N-Terminal modification of Arg-Leu-Tyr-Glu, a VEGFR-2 antagonist, improves anti-tumor activity by increasing its stability against serum peptidases

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a vascular endothelial growth factor (VEGF) receptor-2 antagonist, has been used alone or in combination with chemotherapeutic drugs for treating colorectal cancer in a mouse model. Here, we found that the peptide had a short half-life of 1.2 h in serum, due to degradation by aminopeptidases B and N. Therefore, we designed new modified peptide: The tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a vascular endothelial growth factor (VEGF) receptor-2 antagonist, has been used previously either alone or in combination with chemotherapeutic drugs for treating colorectal cancer in a mouse model. We analyzed the half-life of the peptide and found that due to degradation by aminopeptidases B and N, it had a short half-life of 1.2 h in the serum. Therefore, to increase the stability and potency of the peptide, we designed the modified peptide, N-terminally acetylated RLYE (Ac-RLYE), which had a strongly stabilized half-life of 8.8 h in serum compared with the original parent peptide. The IC50 value of Ac-RLYE for VEGF-A-induced endothelial cell migration decreased to approximately 37.1 pM from 89.1 pM for the parent peptide. Using a mouse xenograft tumor model, we demonstrated that Ac-RLYE was more potent than RLYE in inhibiting tumor angiogenesis and growth, improving vascular integrity and normalization through enhanced endothelial cell junctions and pericyte coverage of the tumor vasculature, and impeding the infiltration of macrophages into tumor and their polarization to the M2 phenotype. Furthermore, combined treatment of Ac-RLYE and irinotecan exhibited synergistic effects on M1-like macrophage activation and apoptosis and growth inhibition of tumor cells. These findings provide evidence that the N-terminal acetylation augments the therapeutic effect of RLYE in solid tumors via inhibition of tumor angiogenesis, improvement of tumor vessel integrity and normalization, and enhancement of delivery and efficacy of the co-administered chemotherapeutic drugs. SIGNIFICANCE STATEMENT The results of this study demonstrate that the N-terminal acetylation of the tetrapeptide RLYE (Ac-RLYE), a novel VEGFR-2 inhibitor, significantly improves its serum stability, anti-angiogenic activity, and vascular normalizing potency, resulting in enhanced therapeutic effect on solid tumors. Furthermore, the combined treatment of Ac-RLYE with the chemotherapeutic drug, irinotecan, synergistically enhanced its anti-tumor efficacy by improving the perfusion and delivery of the drug into the tumors and stimulating the conversion of the tumor-associated macrophages to an immunostimulatory M1-like anti-tumor phenotype.