The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone

Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies

Isolation und Characterisierung des

Angeborene Störungen der Cholesterol-Biosynthese werden durch eine Reihe unterschiedlicher Entwicklungsdefekte charakterisiert. Unter den Enzymen, die in die Cholesterol-Biosynthese mit einbezogen werden, ist die Reduktase des 7 Dehydrocholesterol (DHCR7) das spezifische Enzym des Cholesterins, da es den letzen Schritt der Synthese katalysiert, der direkt zum Cholesterol führt. Mutationen im Gen für die DHCR7 sind die Hauptursache des Smith-Lemli-Opitz Syndroms (SLOS), das durch die Dysmorphogenese verschiedener Organe gekennzeichnet ist. Obwohl Daten zunehmen, die zeigen, dass der SLOS Phänotyp möglicherweise auf einen gestörten Hedgehog-Signalweg zurückzuführen ist, ist der molekulare Mechanismus weiterhin Gegenstand intensiver Forschung, der die Cholesterol-Homöostase in der Entwicklung mit einschließt. Es konnten drei Xenopus EST Klone über eine Durchmusterung entsprechender Genbanken identifiziert werden, die eine hohe Homologie zur menschlichen dhrc7 cDNA besaßen. Mittels RT-PCR konnten drei Isoformen der Xenopus DHCR7 isoliert werden, die alle gemeinsam im Endoplasmatischen Retikulum lokalisierten, wenn diese in Hefen überexprimiert wurden. Die Analyse der embryonalen Expression zeigte eine Expression im Bereich des Spemann Organisators, dem Notochord und später in bestimmten Bereichen des embryonalen Gehirns. Die zeitlich-räumliche Expression sowie die Verteilung der DHCR7-Transkripte in adulten Geweben zeigte eine auffällige Korrelation mit der lokalen Expression von Sonic Hedgehog. Die Überexpression von 2 ng synthetischer RNA der längsten Isoform von DHCR7 führte in der Folge zu einer Entwicklung kleinerer Augen, was einher ging mit dem Verlust placodaler Strukturen, wie durch die Analyse verschiedener neuraler Markergene gezeigt werden konnte. Die Unterdrückung der Translation ("knock-down") durch die Injektion von 1 pmol Xdhr7-Antisense-Oligonukleotide (Morpholinos) führte ebenfalls zur Ausbildung stark verkleinerter Augenanlagen, wahrscheinlich durch eine teilweise Unterdrückung der Neurogenese

Structure-activity relationships of benzbromarone metabolites and derivatives as EYA inhibitory anti-angiogenic agents.

The tyrosine phosphatase activity of the phosphatase-transactivator protein Eyes Absent (EYA) is angiogenic through its roles in endothelial cell migration and tube formation. Benzbromarone, a known anti-gout agent, was previously identified as an inhibitor of EYA with anti-angiogenic properties. Here we show that the major metabolite of BBR, 6-hydroxy benzbromarone, is a significantly more potent inhibitor of cell migration, tubulogenesis and angiogenic sprouting. In contrast, other postulated metabolites of BBR such as 5-hydroxy benzbromaorne and 1'-hydroxy benzbromarone are less potent inhibitors of EYA tyrosine phosphatase activity as well as being less effective in cellular assays for endothelial cell migration and angiogenesis. Longer substituents at the 2 position of the benzofuran ring promoted EYA3 binding and inhibition, but were less effective in cellular assays, likely reflecting non-specific protein binding and a resulting reduction in free, bio-available inhibitor. The observed potency of 6-hydroxy benzbromarone is relevant in the context of the potential re-purposing of benzbromarone and its derivatives as anti-angiogenic agents. 6-hydroxy benzbromarone represents a metabolite with a longer half-life and greater pharmacological potency than the parent compound, suggesting that biotransformation of benzbromarone could contribute to its therapeutic activity

EYA inhibitor Benzbromarone, compound 1.

<p>(<b>a</b>) Compound 1 was identified from a screen of the NCI Diversity Set II library. IC50 values for compound 1 were measured for Eya3(ED), human EYA3 and human EYA2(ED) using as substrates the phosphotyrosine mimic pNPP and a phosphopeptide representing the last 10 amino acids of H2AX, a known EYA substrate. (<b>b</b>) Substrate titration shows that compound 1 is an uncompetitive inhibitor of EYA3(ED). Increasing concentration of substrate does not overcome inhibition. Each point represents the mean and standard deviation of two independent readings. (<b>c</b>) Plots of Vmax and Km as a function of inhibitor concentration show that both values decrease with increased inhibitor concentration. Values in (c) were derived from nonlinear regression analyses of curves in (b) using PRISM (GraphPad Software). (<b>d</b>) Compound 1 does not affect the interaction between EYA3 and SIX2. Recombinant purified EYA3 and His-SIX2 were mixed and treated with either the vehicle control (1% DMSO) (lane 1) or 50 μM compound 1 (lane 5) for 15 minutes at room temperature. The mixture was loaded on a Ni-NTA column. Beads were washed with 3 column volumes of load buffer (last washes, lanes 2 and 6). Proteins retained on the beads are shown in lanes 3 and 7. Lanes 4 and 8 are molecular weight markers.</p

Structure-activity relationships.

<p>Chemical structures of compounds related to Benzbromarone (1a–1j) that were assayed for their ability to inhibit the phosphatase activity of Eya3(ED), EYA3, and PTP1B. IC50 values are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034806#pone-0034806-t001" target="_blank">Table 1</a>.</p

Binding modes of Benzbromarone and the substrate mimic phosphotyrosine differ.

<p>(<b>a</b>) In docking experiments phosphotyrosine (gold) and compound 1 (yellow) bind at the interface of the cap and core subdomains of the catalytic ED domain of EYA3. (<b>b</b>) Phosphotyrosine coordinates the active site metal ion and makes hydrogen bonds with residues in all three conserved motifs in the EYAs. (<b>c</b>) Docked conformation (ICM Score −24.8) of compound 1 docked into the active site of EYA3(ED). The conformation shown here was obtained in multiple docking experiments conducted with different starting conformations for the ligand. Compound 1 inserts its dibromo-phenol group into a hydrophobic cavity adjacent to the phosphotyrosine binding site and is anchored by a hydrogen bond with Tyr 329. The carbonyl group forms a hydrogen bond with Ser 324. Compound 1 is not within bonding distance of the metal ion.</p

Dose-dependent effects of EYA inhibitors on the developing zebrafish vasculature.

<p>(<b>a</b>) Titration of compounds 1, 1a, 1b at the indicated doses. Two independent experiments were performed in most cases using 9–15 embryos per experiment at each dose; the standard error and mean values are shown. Experiments with 0.25 μM compound 1 and 5 μM compound 1b were only performed once using 10 embryos each. The y-axis shows the percentage of embryos showing any defects in vascular development (either intersegmental vessels (ISV) formation or defects in the dorsal aorta/cardinal vein). (<b>b</b>) Images of representative untreated control (CTL), vehicle (DMSO) treated, and EYA inhibitor treated embryos at 28–30 hpf. Note reduced or absent ISV in the inhibitor treated embryos relative to the controls (ISV in controls indicated by white arrows).</p