Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A₁₆₅-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A₁₆₅ to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A₁₆₅. The specific inhibition of VEGF-A₁₆₅ binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A₁₆₅-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA₁₆₅-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.     

Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis,molecular docking study and in vitro biological activities

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A₁₆₅ binding to NRP-1 (IC₅₀ = 39 μM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.     

Synthesis and structure–activity relationship of non-peptidic antagonists of neuropilin-1 receptor

Neuropilins (NRPs) are VEGF-A₁₆₅ co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A₁₆₅/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A₁₆₅/NRP-1 binding. This study confirmed our key structure–activity relationships hypothesis and paved the way to compound 1 ‘hit to lead’ optimization.     

Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR₁₆₅ and molecules interfering with VEGF₁₆₅ binding to NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound, derived from d-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids arginine, and tryptophane or threonine. At 100 μM, all compounds exhibited a weak affinity for NRP-1, the most efficient being the bis-guanidinylated compound 32 (IC₅₀ = 92 μM) which could be considered as a new NRP-1 non-peptidic ligand.     

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n?=?5) and short day (SD; 8L:16D, n?=?5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A ₁₂₀ and VEGF-A ₁₆₄ and our results indicate that VEGF-A ₁₆₄ was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p?<?0.05) and NRP-1 mRNA expression (p?<?0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.     

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC)

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF₁₆₅ binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF₁₆₅ interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF₁₆₅, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF₁₂₁. NaPaC completely inhibited the heparin binding to VEGF₁₆₅, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF₁₆₅, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF₁₆₅. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF₁₆₅.     

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF₁₆₅ binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF₁₆₅/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence.Here, we present a structure–activity relationship study of the systematic optimization of amino acid residues in positions 1–3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF₁₆₅ binding to NRP-1.     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor a<Subscript>165</Subscript>

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A₁₆₅ has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A₁₆₅. We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A₁₆₅ as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A₁₆₅, which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A₁₆₅-dependent pathological neoangiogenesis.     

Neuropilin-1 forms complexes with vascular endothelial growth factor receptor-2 during megakaryocytic differentiation of UT-7/TPO cells

We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF₁₆₅-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF₁₆₅ promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF₁₆₅. These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF₁₆₅ in megakaryocytic cells.     

Molecular modelling,synthesis and biological evaluation of peptide inhibitors as anti-angiogenic agent targeting neuropilin-1 for anticancer application

Vascular endothelial growth factor (VEGF) and its co-receptor neuropilin-1 (NRP-1) are important targets of many pro-angiogenic factors. In this study, nine peptides were synthesized and evaluated for their molecular interaction with NRP-1 and compared to our previous peptide ATWLPPR. Docking study showed that the investigated peptides shared the same binding region as shown by tuftsin known to bind selectively to NRP-1. Four pentapeptides (DKPPR, DKPRR, TKPPR and TKPRR) and a hexapeptide CDKPRR demonstrated good inhibitory activity against NRP-1. In contrast, peptides having arginine residue at sites other than the C-terminus exhibited low activity towards NRP-1 and this is confirmed by their inability to displace the VEGF₁₆₅ binding to NRP-1. Docking study also revealed that replacement of carboxyl to amide group at the C-terminal arginine of the peptide did not affect significantly the binding interaction to NRP-1. However, the molecular affinity study showed that these peptides have marked reduction in the activity against NRP-1. Pentapeptides having C-terminal arginine showed strong interaction and good inhibitory activity with NRP thus may be a good template for anti-angiogenic targeting agent.     

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF₁₆₅ in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF₁₆₅, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.     

Selection of sulfur source for methane production by Methanobacterium thermoautotrophicum

Sulfur sources capable of replacing sulfide were surveyed for biomethanation from H₂ and CO₂ by thermoautotrophic methanogen, Methanobacterium thermoautotrophicum. Among sulfur containing compounds tested, l-cysteine, thiosulfate and coenzyme M gave poor growth when added as sulfur sources, whereas simultaneous addition of two sulfur sources, l-cysteine+thiosulfate, l-cysteine+l-methionine or l-cysteine+coenzyme M stimulated the growth.In a pressure-controlled fermentor system developed to obtain stoichiometry between input and output gases, the ratio of H₂ and CO₂ consumption to CH₄ production was almost stoichiometric, and when l-cysteine and thiosulfate or l-methionine were used in place of sulfide (control) similar growth patterns were observed. In a culture with continuous supply of substrates gases (1.3 vvm) and sulfur sources of 1 mM l-cysteine+2 mM thiosulfate, specific growth rate and specific methane production rate were 0.35 h⁻ and 3.24 l g⁻¹h⁻¹, respectively, compared to 0.22 h⁻¹ and 5.76 l g⁻h⁻¹ with Na₂ S.     

