High-Affinity α 5 β 1 -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 "random design", i.e. the screening of libraries comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop in combina-tion with a randomized sequence (XXX) in the second loop. Screening of 1st generation libraries for α5β1-binding peptides yielded a triple-digit nanomolar bicyclic α5β1-binder (CT3RGDcT3AYGCT3, IC50: 406 nM). Next generation libraries were de-signed by partially varying the structure of the strongest 1st generation lead inhibitor and screened for improved affinities and selectivities for this receptor. In this way, we identified three high-affinity α5β1-binders (CT3RGDcT3AYJCT3, J: D-Leu, IC50: 90 nM; CT3RGDcT3AYaCT3, IC50: 156 nM; CT3RGDcT3AWGCT3, 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 integrin α5β1 was con-firmed by SPFS analysis showing a Kd of 4.1 nM for Cy5-labeled RGD-bicycle CT3RGDcT3AYJCT3 (J: D-Leu), and a some-what higher Kd (9.0 nM) for Cy5-labeled knottin-RGD. The α5β1-bicycles, e.g. CT3RGDcT3AYJCT3 (J: D-Leu), showed excel-lent 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 CT3RGDcT3AWGCT3 to integrins in their natural environ-ment, illustrating the high potential of these RGD-bicycles as markers for α5β1-integrin expression.