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

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.

Small-Molecule Probes as Pharmacological Tools for the Bone Morphogenetic Protein Signaling Pathway

“…It was identified by a chemocentric approach based on the Nanocyclix technology. OD16 presents commendable potency and selectivity for ALK2 ( K d = 17 nM) and ALK1 ( K d = 20 nM) over TGFβ receptors in vitro . Advantageously, no significant off-target inhibition was observed in a kinase profiling assay.…”

“…Advantageously, no significant off-target inhibition was observed in a kinase profiling assay. OD16 was also extensively characterized in various zebrafish assays, demonstrating anti-tumor angiogenesis activity …”

“…OD16 presents commendable potency and selectivity for ALK2 (K d = 17 nM) and ALK1 (K d = 20 nM) over TGFβ receptors in vitro. 61 Advantageously, no significant off-target inhibition was observed in a kinase profiling assay. OD16 was also extensively characterized in various zebrafish assays, demonstrating antitumor angiogenesis activity.…”

“…OD16 was also extensively characterized in various zebrafish assays, demonstrating antitumor angiogenesis activity. 61 2-Aminopyrazine-3-carboxamide Ullrich 23 (Figure 2A) presents an SAR-optimized hit from a kinase inhibitor library screening. It demonstrates high affinity for the FOP mutant ALK2 R206H (IC 50 = 8 nM) as well as the ALK2 wild-type receptor (IC 50 = 12 nM) and is even selective over several other BMP type I receptors.…”

The Dual Effect of the BMP9–ALK1 Pathway in Blood Vessels: An Opportunity for Cancer Therapy Improvement?

“…They have shown higher selectivity than other ALK1/2 inhibitors previously used, like LDN-193189. Both molecules have proved in vitro anti-angiogenic properties that are induced by BMP9 and VEGF [138].…”