Detection of VEGF-Axxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A₁₆₅a, and anti-angiogenic isoforms typified by VEGF-A₁₆₅b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A₁₆₅b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A₁₆₅b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A₁₆₅b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF₁₆₅b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A₁₆₅b antibody. These findings support the conclusion that more information on the biology of VEGF-A₁₆₅b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.     

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC)

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.     

Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

Hemoglobin Rothschild (β37(C3)Trp → Arg): A high/low affinity hemoglobin mutant

In hemoglobin Rothschild arginine replaces the normal tryptophan at β37(C3), at α₁β₂ contact. Residue β37 is in close proximity to Argα92 (FG4). Substitution of Trp by Arg at β37 results in two positively charged Arg residues at FG4 and C3 facing each other, a situation that would destabilize the subunit constraints essential for the tetrameric integrity of the molecule and for the reduced ligand affinity of unliganded normal HB³ compared to isolated chains.Our studies show liganded HbR is extensively dissociated into dimers and has a high ligand affinity in phosphate buffer and a low ligand affinity in bis-Tris at alkaline pH. Kinetic studies indicate that in the T state HbR has a higher ligand affinity than HbA. This is explained by reduced subunit constraints in the T state and dissociation of the monoliganded species (Hb₄L) into dimers. Kinetic studies also show that R state Hb Rothschild has lower ligand affinity than R state HbA. These results are explained on the basis of extensive dissociation of R state Hb Rothschild into dimers and lower ligand affinity of dimers as compared to triliganded tetramers (α₂β₂(O₂)₃). Kinetic data indicate that the lower ligand affinity of dimers (Hb Rothschild) as compared to that of triliganded tetramers (HbA) is due to the increased ligand dissociation rates in the case of oxyhemoglobin and reduced ligand combination in the case of carboxyderivatives. Both the CO combination reaction time-course around 425 nm and the O₂ dissociation rates at 437.8 nm indicate the presence of large α,β-chain differences in Hb Rothschild.     

Identification of Four New Chemical Series of Small Drug-Like Natural Products as Potential Neuropilin-1 Inhibitors by Structure-Based Virtual Screening: Pharmacophore-Based Molecular Docking and Dynamics Simulation

Neuropilin-1 (NRP-1), a surface transmembrane glycoprotein, is one of the most important co-receptors of VEGF-A165 (vascular endothelial growth factor) responsible for pathological angiogenesis. In general, NRP-1 overexpression in cancer correlates with poor prognosis and more tumor aggressiveness. NRP-1 role in cancer has been mainly explained by mediating VEGF-A165-induced effects on tumor angiogenesis. NRP-1 was recently identified as a co-receptor and an independent gateway for SARS-CoV-2 through binding subunit S2 of Spike protein in the same way as VEGF-A165. Thus, NRP-1 is of particular value as a target for cancer therapy and other angiogenesis-dependent diseases as well as for SARS-CoV-2 antiviral intervention. Herein, The Super Natural II, the largest available database of natural products (∼0.33 M), pre-filtered with drug-likeness criteria (absorption, distribution, metabolism and excretion/toxicity), was screened against NRP-1. NRP-1/VEGF-A165 interaction is one of protein-protein interfaces (PPIs) known to be challenging when approached in-silico. Thus, a PPI-suited multi-step virtual screening protocol, incorporating a derived pharmacophore with molecular docking and followed by MD (molecular dynamics) simulation, was designed. Two stages of pharmacophorically constrained molecular docking (standard and extra precisions), a mixed Torsional/Low-mode conformational search and MM-GBSA ΔG binding affinities calculation, resulted in the selection of 100 hits. These 100 hits were subjected to 20 ns MD simulation, that was extended to 100 ns for top hits (20) and followed by post-dynamics analysis (atomic ligand-protein contacts, RMSD, RMSF, MM-GBSA ΔG, Rg, SASA and H-bonds). Post-MD analysis showed that 19 small drug-like nonpeptide natural molecules, grouped in four chemical scaffolds (purine, thiazole, tetrahydropyrimidine and dihydroxyphenyl), well verified the derived pharmacophore and formed stable and compact complexes with NRP-1. The discovered molecules are promising and can serve as a base for further development of new NRP-1 inhibitors.