Targeting vascular endothelial growth factor receptor 2 and protein kinase d1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-alpha -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways

VCC251801 specifically inhibited neutrophil response.

<p>Human neutrophils were pre-treated with VCC251801 for 30 minutes in 37°C at the following concentrations: 0.3 μM, 1 μM, 3 μM, 10 μM. Respiratory burst was induced by immune-complex <b>(A)</b> or 20 ng/ml TNFα on Fibrinogen coating <b>(B)</b>. Every compound was tested at 10 μM using 100 nM PMA stimulation <b>(C)</b>. The dose response curves of VCC251801 and the reference inhibitors are presented using immune complex <b>(D-F)</b> or TNFα on fibrinogen coating <b>(G-I)</b> stimulation. In dose response experiments, each compound was used at the concentrations mentioned above. Every experiment was performed at least 3 times at constant 0.1% DMSO concentration.</p

Treating endothelial cell with VCC251801 did not interfere with cell viability.

<p>The endothelial EA.hy926 cells were incubated with VCC251801 and reference compounds in 0.4–50 μM concentration range for 24 <b>(A)</b> and for 48 <b>(B)</b> hours at constant 0.5% DMSO concentration and cell viability was determined by MTT-assay. <b>(C)</b> Absolute IC₅₀ values from MTT-assays were calculated using non-linear regression from at least three independent experiments.</p

The FcεRI triggered response of mast cells was reduced by VCC251801.

<p>Inhibition of antigen-induced mediator release from Adherent RBL-2H3 mast cells were treated with VCC251801 for 10 minutes then activated by 10 ng/ml antigen and cell degranulation was measured; ***, p < 0.001.</p

VCC251801 inhibited VEGFR2 and PKD1 related signaling pathway in endothelial cells.

<p>EA.hy926 cells were pre-treated with the inhibitors for 1 hour followed by the stimulation of 25 nM PMA, then PKD1 activity was analyzed (<b>A-B</b>). Next, 25 ng/ml VEGF activation was used after 1 hour pre-treatment of the inhibitors and VEGFR2 pathway involving PKD1 was monitored (<b>C-D</b>). Every experiment was carried out at least 3 times at constant 0.2% DMSO concentration; *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

VCC251801 was an effective inhibitor of VEGFR2 and PKD1 in recombinant kinase assays.

<p>The dose response curves of VCC251801 in recombinant VEGFR2 <b>(A)</b> and PKD1 assays <b>(B)</b>. VEGFR2 inhibition was determined by Transcreener assay and PKD1 inhibition was determined applying IMAP assay. Axitinib was used as VEGFR2 reference compound and kb-NB142-70 as PKD1 reference compound. <b>(C)</b> The biochemical IC₅₀ values of the inhibitors (mean ± SD) were calculated from at least three independent experiments. <b>(D)</b> The chemical structure of VCC251801. <b>(E)</b> Recombinant kinase assay against VEGFR isoforms was performed by ProQinase GmbH at single 1 μM concentration. <b>(F)</b> Selectivity study was carried out by SignalChem Ltd. at single 5 μM concentration.</p