Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors

SIMPLE SUMMARY: Anti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to prolonged overall survival of cancer patients, therapy resistance is easily acquired. Therefore, novel inhibitors against other signaling pathways mediating angiogenesis are needed to achieve more efficient and sustainable targeting of the angiogenesis process. Here, we synthesized and identified two compounds belonging to a new class of small molecules termed macrocyclics that selectively inhibit bone morphogenetic protein receptor kinase activity. One compound also inhibits vascular endothelial growth factor-induced signalling. Treatment studies using in vitro cultured cells and zebrafish embryos revealed that both compounds impaired endothelial cell function and decreased normal and tumour-induced angiogenesis. Both compounds might provide a steppingstone for the development of novel-angiogenesis therapeutic agents. ABSTRACT: Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.