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

Abstract

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.

Potential inhibitors interacting in Neuropilin-1 to develop an adjuvant drug against COVID-19, by molecular docking

“…As it was mentioned already, the amino acid Asp320 is very important for NRP1 to interact with different ligands and VEGF-A, 29 , 33 as well as for the CendR region in the SARS-CoV-2, 17 , 32 therefore, the region between the amino acids Thr316, Asp320, Ser346, Thr349 and Tyr353 has a very important role for the S1 region of SARS-CoV-2 to interact with NRP1; in this study was carried out a docking directed to amino acids in the b1 region reported in the NRP1 (Thr316, Asp320, Ser346, Thr349 and Tyr353), 17 that is important to interact with the S-Protein (S1 region) of SARS-CoV-2, 16 since it has been shown that by preventing this interaction, the infectious process caused by this virus could be reduced . 14 , 15 , 17 …”

“…We selected ten compounds depicted here as N1 to N10 from the Express-pick Collection Stock from Chembridge library (ChemBridge Corp.) and the analysis of the interaction of each compound with NRP1 was carried out with the interaction report ( Table 2 and details in Table S1–S11). In addition, it was determined the average interaction for compound EG00229 and EG01377 (with reports of inhibitory effect between NRP1 with VEGF-A 29 , 31 and S-Protein of SARS-CoV-2 32 ), with an average value of −4.95 kcal/mol −1 and −4.86 kcal/mol −1 respectively (interaction details in Tables S1 and S12). Afterwards, the theoretical toxicity for the ten compounds was evaluated with two websites (Prediction of Toxicity and PreADMET web server).…”

“…It was determined that the amino acids Tyr297, Asn300, Trp301, Thr316, Gly318, Glu319, Asp320, Ser321, Arg323, Glu348, Lys351, Tyr353, Trp411, Thr413 and Gly414 are important for the majority of the ten compounds to interact with the NRP1 ( Table 2 ) and the chosen compounds have a better ΔG binding average value than the reference compounds (EG00229 −4.95 kcal/mol −1 and EG01377 −4.86 kcal/mol −1 , Table S1), although this difference obtained theoretically, it does not guarantee that a better ΔG binding value of the chosen compounds causes a greater inhibition when comparing them with EG00229. 30 , 32 To justify the selection of these ten compounds, it is necessary to show that N1 - N10 compounds have a higher probability of interaction with NRP1, according to the results of the docking, the EG00229 and EG01377 compounds, are less specific, since the 30 conformers and the 29 conformers respectively are interacting in a bigger region ( Fig. 2 , Table 2 and Figs.…”

“…The development of new drugs requires a high investment of time and financial resources, so it is necessary to be able to offer potential drugs to development them, even if it is to start in a theoretical way, since this is already developing in the world. 20 , 32 On the other hand, there is still much to know about COVID-19, since the tropism that SARS-CoV-2 has, it is a challenge to understand it. NRP1 has become important to determine the evolution of the infectious process, since it has been identified that this protein can increase the degree of infection when it is present.…”

“…Subsequently, the use of small molecules as inhibitors of NRP1 (EG00299), 29 in which the concentrations and probable sites of interaction have been reported, which are also taken into account to carry out this study, for which the compound EG00299 has shown a selective inhibitory effect on NRP1 with some of its ligands, 20 , 29 in addition evaluating its effects in in vivo tests with cancer cells 30 and as a molecule to perform derivatives 31 (such as EG01377), in which they have reported the important residues to achieve the inhibitory effect and a range of concentrations used between 10 and 30 µM. Therefore, we use data from the references that use the compound EG00299 in NRP1 and its interaction with VEGF-A, there is an antagonist effect on NRP1 that inhibits the binding of VEGF-A, as well as, it was determined that it inhibited the interaction between the b1 domain with the S1 region of SARS-CoV-2, showing that the infectious process is hindered by the virus, 14 , 15 , 17 , 32 since this study seeks to develop an inhibitor (small molecule), which it can accomplish the Lipinski's rule, 26 because these molecules have better pharmacokinetic properties in comparison with peptide-based molecules.…”