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 alpha(5)beta(1)-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 alpha(5)beta(1)-binding peptides yielded a triple-digit nanomolar bicyclic alpha(5)beta(1)-binder (C(T3)RGDc(T3)AYGC(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 alpha(5)beta(1)-binders (C(T3)RGDc(T3)AY]C-T3, J = D-Leu, IC50 = 90 nM; C(T3)RGDc(T3)AYaC(T3), IC50 = 156 nM; C(T3)RGDc(T3)AWGC(T3), IC50 = 173 nM), of which one even showed a higher alpha(5)beta(1)-affinity than the 32 amino acid benchmark peptide knottin-RGD (IC50 = 114 nM). Affinity for alpha(5)beta(1)-integrin was confirmed by SPFS analysis showing a K-d of 4.1 nM for Cy5-labeled RGD-bicycle C(T3)RGDc(T3)AYJC(T3) (J = D-Leu) and a somewhat higher K-d (9.0 nM) for Cy5-labeled knottin-RGD. The alpha(5)beta(1)-bicycles, for example, CT(3)RGDcT(3)AYJC(T3) (J = D-Leu), showed excellent selectivities over alpha(v)beta(5) (IC50 ratio alpha(5)beta(1)/alpha(v)beta(5) between <0.009 and 0.039) and acceptable selectivities over alpha(v)beta(3) (IC50 ratios alpha(5)beta(1)/alpha(v)beta(1) 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 alpha(5)beta(1)-selective bicycle CT(3)RGDc(T3)AWGC(T3) to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for alpha(5)beta(1)-integrin expression.