Bicyclic RGD peptides : Novel high-affinity ligands for selective integrin-binding and integrin-mediated cell adhesion

Bernhagen, Dominik; Möller, Martin (Thesis advisor); Timmerman, Peter (Thesis advisor)

Aachen (2019, 2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019


In this work, bicyclic Arg-Gly-Asp (RGD) peptides are established as novel selective, high-affinity integrin ligands, which can be applied for membrane-integrinvisualization and integrin-mediated cell adhesion in 2D and 3D polymer matrices. For the affinity-based screening and selection of hundreds of potential RGD-bicycles for their binding to integrins avb3, avb5 and a5b1, a fast and cost-efficient ELISA method was developed applying a high-affinity, biotinylated cysteine-knot RGD peptide(knottin-RGD) as a benchmark ligand. Screening and optimization of integrin avb3-, avb5- and a5b1-affinity and -selectivity of RGD-bicycle libraries, comprising the RGD motif in one loop and a random XXX motif in the second, yielded three high-affinity and high-selectivity avb3-binders (CT3HPQcT3RGDcT3, CT3HPQCT3RGDcT3, CT3HSQCT3RGDcT3; IC50:30-42 nM), one medium-affinity and non-selective avb5-binding peptide (CT3RGDcT3NWaCT3;IC50: 650 nM) and three high-affinity and medium-selectivity a5b1-binding peptides (CT3RGDcT3AY(D-Leu)CT3, CT3RGDcT3AWGCT3, CT3RGDcT3AYaCT3;IC50: 90-173 nM). Selected avb3- and a5b1-binders were further characterized via surface plasmon resonance-enhanced fluorescence spectroscopy (SPFS)and 2D NMR spectroscopy. Membrane integrin staining with fluorescently labeled RGD-bicycles, analyzed via confocal microscopy, revealed high staining levels for adipose-derived stem cells when applying the a5b1-selective bicycle CT3RGDcT3AWGCT3, while most efficientHT29 cell membrane staining was observed for the avb3-selective bicycleCT3HPQcT3RGDcT3.Moreover, avb3- and a5b1-selective bicycles were covalently conjugated to elastinlikerecombinamer (ELR) polymers and polyisocyanopeptide hydrogels (PIC) in order to investigate the 2D and 3D cell adhesion and proliferation properties of these materials. Both avb3- and a5b1-selective bicycles promoted HUVEC adhesion and growth on 2D ELR surfaces with much higher efficiencies than linear benchmark GRGDS, and with at least equal or even higher efficiencies as compared to mono-cyclic (cyclo-[KRGDf]) and knottin-RGD benchmark peptides. In 3D PIC hydrogels, the a5b1-selective bicycle CT3RGDcT3AWGCT3 promoted superior cell adhesion and the formation of numerous protrusions already after one day as compared to hydrogels functionalized with linear, monocyclic and knottin-RGD benchmarks. The overall results of this work reveal that bicyclic RGD peptides represent an entirely novel and valuable platform with high potential for the development of new cell integrin biomarkers as well as cell adhesion-promoting compounds.