We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin α v β 3 via high-throughput screening of partially randomized libraries. Peptide libraries (672 different compounds) comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop and a randomized sequence XXX (X being one of 18 canonical L-amino acids) in the second loop, both enclosed by either an L-or D-Cys residue, were converted to bicyclic peptides via reaction with 1,3,5-tris(bromomethyl)benzene (T3). Screening of first-generation libraries yielded lead bicyclic inhibitors displaying submicromolar affinities for integrin α v β 3 (e.g., C T3 HEQc T3 RGDc T3 , IC 50 = 195 nM). Next generation (second and third) libraries were obtained by partially varying the structure of the strongest lead inhibitors and screening for improved affinities and selectivities. In this way, we identified the highly selective bicyclic α v β 3 -binders C T3 HPQc T3 RGDc T3 (IC 50 = 30 nM), C T3 HPQC T3 RGDc T3 (IC 50 = 31 nM), and C T3 HSQC T3 RGDc T3 (IC 50 = 42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC 50 = 38 nM) and outstanding selectivities over integrins α v β 5 (IC 50 > 10000 nM) and α 5 β 1 (IC 50 > 10000 nM). Affinity measurements using surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded K d values of 0.4 and 0.6 nM for the Cy5-labeled bicycle C T3 HPQc T3 RGDc T3 and RGD "knottin" peptide, respectively. In vitro staining of HT29 cells with Cy5labeled bicycles using confocal microscopy revealed strong binding to integrins in their natural environment, which highlights the high potential of these peptides as markers of integrin expression.

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.

aECMs imparting integrin selectivity (α5β1 and αvβ3) and directional guidance cues are developed using MMP degradable PEG hydrogels, functionalized with bicyclic RGD peptides, and including aligned magnetic, short fibers for 3D oriented nerve growth.

We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca and Mg in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αβ but also detectable for both αβ and αβ, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αβ. For assay validation, we finally determined IC values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αβ, αβ, αβ). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.