Architecture and hydration of the arginine‐binding site of neuropilin‐1

Abstract

Neuropilin‐1 (NRP1) is a transmembrane co‐receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C‐terminal arginine of VEGF and residues in the NRP1‐binding site including Tyr297, Tyr353, Asp320, Ser346 and Thr349. We obtained several complexes of the synthetic ligands and the NRP1‐b1 domain and used X‐ray crystallography and computational methods to analyse atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the six new high‐resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand‐binding site hydration map of NRP1 was in agreement with the experimentally derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein–ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands’ C‐terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design and validates the utility of the computational hydration map prediction method in the context of neuropilin.DatabaseThe structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.

Biophysical screening methods for extracellular domain peptide receptors, application to natriuretic peptide receptor C ligands

“…These methods may not only be applicable for GPCRs but for other dissimilar receptors that possess a large and well‐defined ECD . We have shown this to be the case in the application of biophysical screening techniques for discovery of neuropilin‐1 ligands, an immunoglobulin domain protein receptor for the vascular endothelial growth factor (VEGF) …”

In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein

“…The screens were run once without ligand-receptor interaction constraints and repeated with the constraint that compounds form a hydrogen bond to Asp 320, a key residue for coordinating the terminal arginine in the CendR motif 14 . This constraint was used in attempt to select for compounds that interact in a similar way as observed for VEGF-A 14 , known inhibitors 42,54,60,61 and modeled SARS-CoV-219 CendR terminal arginine 6 . However, application of this constraint led to reduced overall scores and strained conformations for most compounds in the DIV and NC1 libraries.…”

“…To enable comparison with small molecules reported by others we docked and calculated physico-chemical properties of seven compounds that also target the NRP-1 CendR site [54][55][56][58][59][60][61] .…”

“…These can form hydrogen bonds with the side chain of the Asn300 amide or the Ser298 hydroxyl. The area between the HBA and these two additional acceptors (where the label A1 is located) is expected to accommodate a structural molecule of water 9,13,60,61 . This water has been proposed to be important in ligand binding as it may bridge interactions to Trp301 60 .…”

“…Due to its role in cancer, NRP-1 has been a target for drug design for over 20 years. During this time, discovery efforts have focused on development of NRP-1 antibody therapies 9,[28][29][30][31][32][33] , including a recent dual-specificity antibody to VEGFA and NRP-1 32,33 , peptides that target transmembrane domain interactions 11,[34][35][36][37] or the CendR interaction site 12,31,[38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] , as well as small-molecules that target the CendR site [54][55][56][57][58][59][60][61] . With one exception 43 , the CendR site targeting peptides contain a CendR motif, even those which are cyclical 39,45,46,49 or branched 51,53 .…”