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

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_T3RGDc_T3AYGC_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_T3RGDc_T3AYJC_T3, J = d-Leu, IC₅₀ = 90 nM; C_T3RGDc_T3AYaC_T3, IC₅₀ = 156 nM; C_T3RGDc_T3AWGC_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_T3RGDc_T3AYJC_T3 (J = d-Leu) and a somewhat higher K_d (9.0 nM) for Cy5-labeled knottin-RGD. The α₅β₁-bicycles, for example, C_T3RGDc_T3AYJC_T3 (J = d-Leu), showed excellent selectivities over α_vβ₅ (IC₅₀ ratio α₅β₁/α_vβ₅ between <0.009 and 0.039) and acceptable selectivities over α_vβ₃ (IC₅₀ ratios α₅β₁/α_vβ₃ 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_T3RGDc_T3AWGC_T3 to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for α₅β₁-integrin expression.