Structure and kinase profile of OD16 and OD29. (A–C) Chemical structure and the activity of a panel of 96 kinases in the presence of 100 nM of OD16 (A), OD29 (B), and LDN-193189 (C). (D) Affinity (Kd) of OD16, OD29, and LDN-193189 for the BMP, activin, and TGF-β type I and type II kinase receptors. (E,F) Structural model of OD16 (E) and OD29 (F) binding to the ALK1 hinge region in the ATP pocket.

OD16 and OD29 inhibit BMP, but not TGF-β signaling in EA.hy926 cells. (A–C) Western blot analysis of the dose-dependent effects of LDN-193189 (A), OD16 (B), and OD29 (C) on BMP9 (1 ng/mL)-induced SMAD1/5 phosphorylation in EA.hy926 cells. (D) Quantification of the BMP9-induced pSMAD1/5 phosphorylation. GAPDH or Tubulin was used for protein loading controls. Results from three biologically independent experiments were integrated. The results are expressed as mean ± SD. (E) IC50 values of LDN-193189, OD16, and OD29 on BMP9-induced pSMAD1/5 response in EA.hy926 cells. (F) Inhibitory effects of LDN-193189, OD16, and OD29 on BMP9-induced BRE transcriptional reporter activity in EA.hy926 cells. Cells were preincubated with the inhibitors at three different concentrations (0.1 µM, 0.5 µM, and 1 µM) for 1 h and then stimulated with BMP9 at 1 ng/mL for 16 h with the presence of the compounds. Representative results from three biologically independent experiments are shown as mean ± SD. * p < 0.05, ** p < 0.005, *** p < 0.001. NS, non-significant. (G–I) RT-qPCR analysis of the effects of LDN-193189, OD16, OD29 (0.5 µM) or vehicle control (DMSO) on BMP9 (1 ng/mL)-induced ID1 (G), ID3 (H), and SMAD6 (I) mRNA expression in EA.hy926 cells. Expression levels were normalized to those of the housekeeping gene GAPDH. Results from three biologically independent experiments are shown as mean ± SD. ** p < 0.005, *** p < 0.001. (J–L) Western blot analysis of the dose-dependent effects of LDN-193189 (J), OD16 (K), OD29 (L), or vehicle control (DMSO) on BMP6 (50 ng/mL)-induced SMAD1/5 phosphorylation in EA.hy926 cells. (M) Quantification of the BMP6-induced SMAD1/5 phosphorylation. GAPDH or Tubulin was used as loading controls. (N–P) Western blot analysis of the dose-dependent effects of LDN-193189 (N), OD16 (O), OD29 (p), or vehicle control (DMSO) on TGF-β2 (1 ng/mL)-induced SMAD2 phosphorylation in EA.hy926 cells. (Q) Quantification of the TGF-β2-induced SMAD2 phosphorylation. Tubulin was used as a loading control. The results from three biologically independent Western blot experiments were integrated. The results are expressed as mean ± SD. Complete Western Blot images of subfigures (A–C) and (J–L,N–P) are available in Figure S9.

OD29 antagonizes VEGF-induced pERK MAP kinase in HUVECs. (A–C) Western blot analysis of dose-dependent effects of small molecule VEGFR kinase inhibitor Axitinib (A), OD16 (B), OD29 (C), or vehicle control (DMSO) on VEGF (40 ng/mL)-induced VEGFR and ERK MAP kinase phosphorylation in HUVECs. (D) Quantification of the Western blot analysis of VEGF-induced p-ERK MAP kinase. Tubulin was used as a loading control. The results from three biologically independent Western blot experiments were integrated. The results are expressed as mean ± SD. (E,F) RT-qPCR analysis of the effects of Axitinib, OD16, OD29 (0.5 µM), or vehicle control (DMSO) on VEGF (40 ng/mL)-induced DLL4 (E) and NR4 A (F) mRNA expression in HUVECs. Results from three biologically independent experiments are shown as mean ± SD. * p < 0.05, ** p < 0.005, *** p < 0.001. NS, not significant. Complete Western Blot images of subfigures (A–C) are available in Figure S10.

