Bicyclic RGD Peptides with Exquisite Selectivity for the Integrin α_vβ₃ Receptor Using a "Random Design" Approach

We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin α_vβ₃ 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_vβ₃ (e.g., C_T3HEQc_T3RGDc_T3, 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 α_vβ₃-binders C_T3HPQc_T3RGDc_T3 (IC₅₀ = 30 nM), C_T3HPQC_T3RGDc_T3 (IC₅₀ = 31 nM), and C_T3HSQC_T3RGDc_T3 (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 α_vβ₅ (IC₅₀ > 10000 nM) and α₅β₁ (IC₅₀ > 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_T3HPQc_T3RGDc_T3 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.