Deciphering RGDechi peptide‐α₅β₁ integrin interaction mode in isolated cell membranes

Abstract

Integrins are a large family of heterodimeric receptors critically engaged in pathological processes such as tumor progression and metastasis. Although they are validated therapeutic targets, the molecular determinants governing integrin‐ligand interactions are not yet fully understood, leading to a scarcity of integrin sub‐type exclusive antagonists. In the past decade, we have investigated the biological behavior of the RGDechi, a chimeric peptide able to specifically bind αvβ3 integrin without cross reacting with αvβ5 and αIIbβ3 integrins. Here we have investigated the capability of the peptide to bind α5β1 integrin and characterized the molecular determinants governing this interaction through a combined experimental and computational approach. The detailed comparison of RGDechi‐α5β1 structural model with that previously determined of RGDechi in complex with αvβ3 shows how the bifunctional nature of the peptide renders the molecule an important tool to recognize integrins with different recognition modalities, providing novel insight on the structural requirements needed to their specific recognition.

Conformational studies of RGDechi peptide by natural‐abundance NMR spectroscopy

“…Indeed, we designed a RGDechi mutant in which hCit in position 15 was replaced with Asp that loses the ability to bind α v β 3 and is able to selectively bind α v β 5 integrin . Finally, since the high sequence similarity between α v β 3 and α 5 β 1 receptors and the sharing of some ligands, (ie, fibronectin and echistatin), very recently, we have also showed the RGDechi is able to interact with α 5 β 1 but with an affinity lower than that to α v β 3 as indicated by biological assays . Using NMR data in combination with computational analysis, we have described the molecular details of the α 5 β 1 molecular recognition, indicating a different binding modality with respect to α v β 3 .…”

Selective Targeting of αvβ5 Integrin in HepG2 Cell Line by RGDechi15D Peptide

“…The cells were seeded both onto fibronectin, the main ECM protein that binds α5β1 receptor, and onto α5β1 antibody coated plates. The results obtained indicate that RGDechi15D is not able to inhibit K562 adhesion neither on fibronectin nor that of the α5β1 specific antibody; in this experiment the RGDechi peptide [ 33 ] was used as a control to recognize αvβ3 integrin [ 32 ] thus proving the validity of experimental data ( Figure 3 A,B). The competition experiments here reported are all in the direction of corroborating RGDechi15D specificity towards αvβ5 with respect to αvβ3 or α5β1 integrins.…”

“…Furthermore, it is worth noting that the inhibition occurred in a concentration-dependent manner with an IC 50 of 31.6 µM ( Figure 2 B). With the aim to evaluate the capability of the peptide to discriminate between αvβ5 and α5β1, cell adhesion assays on K562 cells, displaying α5β1 at high levels and αvβ3 and αvβ5 at very low levels [ 32 ], were carried out. The cells were seeded both onto fibronectin, the main ECM protein that binds α5β1 receptor, and onto α5β1 antibody coated plates.…”

“…In the last few years, our research activities have mainly focused on the identification of integrin selective peptide ligands by rational design [ 32 , 33 , 34 , 35 ]. In 2016 we reported NMR and computational studies on the αvβ3 selective peptide, RGDechi, aiming to identify the molecular basis that regulates receptor recognition mechanism.…”

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

“…The effect of APQ30 peptide on the adhesion of K562 cells, where α 5 β 1 results predominantly present with respect to α v β 3 and α v β 5 (reported in the supplementary material of Russo et al) on fibronectin coated plates, was investigated to understand a possible involvement also of this integrin. Such explorative experiments showed no effect (data no shown) letting hypothesize no interaction between peptide and α 5 β 1 .…”