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. 2010 Mar 11;53(5):2215-26.
doi: 10.1021/jm901755g.

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction

Ashley Jarvis  1 , Charles K Allerston, Haiyan Jia, Birger Herzog, Acely Garza-Garcia, Natalie Winfield, Katie Ellard, Rehan Aqil, Rosemary Lynch, Chris Chapman, Basil Hartzoulakis, James Nally, Mark Stewart, Lili Cheng, Malini Menon, Michelle Tickner, Snezana Djordjevic, Paul C Driscoll, Ian Zachary, David L Selwood
Affiliations
Free PMC article

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction

Ashley Jarvis et al. J Med Chem. .
Free PMC article

Abstract

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

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Figures

Figure 1
Figure 1
Model for binding of VEGF-A165 to NRP1. NRP1 has a large extracellular (Ex) domain comprising tandem a1/a2, b1/2, and a c domain, a single membrane-spanning domain, and a small cytosolic domain (Cyt). The VEGF-A165 C-terminal domain encoded by exons 7 and 8 (yellow and blue oblongs, respectively) binds to the extracellular NRP1 b1 domain. Concomitant binding of the VEGF homology domain of VEGF-A165 (solid red ovals) to VEGFR2 results in formation of a receptor complex of NRP1 with VEGF-A165 and VEGFR2 and enhanced intracellular signaling, essential for optimal migration and angiogenesis in development and in tumors.
Figure 2
Figure 2
Bicyclic peptide 1 (C-terminus of VEGF) and small molecule neuropilin inhibitor 2.
Figure 3
Figure 3
(a) VEGF/tuftsin binding site of NRP1 b1 domain (black arrow), with the protein surface and the loops L1 (green), L2 (yellow), L3 (cyan), L4 (pink), L5 (red), and L6 (black) shown. Model constructed from PDB code 2ORZ. (b) Protein sequence alignment of human, mouse, and rat NRP1 (hNRP1, mNRP1, rNRP1) with human NRP2 (hNRP2). Highlighted residues were predicted to be in close contact with bound ligand from the model in panel a.
Figure 4
Figure 4
(a) Mutational analysis of the NRP1 pocket. COS-7 cells were transfected with expression plasmids for wild-type (WT) or mutant NRP1 as indicated. Binding assays using bt-VEGF-A165 were performed 48 h after transfection. Values presented are the means ± SD obtained from three to six independent experiments each performed in duplicate. (b) An excerpted region of the 2D 15N,1H HSQC NMR spectrum of human NRP1 b1 in the free state (black contours) and with saturating concentrations of tuftsin (blue) and 1 (green). The cross-peak assignments for the free-state NRP1 b1 are indicated in bold. Arrows depict significant ligand-dependent chemical shift changes. (c) Solvent accessible surface representations of the structure NRP1 b1 with color highlighting showing those residues whose backbone amide NH cross-peak exhibits a significant chemical shift perturbation (compound Δδ > 0.15; see Experimental Section) upon addition of a saturating concentration of tuftsin. (d) As for (c) but with 1 as the ligand. The intensity of the color is scaled to the residue-by-residue value of the compound Δδ. (e) Pymol diagram showing positions of mutated residues on the NRP1 b1 protein surface. Mutations giving 100% inhibition of binding are shown as red, and moderate inhibition is shown as salmon (K351).
Scheme 1
Scheme 1. Mixed Solid Phase−Solution Phase Synthetic Strategy
Scheme 2
Scheme 2. Typical Analogue Synthesis
Reagents: (a) Pd2(dba)3, Pd(PPh3)4, KF, dioxane, reflux 24 h; (b) KMnO4, py, H2O, reflux 3 h; (c) HOBt, DCI, DMF, Wang-Arg-OH; (d)SnCl2·H2O, DMF; (e) PyBrOP, RCOOH, 2,6-lutidine, CH2Cl2; (f) TFA, TIPS, H2O.
Scheme 3
Scheme 3. Solution Phase Methodology
Reagents: (a) pyridine, 20 °C; (b) LiOH·H2O, 50 °C, THF, MeOH, H2O; (c) PyBrOP, DIPEA, DCM, 20 °C; (d) TFA/H2O 9:1.
Figure 5
Figure 5
X-ray analysis of 2. (a) FOM weighted difference map of 2-bound NRP1 b1 crystal structure calculated from the model refined without atomic coordinates for 2. The calculated electron density (green mesh) clearly shows the presence of the ligand in two different conformations in the binding sites A and B. Final stick models for 2 in chain A (left) and chain B (right) are overlaid over the density. Colors are green, red, blue, and yellow for carbon, oxygen, nitrogen, and sulfur, respectively. (b) Detail of conformation of chain A bound 2. The position of the (putative) shared hydrogen is indicated by a magenta sphere and arrow.
Figure 6
Figure 6
Binding and functional activity of 2. (a) 2 displaces biotinylated VEGF from NRP1 b1 in a cell-free assay. Plates precoated with purified recombinant NRP1 b1 domain were incubated for 2 h at room temperature with 0.25 nM bt-VEGF-A165 in the presence of the indicated concentrations of 2, and specific VEGF binding was determined. Values presented are the means ± SEM obtained from three independent experiments each performed in triplicate. R2 = 0.9965. (b) 2 inhibits VEGF-stimulated VEGFR2 phosphorylation. Confluent HUVECs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent (DMSO) and were then treated with or without 25 ng/mL VEGF-A165 for 5 min at 37 °C. Total VEGFR2 and VEGFR2 phosphorylated at Tyr1175 were then determined in treated cell lysates using a specific ELISA. Values presented are the means ± SEM obtained from three independent experiments each performed in duplicate: (∗) p < 0.05 versus no 2; (∗∗) p < 0.01 versus no 2; (∗∗∗) p < 0.001 versus no 2. (c) 2 decreases chemotactic responses to VEGF. HUVECs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent (DMSO) and placed into top inserts. The chemotaxis of cells toward either VEGF at 25 ng/mL or medium control (C) in bottom wells was determined after 4 h of incubation. Values presented are the means ± SEM obtained from three independent experiments each performed in duplicate: (∗) p < 0.05 for 100 μM versus 0 μM 2 pretreated cells toward VEGF. (d) Sensitization of carcinoma cells to a chemotherapeutic agent by 2. A549 cells were incubated in serum-free medium containing paclitaxel at the indicated concentrations in the absence or presence of 100 μM 2. Cell viability was measured after 48 h of treatment. Values presented are the means ± SEM obtained from three independent experiments each performed in triplicate: (∗∗) p < 0.01 for the chemotherapeutic drug alone versus drug plus 2.
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