From combinatorial peptide selection to drug prototype (I): targeting the vascular endothelial growth factor receptor pathway

Abstract

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated (D)(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, (D)(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.

Fig. 1.

Drug design and protease degradation-resistance assay. (A) Schematic representation of Arg-Pro-Leu (RPL) retro-inversion. (B and C) Mass-spectroscopy analysis (MALDI-TOF) of RPL and _D(LPR) pre- and postincubation with pancreatic enzymes. Peaks corresponding to the intact peptides and enzymatic degradation products are color-coded [red, RPL; blue, _D(LPR)] and indicated by arrows.

Fig. 2.

The tripeptide RPL and the drug _D(LPR) target VEGFR-1 and NRP-1. (A) In a phage-competition assay, increasing concentrations of RPL (black circles) or _D(LPR) (open squares) inhibit binding of CPQPRPLC-displaying phage to the immobilized receptors VEGFR-1 or NRP-1. (B) Chemical-shift changes induced on the _D(LPR) peptidomimetic resonances by its binding to VEGFR-1 or NRP-1 at 25 °C are shown. 2D TOCSY spectra of _D(LPR) alone (black color) or in the presence of the individual receptors (red color) are shown. Different regions of the spectra of _D(LPR) are shown to indicate chemical-shift changes in individual _D(LPR) residues.

Fig. 3.

Inhibition of neovascularization in vivo by _D(LPR) treatment. (A) Representative pictures of Matrigel plugs containing 500 μg/mL of _D(LPR) or control after 7 days of implantation (Lower). Matrigel plugs were excised, and angiogenesis was quantified by measurement of the hemoglobin content within the matrix. The bar graph shows representative mice from the experiment (Upper). (B) Immunostaining with anti-human factor VIII antibody of scaffolds containing human microvascular endothelial cells (HDMEC) implanted into SCID mice that received 25 mg/kg i.p. daily of _D(LPR) or control. (C) Number of human factor VIII-positive blood vessels at 200× magnification. *Student’s t test (P < 0.01).

Fig. 4.

Systemic and topical treatment with _D(LPR) inhibits retinal angiogenesis in a mouse retinopathy-of-prematurity model. Retinal neovascularization was induced in C57BL/6 neonatal mice by exposure to 75% oxygen (P7–P12) followed by daily i.p. administration of _D(LPR) or control (20 mg/kg per day). (A) H&E-stained retinal sections (P19) showed new blood vessels at the retinal inner surface (arrows) in control animals (Top and Center). There was marked reduction in _D(LPR)-treated mice (Bottom). (B) P19 quantification of neovascular nuclei protruding into the vitreous space. Serial sections (n > 5) of eyes (n > 10) were quantified in each group. Treatment with _D(LPR) yielded a significant reduction in nuclei relative to vehicle or control. (C) In systemic (i.p.) delivery, the magnitude of _D(LPR)-induced neovascularization inhibition was similar to that observed by treatment with the monoclonal antibody bevacizumab. (D) Topical delivery of _D(LPR) in an eye-drop formulation (200 μg e.d. three times daily) also induced a significant reduction in abnormal retinal angiogenesis. Shown is mean ± SEM in each experiment.*, Student’s t test (P < 0.05).

Fig. 5.

CPQPRPLC-targeted phage homed to tumors, and _D(LPR) treatment reduced tumor growth. (A) Tumor-bearing mice received 10¹⁰ transducing units (TU) of either CPQPRPLC phage or insertless phage (negative control). After 24 h of circulation, phage homing to tissues was evaluated by counting the relative TU. The brain served as a negative control organ. (B) Tumor-bearing mice (n = 7 per cohort) were treated with vehicle alone, _D(LPR), or control peptide (50 mg/kg/day for 5 days). Two independent experiments were performed with similar results. A representative experiment is shown. *Student’s t test (P = 0.02).

Fig. 6.

Effect of _D(LPR) on VEGFR-1–meditated endothelial cell proliferation and signaling. (A) Dose-dependent effect of _D(LPR) on PlGF-induced HUVEC proliferation. Results are presented as values relative to BSA. (B) Immunoblot analysis of phosphorylated and total forms of VEGFR-1, ERK, and Akt. P1GF was used at 100 ng/mL, and _D(LPR) or control was used at 10 μg/mL.