France 14Institute of Molecular Biology, Hannover Medical School, Hannover, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 3319775540; E-mail: [email protected] EMBO Mol Med (2018)10:e9155https://doi.org/10.15252/emmm.201809155 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non-invasive pharmacological therapies. Here, we present the results of several unbiased small-molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology-based target prediction tool to integrate the results with the whole-transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small-molecule-based therapies. We found indirubin-3-monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss-of-CCM phenotypes in human endothelial cells. Our multi-organism-based approach reveals new components of the CCM regulatory network and foreshadows novel small-molecule-based therapeutic applications for suppressing this devastating disease in patients. Synopsis Currently, the only treatment for cerebral cavernous malformation (CCM) vasculature lesions is surgery. This study by Otten et al establishes a multi-organismal pharmacological approach to suppress the formation of new lesions or to regress existing ones. Many small molecule compounds alleviating the loss of CCM proteins were identified in suppression screens in C. elegans and zebrafish. DePick, a prediction programme for relevant protein targets of active compounds, was used to identify relevant molecular pathways and CCM-related drug targets. Indirubin-3-monoxime (IR3mo) alleviated the lesion burden in preclinical CCM mouse models. Future experiments with IR3mo will help to unravel the CCM pathobiology. Introduction Cerebral cavernous malformations are characterized by the presence of vascular lesions that are most prevalent in the brain and can occur sporadically or through a familial condition from mutations in CCM1/KRIT1, CCM2/Malcavernin, or CCM3/PDCD10 [reviewed in (Chan et al, 2010; Riant et al, 2010; Fischer et al, 2013)]. The loss of CCM3 has particularly been associated with an early onset and severe progression of the pathology (Riant et al, 2013; Shenkar et al, 2015). Surgical resection is currently the only curative option for CCM patients, but when lesions are seated deeply in regions of the brain not accessible to neurosurgery, the condition may cause severe morbidity or even lethality due to recurrent cerebral hemorrhages. Hence, pharmacological interventions are desperately needed to (i) prevent the growth and bleeding of existing lesions and (ii) to suppress the formation of new ones. Loss of CCM proteins in Caenorhabditis elegans, zebrafish, and mouse leads to penetrant defects in vascular structures and conserved signaling pathways. Importantly, these model organisms offer many advantages for suppressing these phenotypes using small molecules or genetic methods. In the nematode C. elegans, complete loss of the CCM1 homologous gene kri-1 causes resistance to apoptosis (Ito et al, 2010), whereas loss of ccm-3 causes defects in biological tube development (Lant et al, 2015; Pal et al, 2017). Similarly, loss of any of the Ccm proteins in zebrafish and mouse causes cardiovascular malformations that result in cardiac defects, including abnormal cardiac chamber ballooning, a failure of endocardial cushions to form at the atrioventricular canal, and defects in blood vessel formation (Mably et al, 2003, 2006; Hogan et al, 2008; Boulday et al, 2009; Kleaveland et al, 2009; Zheng et al, 2010; Yoruk et al, 2012; Renz et al, 2015; Zhou et al, 2015). In mice, the endothelial-specific deletion of Ccm1-3 at postnatal day 1 leads to lesions in the CNS and retinal vasculature which resemble CCM lesions in patients (Boulday et al, 2011). Several molecular pathways have been implicated mechanistically in the pathological changes that occur within endothelial cells upon the loss of CCM proteins. These include signaling via transcription factors KLF2/4 (Maddaluno et al, 2013; Renz et al, 2015; Zhou et al, 2015, 2016), the innate immunity TLR4 receptor (Tang et al, 2017), MAPK (Uhlik et al, 2003; Fisher et al, 2015; Zhou et al, 2015, 2016; Cuttano et al, 2016), β1-integrin (Brütsch et al, 2010; Faurobert et al, 2013; Renz et al, 2015), angiogenesis and/or Notch (Boulday et al, 2009, 2011; Brütsch et al, 2010; Wüstehube et al, 2010; Zhu et al, 2010; You et al, 2013, 2017; Renz et al, 2015; Schulz et al, 2015; Lopez-Ramirez et al, 2017), Rho/ROCK (Glading et al, 2007; Whitehead et al, 2009; Borikova et al, 2010; Stockton et al, 