Structural insights into molecular-targeting helix-loop-helix peptide against vascular endothelial growth factor-A.

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix-Loop-Helix Peptide to Bind VEGF for Tumor Growth Inhibition.

As a small affinity molecule to serve as an alternative to antibodies, we have developed a conformationally constrained peptide with a de novo designed helix-loop-helix (HLH) scaffold. To evaluate its potential for biomedical applications, we performed directed evolution of HLH peptides to obtain an inhibitor for vascular endothelial growth factor-A (VEGF). A phage-displayed library of HLH peptides was constructed and screened against VEGF, giving the peptide VS42 that inhibits the VEGF/VEGF receptor-2 interaction (IC50 = 210 nM), which was further improved by in vitro affinity maturation using a yeast-displayed library. An identified HLH peptide, VS42-LR3, exhibited improved inhibitory activity (IC50 = 37 nM), high thermal stability, and excellent resistance against chemical denaturation. In biological activity tests, the HLH peptide was found to block VEGF-induced proliferation of human umbilical vein endothelial cells and suppress tumor growth in a murine xenograft model of human colorectal cancer.