Bicyclic RGD Peptides with Exquisite Selectivity for the Integrin αvβ3 Receptor Using a "Random Design" Approach

ACS Comb Sci. 2019 Mar 11;21(3):198-206. doi: 10.1021/acscombsci.8b00144. Epub 2019 Jan 26.


We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin αvβ3 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 αvβ3 (e.g., CT3HEQcT3RGDcT3, IC50 = 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 αvβ3-binders CT3HPQcT3RGDcT3 (IC50 = 30 nM), CT3HPQCT3RGDcT3 (IC50 = 31 nM), and CT3HSQCT3RGDcT3 (IC50 = 42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC50 = 38 nM) and outstanding selectivities over integrins αvβ5 (IC50 > 10000 nM) and α5β1 (IC50 > 10000 nM). Affinity measurements using surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded Kd values of 0.4 and 0.6 nM for the Cy5-labeled bicycle CT3HPQcT3RGDcT3 and RGD "knottin" peptide, respectively. In vitro staining of HT29 cells with Cy5-labeled 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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Gene Expression Regulation
  • HT29 Cells
  • High-Throughput Screening Assays / methods
  • Humans
  • Integrin alphaVbeta3 / chemistry*
  • Integrin alphaVbeta3 / genetics
  • Oligopeptides / chemistry*
  • Optical Imaging / methods
  • Peptide Library
  • Protein Binding
  • Protein Conformation
  • Structure-Activity Relationship
  • Surface Properties


  • Amino Acids
  • Integrin alphaVbeta3
  • Oligopeptides
  • Peptide Library
  • arginyl-glycyl-aspartic acid