2010; Richardson et al, 2013), and BMP/TGFβ/endoMT or Wnt (Maddaluno et al, 2013; Bravi et al, 2015, 2016). In addition, the pathology may be accompanied by increases in the production of ECM-degrading metalloproteases (Zhou et al, 2015), the secretion of angiopoietin-2 (Jenny Zhou et al, 2016), and oxidative stress (reviewed in Retta & Glading, 2016), or defective autophagy (Marchi et al, 2015) and apoptosis (Ito et al, 2010). The identification of these misregulated signaling pathways has suggested potential routes for pharmacological interventions, and molecules that modulate these pathways have been tested in preclinical studies of murine endothelial-specific inducible CCM models and even in clinical studies. These studies investigated the potential of the Rho/Rock signaling inhibitors fasudil or simvastatin (Zhou et al, 2015; Shenkar et al, 2017), the blood pressure lowering drug propranolol (Reinhard et al, 2016), the Wnt signaling inhibitor sulindac sulfone (Bravi et al, 2015), the TGFβ and pSMAD inhibitors LY-364947 and SB-431542 (Maddaluno et al, 2013), the TLR4 antagonist TAK-242 (resatorvid; Tang et al, 2017), or treatment with antibiotics (Tang et al, 2017). However, since many of these candidate drugs may cause severe side effects in patients and because a comprehensive overview of CCM-relevant molecular pathways is still lacking, the scientific community has recognized the need of performing more systematic small-molecule screens. The fastest route to clinical trials for drugs in the treatment of CCMs would be repurposed substances already on the market. Recently, two screens based on Food and Drug Administration (FDA)-approved small molecules have been performed: the first, carried out on human CCM2-deficient endothelial cells, led to the identification of several molecules including tempol, a free radical scavenger, and vitamin D, neither of which had previously been implicated in the treatment of this disease (Gibson et al, 2015). Another screen assayed CCM3-deficient mouse primary astrocytes and Drosophila glial cells for the suppression of overproliferation. This led to the identification of compounds affecting the mevalonate pathway (Nishimura et al, 2017). While these studies have provided important first insights into potential therapeutic approaches, an unbiased, integrative, and multi-organismic screen has not previously been attempted. Performing such compound screens in the context of the complex multi-tissue comprising CCM-deficient organisms may provide comprehensive insights into conserved druggable pathways. Vertebrate models with a cardiovascular system such as zebrafish offer additional advantages that may not be available by screening cultured cells or invertebrates. Hence, combined screens using multiple systems are more likely to provide a comprehensive list of CCM-relevant compounds. Here, we present the results of a repurposed drug screen that assayed the efficacy of suppressing cardiovascular defects in zebrafish ccm2m201 mutants or synthetic lethality in kri-1; ccm-3 double mutants in C. elegans. Our study combines system biological analyses integrating ccm2m201 mutant transcriptional data with molecular pathways that have been modulated using small-molecule compounds. These analyses pinpoint particular disease signatures as critical hubs that could be targeted by therapies. In addition to many previously identified compounds, our unbiased screen provides a range of new candidates that affect angiogenesis, vitamin D and retinoic acid signaling, blood pressure, ion channels, neurotransmitters, the oxidative stress/redox system, inflammation, and the innate immune system. These findings provide an unbiased framework for therapeutic approaches to tackle this debilitating disease. The relevance of this unbiased screen for CCM therapeutics is well illustrated by the identification of indirubin-3-monoxime as a compound showing a rescue in human endothelial cells and a strong preventive effect in CCM mouse models. Results Repurposed drug screens identify compounds that suppress CCM mutant phenotypes in zebrafish and C. elegans Most screens in the past have been primarily based on simplified in vitro models that had only a limited ability to recreate the complexity of the cardiovascular system or of the complex whole organismal interactions that may be affected in the CCM pathology (Gibson et al, 2015; Nishimura et al, 2017). To identify compounds for a pharmacological suppression of CCM phenotypes, we employed diverse assays on