2-Desaza-annomontine (C81) impedes angiogenesis through inhibition of CDC2-like kinases (CLKs) and β-catenin activity.

Angiogenesis is the creation of blood-vessels from pre-existing ones. In multiple pathologies, such as cancer or chronic inflammation, angiogenesis contributes to the progression of the underlying condition. While therapies have been developed and applied, their efficacy is still not adequate, especially in the treatment of aberrant angiogenesis in inflammation-related diseases.

C81 is a derivative of the alkaloid annomontine and impedes inflammatory processes in the endothelium. Accordingly, the initial aim of this project was to evaluate, whether C81 also impairs angiogenesis, and thus possesses a dual mechanism. Moreover, C81 inhibits the kinases DYRK1B, DYRK2, PIM3, and the splicing kinases CLK1-4. Therefore, a further aim was to relate the targeted proteins to the effects of C81.

Indeed, in an in vivo laser-induced choroidal neovascularization (CNV) model we observed that C81 potently reduced angiogenic activation of the endothelium. Functionally, this was caused by C81 reducing the VEGF-induced angiogenic activity of endothelial cells (ECs), which was derived from decreased VEGFR2 expression.

To find the responsible targets, we used inhibitors of the addressed kinases and observed that only CLK inhibition phenocopied the C81-derived effects in ECs. Moreover, knockdowns of the individual CLK isoforms were also able to phenocopy C81-derived effects on VEGFR2 expression, proving the involvement of these kinases.

When investigating the mechanism, RNA-seq revealed that CLK inhibition does not alter the splicing of VEGFR2. However, a following GO-term analysis and reporter gene assay confirmed that CLK inhibitors are potent inhibitors of β-catenin activity, a pathway that is known to induce VEGFR2 expression and angiogenesis. Finally, the relevance of this pathway was confirmed through activation of β-catenin signaling, which increased VEGFR2 expression, while a knockdown of β-catenin decreased VEGFR2 expression.