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...