Fig 1.
Chemical structures of the three TKIs included in this study.
Structures were created using KingDraw (Qingdao, China). TKI, tyrosine kinase inhibitor.
Fig 2.
Kinase assay screen using 10μM of each TKI.
(A) Control (baseline) TREEspot image depicting what 100% inhibition of all receptors associated with angiogenesis (blue) would look like. TIE2 receptor signaling is essential to maintain blood vessel stability and 100% inhibition of the TIE2 receptor is depicted (red). (B) In comparison to anti-VEGF antibodies, the three tested TKIs effectively inhibit all the receptors involved in pathological angiogenesis; the TKIs differ from each other as axitinib strongly inhibits TIE2, which is undesirable as maintained TIE2 function is essential for vascular stability. TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor.
Fig 3.
(A) IC50 curves for the three TKIs inhibiting VEGFR1, VEGFR2, and VEGFR3. (B) TIE2 IC50 curves for the three TKIs. The TIE2 IC50 confirmed the kinase screen data as the three tested TKIs differed greatly in their ability to inhibit TIE2 receptor. Note that all TKI compounds were prepared and tested in duplicate in a ten-dose IC50 model. (C) Computer modeling of axitinib with TIE2 receptor. On the left, the protein is displayed using ribbons while the protein surface is displayed using a white transparent pattern. The small molecule axitinib is shown as a teal stick image. The middle and left side of the figure shows the detailed analysis of the binding mode of small molecule simulation with TIE2 in a steady state. In the middle are 3D diagrams of interaction with the hydrogen bonds shown by a dotted line. On the right side is a 2D diagram depicting the axitinib interaction with hydrogen bonds indicated with a red arrow. IC50, half-maximal inhibitory concentration; TIE2, tyrosine kinase with immunoglobulin-like and EGF-like domains 2; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
Table 1.
Postulated TIE2 in vivo inhibition based on clinical TKI dosing and tested models.
To understand the relevance of TIE2 inhibition, observed and reported TKI levels in retina/choroid of pre-clinical in vivo models treated with a clinical dosage of the TKIs were analyzed. The observed/reported TKI information in this table was extrapolated from the indicated references, which are publicly available presentations/publications, and that information did not provide statistical data. TIE2 IC50 values for each TKI were determined by Reaction Biology (US) and shown in Fig 3B.
Fig 4.
HUVEC sprouting assay results.
(A) Representative photos of the spheroids when treated with two different concentrations of each TKI. (B) Quantitative analysis of the cumulative sprout length per spheroid (CSL) for each TKI at the different test concentrations. Results are (mean ± SD). Controls were ± 25 ng/mL VEGF-A. Results are (mean ± SD). Statistical differences were made visible by the presence of stars: *0.05 ≥ p > 0.01; **0.01 ≥ p > 0.001; ***0.001 ≥ p ≥ 0.0001; ****0.0001 ≥ p. CSL, cumulative sprout length; HUVEC, human umbilical vein endothelial cell; SD, standard deviation; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor.
Fig 5.
CAM assay results for the three TKIs tested at their VEGFR2 IC50 value (vorolanib = 52nM, sunitinib = 43nM, and axitinib = 0.2nM).
(A) Representative images of gelatin sponge and surrounding vessels in ovo for negative control, positive control (anti-VEGF antibody, bevacizumab) and the three tested TKIs. Black arrows point at the white spot which is the location of the gelatin sponge. Scale bar is 1000 μm. (B) The number of macroscopic blood vessels perfusing the gelatin sponge for each TKI was determined (mean ± SEM). (C) The individual semi-quantitative scoring of vessel size for each TKI is shown (mean ± SEM). For panels B and C, statistical differences were made visible by the presence of stars: *0.01 < p ≤ 0.05; **0.001 < p ≤ 0.01; ***0.0001 ≤ p ≤ 0.001; ****p ≤ 0.0001. CAM, chorioallantoic membrane; IC50, half-maximal inhibitory concentration; SEM, standard error of the mean; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
Table 2.
Melanin-binding data using a 0.06 to 25.0μM concentration range for all TKIs.
Sunitinib was the only TKI of the three tested TKIs to exhibit definitive characteristics of melanin binding. SEM is indicated in the table.
Fig 6.
Computer modeling of vorolanib bound to VEGFRs.
(A) VEGFR1; (B) VEGFR2; and (C) VEGFR3. Left panels, protein displayed using ribbons while the protein surface is displayed using a white transparent pattern. Vorolanib is shown as a purple stick image and image is at 50 ns stimulation. Left and center panels, binding of vorolanib with VEGFR at 50 ns stimulation. Center panels, 3D diagrams of interaction with hydrogen bonds indicated by yellow dotted lines and π-π stacking is indicated by green dotted lines. Right panels, 2D diagrams depicting the interaction of vorolanib with VEGFRs. Hydrogen bonds are indicated using purple arrows, and π-π stacking is indicated with a green line. VEGFR, vascular endothelial growth factor receptor.
Fig 7.
Schematic of cell membrane RTKs involved in angiogenesis and vascular stability, including stimulating ligands, anti-VEGF antibody therapies, and TKIs.
All three TKIs showed pan-VEGFR inhibition and effectively inhibited all the receptors that participate in pathological angiogenesis. Axitinib was the only TKI identified as a potent inhibitor of TIE2 which is not desired as normal TIE2 function is essential as it functions to maintain vascular stability. This image was created using BioRender software. Ang, angiopoietin; FGFR, fibroblast growth factor receptor; PDGFR, platelet-derived growth factor receptor; PLGF, placental growth factor; RTK, receptor tyrosine kinase; TIE2, tyrosine kinase with immunoglobulin-like and EGF-like domains 2; TKI, tyrosine kinase inhibitor; VE-PTP, vascular endothelial cell-specific protein tyrosine phosphatase; VEGFR, vascular endothelial growth factor receptor.