Does Cysteine Rule (CysR) Complete the CendR Principle? Increase in Affinity of Peptide Ligands for NRP-1 Through the Presence of N-Terminal Cysteine

Abstract

The structure-activity relationship of branched H-Lys(hArg)-Dab-Dhp-Arg-OH sequence analogues, modified with Cys-Asp or Cys at N-terminal amino acids (Lys, hArg), in VEGF-A 165 /Neuropilin-1 complex inhibition is presented. The addition of Cys residue led to a 100-fold decrease in the IC 50 value, compared to the parent peptide. The change occurred regardless of coupling Cys to the free N-terminal amino group present in the main or the side chain. A few analogues extended by the attachment of Cys at the N-terminus of several potent NRP-1 peptide ligands documented in the literature are also presented. In all studied cases, the enhancement of inhibitory properties after the addition of Cys at the N-terminus is observed. It is particularly evident for the tetrapeptide derived from the C-terminus of VEGF-A 165 (KPRR), suggesting that extending the K/RXXK/R motif (CendR) with the Cys moiety can significantly improve affinity to NRP-1 of CendR peptides.Biomolecules 2020, 10, 448 2 of 11 development in cancerous tumors and that its expression on the surface of cancerous cells is increased and often associated with poor prognosis [10][11][12][13]. These results indicate that searching for new NRP-1 inhibitors of pathological angiogenesis, including functional peptides, could play a crucial role in drug development (e.g., in oncology).The VEGF-A 165 fragment encoded by exons 7 and 8 is mainly responsible for protein interaction with NRP-1 [14-17]. The binding pocket of the NRP-1 b1 domain is shallow, and only the C-terminal Arg of the ligand is able to directly enter into it [6,18]. Screening of phage libraries led to the identification of peptides possessing a R/KXXR/K consensus motif embedded in their terminal sequence, which showed a high binding affinity to NRP-1 [18]. As with VEGF-A 165 , the C-terminal Arg (or rarely, Lys) was important for NRP-1-binding. Blocking the free carboxyl group of this Arg residue by amidation or adding another amino acid eliminated ligand binding to the receptor. The strict requirements of the R/KXXR/K C-terminal structural motif in NRP-1 peptidic ligands is called the C-end rule (CendR) [19]. The CendR fragment is present in the structure of active peptidic NRP-1 ligands described in the literature, e.g., heptapeptide A7R (ATWLPPR) [19][20][21], shorter analogues of A7R [22], tuftsin [23], and penetrating iRGD peptide [24] as well as the C-terminus of VEGF-A 165 (CDKPRR) encoded by the exon 8 and multiple viruses' capsid proteins, e.g., human T-cell lymphotropic virus type 1 (HTLV-1) [25,26] and Epstein-Barr virus (EBV) [27].Another characteristic feature of the VEGF-A 165 structure is the presence of eight invariant cysteine (Cys) residues associated with intermolecular and intramolecular disulfide bonds [28]. The N-terminal fragment of the VEGF-A 165 domain ERTCDKPRR encoded by exons 7 and 8, as well as its shorter analogue CDKPRR, showed nearly 100% inhibition of the formation of the VEGF-A 165 /NRP-1 complex at a concentration of 100 µM [29]. The replacement of Cys with Ser at posit...

The Role of VEGF Receptors as Molecular Target in Nuclear Medicine for Cancer Diagnosis and Combination Therapy

“…Radiolabelled small molecular inhibitors of VEGFR tyrosine kinases constitute a relatively large group of radiotracers used for VEGF/VEGFR system imaging [ 8 , 16 , 18 , 25 , 26 , 27 , 28 , 40 , 41 , 253 ]. These molecules should exhibit an extremely high affinity towards VEGFR-TKs (Kd in low nanomolar range) and be characterised with logD values from 1 to 3, which allows crossing the cellular membrane by passive diffusion and reaching the intracellular RTK targets [ 254 ].…”

“…Additionally, neuropilin 1 (NRP-1) a major co-receptor of VEGFR-2, as well as an independent receptor, is involved in the regulation of physiological and pathological angiogenic processes. For this reason, current research has focused on various neuropilin-targeting substances due to its possible application of anticancer therapy [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ].…”

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties

“…This method was previously described for NRP-1 [44][45][46]. Briefly, overnight coating at 4 • C of the flat bottom surface of a 96-well plate with 100 µL (200 ng/well) recombinant human NRP-1, NRP-2, VEGFR-1 or VEGFR-2 was followed by non-specific interactions blocking using 0.5% bovine serum albumin (BSA) in PBS.…”

“…A degradation assay was adapted from a method described previously [42,[44][45][46]. Human blood serum was preactivated in Eppendorf ThermoMixer ® Comfort (Hamburg, Germany) at 37 • C for 20 min (350 rpm).…”

“…Strategies of blocking VEGF-A 165 /NRP-1 complex formation to modulate angiogenesis are currently extensively studied. Designed inhibitors include small molecules [22][23][24][25][26][27][28][29] and linear [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] and cyclic [47][48][49][50] peptides or peptidomimetics. Peptides are generally selective molecules that bind to specific cell surface receptors and are capable of initiating intracellular effects.…”