Combined Use of Oligopeptides, Fragment Libraries, and Natural Compounds: A Comprehensive Approach To Sample the Druggability of Vascular Endothelial Growth Factor

Abstract

The modulation of protein-protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide-mediated PPIs efficiently, the inhibition of domain-domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF-VEGFR interaction is a typical domain-domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR-spectroscopy-based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.

Keywords: drug discovery; fragment screening; growth factors; peptides; protein-protein interactions.

Figure 1

a) Comparison of the ¹H–¹³C HSQC spectra of methyl‐¹³C‐Met labeled VEGF (250 μm) in the absence (black) and in the presence of 2 equivalents of peptide v107 (red). The (His)₆‐VEGF fusion protein used in this experiment contains a methionine outside of the VEGF sequence that is encoded by the starting codon of the gene expressing the fusion protein. An asterisk marks the cross signals of the (His)₆‐VEGF initial Met. b) Expanded view of the structure of the VEGF–v107 complex (PDB ID: 1KAT). Homodimeric VEGF and v107 are shown in gray and blue, respectively. Methionine residues of VEGF are shown as stick models.

Figure 2

a) Chemical structure of the cyclic hexapeptide &d‐Pro‐Trp‐Glu‐d‐Pro‐Trp‐Glu&. b) STD NMR spectrum (T=278 K) of a sample containing 1 mm cyclic hexapeptide &d‐Pro‐Trp‐Glu‐d‐Pro‐Trp‐Glu& and 10 μm VEGF. c) Overlay of the ¹H–¹³C HSQC spectra (T=318 K) of methyl‐¹³C‐Met‐labeled VEGF (50 μm) in the absence (black) and in the presence of 2.5 mm cyclopeptide &d‐Pro‐Trp‐Glu‐d‐Pro‐Trp‐Glu& (red). The cross signals of Met78 are folded. Resonances corresponding to ¹³C natural abundance peptide are marked by an asterisk.

Figure 3

Overview of the strategy used for fragment selection.

Figure 4

a) Analysis of the SiteMap computations reveals druggability mainly in the channel formed between the two chains of the VEGF homodimer (sites 1, 2, 3, and 4). Red, blue, and yellow surfaces show sites for potential H‐bond acceptor, H‐bond donor, and hydrophobic interactions, respectively. White dots indicate the presence of small cavities. Receptor binding sites are indicated by 5. The number size correlates with the size of the binding sites. b) Representation of the XP GLIDE docking results. A selected library of approximately 500 compounds (gray carbon atoms) was docked against the VEGF homodimer (black ribbons). Sites 1 and 2 (panel a) were most populated.

Figure 5

Chromatographic profile of representative water extracts of a) Medulla Junci and c) Radix scutellariae. ¹³C‐Filtered–¹³C decoupled ¹H NMR spectra of a 30 μm methyl‐¹³C‐Met‐labeled VEGF sample in the absence (b and d, top) and in the presence of 3 μL (b and d, middle) and 6 μL (b and d, bottom) of a stock solution, prepared as described in the Experimental Section, of either b) Medulla Junci or d) Radix scutellariae extracts.

Figure 6

a) HPLC chromatogram of baicalin extracted from the Radix scutellariae plant extract. The chemical structure of baicalin is shown (right). b) STD NMR spectrum (T=298 K) of a sample containing 1 mm baicalin and 10 μm VEGF. STD spectra of the same sample in the presence of c) 50 μm and d) 100 μm v107 peptide showing the reduction of the STD signal intensities of baicalin in the presence of v107.

Figure 7

a) Chemical structure of the flavonoid molecules tested. b) Histogram showing weighted average chemical shift changes for VEGF amide resonances between free ¹⁵N‐labeled VEGF (250 μm) and ¹⁵N‐labeled VEGF (250 μm) in the presence of either 7.7 mm baicalin (black) or 4.4 mm quercetin‐3‐β‐glucoside (gray). c) Model of the VEGF–baicalin complex. VEGF is shown as a cartoon representation and baicalin as a ball and stick model.