2-Desaza-annomontine impedes angiogenesis by inhibiting CDC2-like-kinases and β-catenin activity
Authors:   , Zech Thomas 
Published: December 2024

Angiogenesis, the formation of blood vessels from pre-existing ones, is a vital process in growth, development, and wound healing. However, in the context of solid tumors, chronic inflammation, and wAMD, angiogenesis is often dysregulated, contributing to the progression of the disease. As a result, several anti-angiogenic therapies have been developed and used with some success in the treatment of wAMD and cancer. However, while these treatments have significantly improved the outcome of these diseases, their efficacy is still limited. This is because resistance is common in cancer, while in wAMD, the underlying inflammatory processes can still drive the disease. In addition, angiogenesis is still not routinely targeted in chronic inflammatory diseases. Thus, there is still a great need for anti-angiogenic agents, and combined inhibition of angiogenesis and inflammation is desirable.
2-Desaza-annomontine, also called C81, is a synthetically available derivative of the alkaloid annomontine, which is found in the plant family Annonaceae. Derivatives of annomontine have previously been characterized as bioactive compounds. As one of them, C81 has been shown to affect inflammatory processes of the endothelium and restore endothelial barrier function against immune cells by Krishnathas et al. (2021), who also discovered that C81 inhibits DYRK2, PIM3, and the splicing kinases CLK1 and CLK4. Therefore, we hypothesized that C81 could also inhibit endothelial angiogenic functionality, making it a promising compound for the treatment of diseases that depend on angiogenesis and inflammation.
As an initial test of this hypothesis, a murine laser-induced choroidal neovascularization (CNV) model was used. In this model, 3 and 10 µM C81 strongly inhibited vascular permeability and endothelial infiltration into the choroid, indicating that C81 inhibits angiogenesis. Since VEGF is one of the most important pro-angiogenic signaling molecules in CNV, we next tested whether C81 specifically affects VEGF-induced angiogenesis. A murine aortic ring model was used for this purpose. In this model, 3 µM C81 significantly inhibited aortic sprouting, demonstrating that C81 is an inhibitor of VEGF-induced angiogenesis.
To elucidate the processes by which C81 affects angiogenesis, assays testing individual steps of the angiogenic cascade were performed in human umbilical vein endothelial cells (HUVECs). In these assays, C81 inhibited the ability of HUVECs to form VEGF-induced sprouts from spheroids, reduced the formation of capillary-like structures on Matrigel, abrogated proliferation, and inhibited migration. Because angiogenesis in vivo is mediated by the microvascular endothelium, key experiments were repeated in the microvascular cell line HMEC-1. Since C81 also reduced proliferation, sprouting, and migration of HMEC-1, we concluded that C81 impedes angiogenesis by inhibiting angiogenesis-related cellular functions of the endothelium.
We then investigated the pathways by which C81 affects angiogenic cellular functions. We found that C81 reduced the activity of the VEGF/VEGFR2 signaling pathway by abrogating VEGFR2 protein expression. This reduction in VEGFR2 protein expression was, in turn, caused by impaired VEGFR2 mRNA expression in C81-treated cells. Taken together, these results indicate that C81 impairs angiogenesis by downregulating VEGFR2 expression.
To find the targets responsible for the C81-induced effects, we tested established chemical probes against the targeted kinases. This revealed that only inhibition of CLKs resulted in a significant downregulation of VEGFR2. Subsequently, we discovered that C81 is a pan-CLK inhibitor. Moreover, the CLK inhibitors MU1210 and T3-CLK were also able to phenocopy C81-derived effects on the sprouting of VEGF-stimulated HUVEC spheroids. These results suggest that the CLKs are the targets responsible for the anti-angiogenic effects of C81. This was confirmed by the knockdown of individual CLKs. Knockdowns of all CLK isoforms inhibited VEGFR2 mRNA expression and VEGF-induced sprouting of HUVEC spheroids. We could not identify an alternative splicing event associated with VEGFR2 in C81- or MU1210-treated HUVECs. Therefore, alternative splicing of VEGFR2 is unlikely to be the responsible mechanism. However, a GO term analysis followed by a reporter gene assay confirmed the previously published finding that CLK inhibitors abolish the WNT/β-catenin signaling pathway activity. This pathway has been shown to affect VEGFR2 expression in HUVECs and CNS endothelial cells. Therefore, we induced the activity of this pathway by using a GSK3β inhibitor, which increased the expression of VEGFR2. Conversely, knockdown of β-catenin inhibited VEGFR2 expression, confirming the involvement of this pathway.
In summary, this work shows that CLK inhibitors are promising compounds in the treatment of angiogenesis related diseases. Especially in the therapy of inflammation related diseases and tumors they could be relevant, as CLK inhibitors have previously shown promising disease modifying properties in this context.