Peptides with affinity for NRP-1

Angiogenesis is the basic process of creating new capillaries from existing blood vessels, necessary for a number of processes related to the life functions of the human body (physiological processes) as well as the development, mechanism and effect of diseases (pathological processes). In a healthy organism, chemical "switches", capable of regulating angiogenesis by stimulating or inhibiting it, are produced in a controlled manner. The development of many diseases is associated with the disturbance of the balance between the regulatory factors of this process. The perspective of influencing this process is a significant challenge in modern medicine. The most important 'switch', that stimulates the growth of new capillaries, is the vascular endothelial growth factor (VEGF-A165), which binds to receptors from the VEGF-R and neuropilins family (NRP), including one of its most important variant (isoforms), i.e. neuropilin-1. NRP-1 is a co-receptor enhancing the signals sent to the cell by the VEGF-A165/VEGF-R2 complex, and this signalling is crucial for angiogenesis process. It regulates cell proliferation and migration as well as the permeability of blood vessels. In addition to the fundamental NRP-1/VEGF-A165/VEGFR complex formation, NRP-1 may also act alone as an intermediary in the development and growth of tumours, as it is secreted to an increased extent (overexpressed) on the surface of many cancer cells, associated with tumour growth, spread and lead to poor clinical prognosis. Blocking the NRP-1 signalling pathway could be one of the effective ways to fight angiogenesis-linked diseases.

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The aim of the project was the synthesis, stability analysis and evaluation of the possibility of the angiogenesis process blockage (inhibition) using short peptides and compounds imitating the structure of peptides (peptidomimetics), based on the sequence with high affinity to NRP-1, developed in our laboratory. The research was carried out in two directions, analyzing the influence of the thiol group (-SH), present in one of the peptides, on its inhibition of the VEGF-A165/NRP-1 complex formation, as well as improving resistance of prepared molecules for to enzymatic degradation in human serum, by replacing selected peptide bonds with urea mimetics.

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The preliminary studies resulted in the design of a peptide with a 50-fold higher inhibitory activity against VEGF-A165/NRP-1 complex formation, compared to the parent compound, by extending its sequence with an additional amino acid at the N-terminus - cysteine ​​(containing the a for mentioned thiol group in its side chain). As part of the project, the activity-structure relationship of peptides with various analogs, homologues and cysteine ​​derivatives was also analysed. The hypothesis that the thiol group of cysteine, as well as its location in the peptide sequence and the length of the side chain of the thiol-containing amino acid side chain, determine the increase in receptor affinity, has thus been proven. Urea-peptide hybrids were also designed and tested, as the premise was that it will significantly extended half-life (the period in which the compound concentration drops to half of the initial value) in the serum, due to the unrecognized by enzymes urea bond. Moreover, one of the designed compounds has a 10-fold higher affinity for NRP-1, compared to the parent peptide.

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The obtained results were used in the next stage to design the so-called "super-sequences", which contain in their structure all the key elements for increased activity and stability. Thanks to this procedure, it was possible to create hybrids with the highest known VEGF-A165/NRP-1 complex formation inhibitory activity among peptides. Most promising compounds are currently being tested on cell lines which overexpress NRP-1, in order to verify their biological activity.

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Results acquired during the project implementation provided many information on the interactions of peptidic ligands with NRP-1, as well as new insight into the structural requirements for inhibiting VEGF-A165/NRP-1 binding. The obtained results have a chance to contribute in the progress fighting against diseases associated with the angiogenesis process, including cancer. Despite very large advances in therapies, there is still a strong demand to design innovative pharmaceuticals, that will define new pathways of action or improve the effectiveness of applied therapies. Stable and active peptidic and urea-peptidic inhibitors have a chance to be used in the future treatment of diseases related to the process of blood vessel development.