multiple organisms that can help to discriminate distinct effects on the cardiovascular system or cell biology upon loss of CCM proteins (Fig 1A). These assays included screening small compound libraries at concentrations of 10 μM for 24 h in zebrafish ccm2m201 mutant embryos carrying the endothelial-specific reporter transgene Tg (kdrl:GFP)s843 and probing for the suppression of the ballooning heart phenotype at 48 h postfertilization (hpf) (Mably et al, 2006; Materials and Methods). In parallel, we took advantage of the synthetic lethality caused by the co-ablation of kri-1 (CCM1) and ccm-3 in C. elegans (Lant et al, 2015; Materials and Methods) and screened for a restoration of viability, which led to insights into cellular processes affected by the loss of CCM proteins that are conserved between the species. Figure 1. Small-molecule drug screens identify compounds relevant for CCM A. Overview of the four different screening assays used in this study. Zebrafish embryos and C. elegans are screened in 24-well and 96-well plates, respectively. The most promising active compounds are retested in shCCM2 HUVECs. One compound is tested for suppression of vascular lesion formation in the cerebellum of iCCM2 and iCCM3 mouse models. B. Overlap of rescue compounds screened in the different assays. C–E. Examples of rescue of cardiovascular defects of the zebrafish ccm2m201 mutant. Inverted images of confocal z-scan projections of the 46 hpf head region and heart (endocardium) of wild-type (WT) and ccm2m201 mutant zebrafish embryos carrying the endothelial Tg(kdrl:GFP)s843 reporter transgene. Embryos are untreated (C) or treated between 17 and 48 hpf with 10 μM of the Lck inhibitor C8863 (D) or with 10 μM of the ERK5 inhibitor XMD8-92 (E). Both compounds resulted in a reduction in heart size and narrowing of the heart tube at the atrioventricular canal (arrowheads). Scale bar is 100 μm. Download figure Download PowerPoint We screened a total of 1,600 unique compounds in zebrafish (LOPAC/Selleck libraries), 8.4% of which (134/1,600) alleviated the ccm2m201 mutant heart phenotype (Fig 1B–E; Dataset EV1). Concurrently, we screened 4,748 unique compounds [LOPAC/Selleck, Spectrum, and GlaxoSmithKline protein kinase inhibitors (GSK-PKIs)] in C. elegans kri-1(ok1251) mutants fed ccm-3 RNAi, 7.4% of which (350/4748) rescued the synthetic lethal phenotype (Figs 1B and EV1; Dataset EV1). The two screens identified six compounds that had already been implicated in alleviating CCM loss-of-function phenotypes in other models: sulindac sulfone (Bravi et al, 2015), XMD8-92 (Cuttano et al, 2016), cholecalciferol (Gibson et al, 2015), propranolol hydrochloride (Moschovi et al, 2010), simvastatin (Whitehead et al, 2009), and sorafenib tosylate (Wüstehube et al, 2010; Table EV1). Click here to expand this figure. Figure EV1. Small-molecule drug screen in C. elegans identifies compounds relevant for CCM A. kri-1 mutant worms treated with control L4440 RNAi and with DMSO are viable. Shown is a representative picture of a control well from a 96-well plate. B. Treatment of kri-1 mutants with ccm-3 RNAi causes synthetic lethality. Incubation of this strain with DMSO (control) has no further effect on the synthetic lethality. C. Incubation of the ccm-3 RNAi-treated kri-1 mutants with the phospholipase C inhibitor ET-18-OCH3 results in a mild rescue, as seen by the presence of a few worms. D. Incubation of the ccm-3 RNAi-treated kri-1 mutants with the GSK-3/PI3K/Akt/mTOR inhibitor TWS119 strongly rescues synthetic as indicated by the high number of worms. Data information: All scale bars are 1 mm. Download figure Download PowerPoint Among the 1,080 compounds that were screened in both zebrafish and C. elegans, 32 suppressed CCM phenotypes in both models (Fig 1B; Datasets EV1 and EV2). This overlap is highly significant (P-value = 0) when compared to a random scenario. Finally, we used human umbilical cord venous endothelial cells (HUVECs) treated with CCM2 shRNA to screen 31 compounds that showed rescue both in zebrafish and C. elegans and another 131 compounds that had been particularly effective in one animal model or the other. Of these 162 compounds, 26 rescued at least some features of the CCM2 phenotype, which is characterized by an increase in the formation of stress fibers, reduced levels of cortical ACTIN, and cell shapes that are more elongated than control shRNA-treated HUVECs (Faurobert et al, 2013; Materials and Methods; Fig 1B; Datasets EV1 and EV2). For example, indirubin-3-monoxime (IR3mo), which was identified in the zebrafish screen, also gave a rescue in the HUVECs screen (Datasets EV1 and EV2). Five compounds showed some degree of rescue in all three CCM models (Dataset EV2): the FLT3 angiogenesis inhibitor ENMD-2076, the PKC/phospholipase A2/D inhibitor DL-erythro-dihydrosphingosine, the PI3K/Akt/mTor pathway inhibitor ridaforolimus, the muscarinic acetylcholine receptor antagonist DL-homatropine hydrobromide, and 13-cis-retinoic acid, which has anti-inflammatory and anti-tumorigenic effects. Strikingly, most of the molecular pathways targeted by these small molecules had not previously been implicated in CCM. Classification of compound activities A functional annotation analysis based on the 18% (24/134) of the zebrafish-active compounds and 20% (70/350) of the C. elegans-active compounds with at least one Medical Subject Headings (MeSH) term assignment revealed that they have a wide range of therapeutic uses (Fig EV2), physiological effects (Fig EV3A), and affect distinct molecular mechanisms (Fig EV3B). The comparison of 102 different therapeutic, physiological, or molecular terms according to which the compounds active in zebrafish and C. elegans were classified revealed an enrichment of anti-inflammatory, anti-hypertensive, neurotransmission modulatory, anti-oxidative, or anti-neoplastic functions. Importantly, we found a number of examples where different compounds with a shared pharmacological functional annotation alleviated CCM phenotypes only in either one or the other animal model. These included vasodilatory agents, for which felodipine (F 9677) affected only zebrafish and carvedilol (C 3993) affected only C. elegans, or the sensory system agents for which niflumic acid (N 0630) affected only zebrafish and loxoprofen (L 0664) only C. elegans (Table EV2). Click here to expand this figure. Figure EV2. Representation of MeSH terms for therapeutic uses obtained for some of the active compounds identified in the zebrafish and C. elegans screens Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Representation of MeSH terms obtained for some of the active compounds identified in the zebrafish and C. elegans screens A. MeSH terms for physiological effects. B. MeSH terms for molecular mechanisms. Download figure Download PowerPoint Target protein predictions reveal networks involved in CCM Clustering analyses based on MeSH term assignments did not help characterize the potential therapeutic, physiological, or molecular activities of many compounds because their mode of action had not yet been sufficiently defined to be assigned a MeSH term. To predict additional protein targets of active compounds, we applied the DePick computational target deconvolution tool. This draws on an extended version of the human drug target prediction tool HitPick to determine protein targets of small compounds identified in phenotypic screens (Liu et al, 2016; Materials and Methods). Based on 1,472 of 1,600 compounds with annotated targets in zebrafish and 4,170 of 4,748 in C. elegans, DePick predicted 47 and 134 human proteins as statistically significant targets of the compounds identified in the zebrafish and in the C. elegans screens, respectively (Table EV3; Dataset EV3). Several of the targets identified in the C. elegans screen had previously been implicated in CCM; these included TLR4 (Tang et al, 2017), metalloproteinases (MMP2, MMP7, MMP13, MMP14; Zhou et al, 2015), and HMGCR (Nishimura et al, 2017), which is a strong validation of the DePick method (Table EV3). DePick analyses revealed a number of important insights into the regulatory network involved in CCM. First, DePick datasets identified a number of specific processes that were targeted in both zebrafish and C. elegans CCM mutants. We carried out comparative Gene Ontology (GO) term analyses for biological processes (GO-BP) based on the zebrafish and C. elegans DePick datasets (Ashburner et al, 2000; The Gene Ontology Consortium, 2017). We identified 42 significantly targeted GO-BP terms based on the zebrafish DePick dataset; the analysis of the C. elegans dataset using these terms revealed that 20 of those were also statistically significant (Dataset EV4). Direct comparison of the zebrafish and C. elegans datasets revealed that among the most significantly targeted proteins in both compound screens were nine proteins with a role in vitamin D or retinoic acid signaling (Table EV3; Datasets EV3 and EV4). In addition, GO-BP te
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