OD16 and OD29 attenuate EC migration, cord formation, and invasion in vitro. (A–C) Analysis of the real-time migration behavior of EA.hy926 cells exposed to (A) BMP9 (1 ng/mL), (B) VEGF (15 ng/mL), and (C) 1% FBS in the presence of vehicle control (DMSO), LDN-193189, OD16, or OD29 (0.5 µM) within 24 h. Representative results from three biologically independent experiments are shown as mean ± SD. (D) Cord formation assay of VEGF (15 ng/mL)-stimulated HUVEC-eGFP cells co-cultured with human dermal fibroblasts for 6 days to assess the effects of vehicle control (DMSO), OD16, or OD29 (0.5 µM). The small molecule VEGFR kinase inhibitor Sunitinib (1 μM) and BMPR-I kinase inhibitor LDN-193189 (0.5 µM) were used for comparison. Representative images are shown. Scale bar represents 400 μm. (E) Quantification the effects of OD16 or OD29 (0.5 µM) on VEGF (20 ng/mL)-induced cord formation HUVEC-eGFP cells co-cultured with human dermal fibroblasts over 9 days. The VEGFR kinase inhibitor Axitinib was included for comparison. The cord length is shown as mean ± SD. (F) Effects of vehicle control (DMSO), Axitinib, LDN-193189, OD16, or OD29 (0.5 µM) on HUVEC chemotactic cell invasion (towards full culture medium with 20% FBS and supplements). NS, not significant; * p < 0.05; ** p < 0.005, *** p < 0.001.

OD16 and OD29 attenuate EC migration, cord formation, and invasion in vitro. (A–C) Analysis of the real-time migration behavior of EA.hy926 cells exposed to (A) BMP9 (1 ng/mL), (B) VEGF (15 ng/mL), and (C) 1% FBS in the presence of vehicle control (DMSO), LDN-193189, OD16, or OD29 (0.5 µM) within 24 h. Representative results from three biologically independent experiments are shown as mean ± SD. (D) Cord formation assay of VEGF (15 ng/mL)-stimulated HUVEC-eGFP cells co-cultured with human dermal fibroblasts for 6 days to assess the effects of vehicle control (DMSO), OD16, or OD29 (0.5 µM). The small molecule VEGFR kinase inhibitor Sunitinib (1 μM) and BMPR-I kinase inhibitor LDN-193189 (0.5 µM) were used for comparison. Representative images are shown. Scale bar represents 400 μm. (E) Quantification the effects of OD16 or OD29 (0.5 µM) on VEGF (20 ng/mL)-induced cord formation HUVEC-eGFP cells co-cultured with human dermal fibroblasts over 9 days. The VEGFR kinase inhibitor Axitinib was included for comparison. The cord length is shown as mean ± SD. (F) Effects of vehicle control (DMSO), Axitinib, LDN-193189, OD16, or OD29 (0.5 µM) on HUVEC chemotactic cell invasion (towards full culture medium with 20% FBS and supplements). NS, not significant; * p < 0.05; ** p < 0.005, *** p < 0.001.

OD16 and OD29 inhibit subintestinal vessel (SIV) formation in transgenic Casper zebrafish line Tg (fli:EGFP). (A) Schematic representation of the experimental procedure. Freshly collected 2.5 hpf embryos were challenged with testing compounds in egg water. The SIV vascularization was analyzed after 3 days of treatment. (B) Effects of OD16, OD29, Axitinib, and LDN-193189 at 0.5 µM, or vehicle control (DMSO) on zebrafish SIV development. Compounds were added to egg water for 3 days before observing the SIV vascularization. Representative images of overview (left panel) and high magnification (white rectangle areas from left) are shown. Asterisks represent sprouting vessels. Scale bars represent 100 and 50 µm. (C) Quantification of the SIV vessel length of the zebrafish embryos in each group from the experiment shown in (B). * p < 0.05, ** p < 0.005, *** p < 0.001. (D) Quantification of the number of leading buds (indicated as asterisks in panel B). *** p < 0.001. NS, not significant.

OD16 and OD29 inhibit vessel invasion in a breast cancer xenograft model in zebrafish. (A) Schematic representation of the experimental procedure. Two dpf zebrafish embryos were injected with mCherry (red) labeled human MDA-MB-231 breast cancer cells in the perivitelline space. The injected zebrafish embryos were challenged with testing compounds in egg water for 3 days, and the newly formed vessels within the perivitelline were analyzed. (B) Representative images of the eGFP expressing blood vessels (green) and mCherry MDA-MB-231 cells (red) after treatment of the zebrafish embryos with vehicle control (DMSO), Axitinib, LDN-193189, OD16, or OD29 (0.5 µM) for 3 days. The medium (vessels and tumor cells are visualized) and right panels (only vessels are visualized) represent high magnification views of the area, indicated by white rectangles in the overviews (left panel) images. Scale bars represent 200 µm. (C) Quantification of the relative vasculature density induced by the injected MDA-MB-231 cells within the perivitelline space of the zebrafishes in each group. * p < 0.05, ** p < 0.005, *** p < 0.001.