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

ACS Comb Sci. 2019 Aug 12;21(8):598-607. doi: 10.1021/acscombsci.9b00081. Epub 2019 Aug 1.


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 (CT3RGDcT3AYGCT3, 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 (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 α5β1-integrin was confirmed by SPFS analysis showing a Kd of 4.1 nM for Cy5-labeled RGD-bicycle CT3RGDcT3AYJCT3 (J = d-Leu) and a somewhat higher Kd (9.0 nM) for Cy5-labeled knottin-RGD. The α5β1-bicycles, for example, CT3RGDcT3AYJCT3 (J = d-Leu), showed excellent selectivities over αvβ5 (IC50 ratio α5β1vβ5 between <0.009 and 0.039) and acceptable selectivities over αvβ3 (IC50 ratios α5β1vβ3 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 environment, illustrating the high potential of these RGD bicycles as markers for α5β1-integrin expression.

Keywords: ELISA; RGD; SPFS; bicyclic peptide; integrin; library screening; peptide−protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Combinatorial Chemistry Techniques
  • Oligopeptides / analysis*
  • Oligopeptides / chemical synthesis
  • Peptide Library*
  • Receptors, Vitronectin / chemistry*


  • Oligopeptides
  • Peptide Library
  • Receptors, Vitronectin
  • integrin alphavbeta1
  • arginyl-glycyl-aspartic acid