Imaging of Tie2 with a Fluorescently Labeled Small Molecule Affinity Ligand

Abstract

The receptor tyrosine kinase inhibitor, Tie2, has significant roles in endothelial signaling and angiogenesis and is relevant in the pathophysiology of several diseases. However, there are relatively few small molecule probes available to study Tie2, making the evaluation of its activity in vivo difficult. Recently, it was discovered that the small molecule rebastinib (DCC-2036) is a potent Tie2 inhibitor. We hypothesized that fluorescent derivatives of rebastinib could be used as imaging agents for Tie2. On the basis of crystallography structures, we synthesized three fluorescent derivatives, which we then evaluated in both in vitro and in vivo assays. We found that the Rebastinib-BODIPY TMR (Reb-TMR) derivative has superior imaging characteristics in vitro, and we successfully labeled endothelial cells in vivo. We propose that this probe could be further used in in vivo applications for studying the role of Tie2 in disease.

Figure 1:

Synthesis of fluorescently labeled rebastinib derivatives. The synthesis of labeled rebastinib was completed using the scheme depicted above.

Figure 2:

Modeling. Addition of a linker to the amide of rebastinib allows for coupling of a fluorophore to the small molecule. Based on a previously published structure of rebastinib in complex with Tie2 (PDB: 6MWE), we predicted this addition should not interfere with binding of rebastinib to Tie2.

Figure 3:

In vitro testing. Staining of HUVEC and HEK293T cells with the three fluorescent analogs of rebastinib. (a). Panel of representative images of Reb-TMR (100 nM), Reb-650 (100 nM), and Reb-SiR (1 uM) in HUVEC (Tie2+) and HEK293T (Tie2−) cell lines. Reb-TMR and Reb-650 stain HUVEC cells strongly, whereas the Reb-SiR analog stains cells weakly. (b) Quantification of fluorescent intensities in HUVEC and HEK293 cells at single cell resolution. Reb-TMR shows the strongest differential between HUVEC and HEK293T cells.

Figure 4:

Quantitative binding data and inhibition of Reb-TMR (a) Representative images of Reb-TMR in HUVEC cells in the presence (below) or absence (top) of unlabeled rebastinib (10 uM). (b) Quantification of Reb-TMR fluorescent intensity as a function of increasing concentration of unlabeled rebastinib. Reported is the mean single cell fluorescent intensity per well, N = 2 wells. Error bars correspond to S.D. (c) Biochemical fluorescent polarization assay of rebastinib-bodipy TMR (0.8 uM) on enzymatic domain of Tie2. Leftmost point corresponds to polarization of Reb-TMR in PBS, and right points correspond to polarization of Reb-TMR with Tie2, and in increasing concentrations of unlabeled rebastinib (0,0.1,1,10 uM). Reported is mean over N = 2 wells. Error bars correspond to S.D.

Figure 5:

Co-localization (a) High resolution imaging of HUVECs. Treatment of HUVECs with Reb-TMR (100 nM) followed by a stain for Tie2 shows colocalization of the two markers in the cytoplasm (b) Correlation of Reb-TMR and Tie2 immunofluorescence signals (Pearson r² = 0.88)

Figure 6:

Intravital Microscopy. Tie2 reporter mouse, with implanted dorsal skin chambers, were injected with Reb-TMR (20 mg/kg, tail vein I.V.). About 1 hour after injection, select sites in the vascularized region were imaged. The two sites above show Reb-TMR lining the endothelia of the blood vessels, overlaying Tie2.