High-Affinity α₅β₁-Integrin-Selective Bicyclic RGD Peptides Identified via Screening of Designed Random Libraries

Abstract

We report the identification of high-affinity and selectivity integrin α5β1-binding bicyclic peptides via “designed random libraries”, that is, the screening of libraries comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop in combination with a randomized sequence (XXX) in the second loop. Screening of first-generation libraries for α5β1-binding peptides yielded a triple-digit nanomolar bicyclic α5β1-binder (C T3 RGDc T3 AYGC T3 , IC50 = 406 nM). Next-generation libraries were designed by partially varying the structure of the strongest first-generation lead inhibitor and screened for improved affinities and selectivities for this receptor. In this way, we identified three high-affinity α5β1-binders (C T3 RGDc T3 AYJC T3 , J = d-Leu, IC50 = 90 nM; C T3 RGDc T3 AYaC T3 , IC50 = 156 nM; C T3 RGDc T3 AWGC T3 , IC50 = 173 nM), of which one even showed a higher α5β1-affinity than the 32 amino acid benchmark peptide knottin-RGD (IC50 = 114 nM). Affinity for α5β1-integrin was confirmed by SPFS analysis showing a K d of 4.1 nM for Cy5-labeled RGD-bicycle C T3 RGDc T3 AYJC T3 (J = d-Leu) and a somewhat higher K d (9.0 nM) for Cy5-labeled knottin-RGD. The α5β1-bicycles, for example, C T3 RGDc T3 AYJC T3 (J = d-Leu), showed excellent selectivities over αvβ5 (IC50 ratio α5β1/αvβ5 between <0.009 and 0.039) and acceptable selectivities over αvβ3 (IC50 ratios α5β1/αvβ3 between 0.090 and 0.157). In vitro staining of adipose-derived stem cells with Cy5-labeled peptides using confocal microscopy revealed strong binding of the α5β1-selective bicycle C T3 RGDc T3 AWGC T3 to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for α5β1-integrin expression.

Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels

“…Bicyclic RGD peptides ( Fig. 1c ) (K-linker-C T3 RGD c T3 AWGC T3 , 43 NQEQVSPL-C T3 RGDc T3 AYJC T3 G, K-linker-C T3 HPQc T3 RGD c T3 , 42 NQEQVSPLC T3 HPQc T3 RGD c T3 G,), cyclic RGD peptides (cyclo- RGD fK, 35 cyclo- D fK[K-linker] RG ), and linear RGD peptides (K-linker-G RGD S, NQEQVSPL RGDS PG, G RGD S 35 ) are used as cell adhesive domains in this study with controls, such as scrambled second loops (K-linker-C T3 RGD c T3 WGAC T3 , K-linker-C T3 PQHc T3 RGD c T3 ) and scrambled RGD peptides (K-linker-C T3 GDRc T3 AWGC T3 , K-linker-C T3 HPQc T3 GRDc T3 ). Fibronectin fragment FNIII 9*-10/12-14 is used as positive control and PEG gels without RGD as another negative control.…”

“…The secondary loop consists of a tri aminoacid sequence, which is screened using a 'random approach' and imparts the potential to affect the RGD selectivity towards different integrin subunits (α v β 3 or α 5 β 1 ). 42,43 Elastin-like recombinamers (ELR), produced with recombinant proteins and coupled with bicyclic RGD peptides selective for α 5 β 1 show an increase in HUVEC proliferation at early periods of culture. 44 Here, we describe for the first time the effect of bicyclic RGD peptides for nerve growth in synthetic 3D PEG hydrogels to study the affinity towards selective integrin sites.…”

“…These peptides are classified based on their structure as linear, cyclic, and bicyclic, reporting an increase in integrin affinity and selectivity based on IC 50 from former to the latter. 35,43,47 L929 mouse fibroblasts are encapsulated within the hydrogel at a fixed cell concentration. 3D cell growth in multiple planes is quantified after staining for actin filaments and imaging as z-stacks.…”

“…35 In addition, none of the RGD peptides have the synergistic domain PHSRN, which is required for efficient α 5 β 1 binding. 48 This could explain the fact that the bicyclic peptide P2 shows a higher binding affinity towards α v β 3 (IC 50 : 30 nM 47 ) compared to peptide P1 towards α 5 β 1 (IC 50 : 173 nM 43 ). Among the bicyclic RGD peptides tethered via K, the peptide K-P2 show significantly higher cell invasion compared to K-P1, however, no such difference is observed in the case of bicyclic RGD tethered via Q.…”

Small and Simple, yet Sturdy: Conformationally Constrained Peptides with Remarkable Properties

“…An identical approach was employed in the discovery of a macrocyclic peptide disruptor of NEMO-IKKβ interaction, inhibiting proliferation of cisplatin-resistant ovarian cancer cells [21]. A related principle was adopted by Bernhagen et al [106,107]; they have screened bicyclic libraries with built-in universal integrin-binding sequence Arg-Gly-Asp in the first loop and a randomized tripeptide sequence in the second loop. With the 'guided' library, they have probed adjacent binding sites on α 5 β 1 , and identified high-affinity integrin binders with potential as therapeutics or delivery vehicles in diagnostics and treatment of breast cancer.…”