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 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 environment, illustrating the high potential of these RGD bicycles as markers for α5β1-integrin expression.

中文翻译：

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通过筛选随机库鉴定出的高亲和力α5β1-整联蛋白选择性双环RGD肽。

我们报告了通过“设计的随机文库”鉴定高亲和力和选择性整合素α5β1结合双环肽的鉴定，也就是说，在第一环中筛选包含通用整合素结合序列Arg-Gly-Asp（RGD）的文库。与第二个循环中的随机序列（XXX）组合。对第一代文库中α5β1结合肽的筛选产生了三位数的纳摩尔双环α5β1结合剂（CT3RGDcT3AYGCT3，IC50 = 406 nM）。通过部分改变最强的第一代铅抑制剂的结构来设计下一代文库，并筛选该受体的亲和力和选择性得到改善。这样，我们确定了三种高亲和力的α5β1结合剂（CT3RGDcT3AYJCT3，J = d-Leu，IC50 = 90 nM; CT3RGDcT3AYaCT3，IC50 = 156 nM; CT3RGDcT3AWGCT3，IC50 = 173 nM），其中一个甚至显示出比32个氨基酸基准肽Knottin-RGD（IC50 = 114 nM）更高的α5β1-亲和力。通过SPFS分析确认了对α5β1-整联蛋白的亲和力，显示Cy5标记的RGD自行车CT3RGDcT3AYJCT3（J = d-Leu）的Kd为4.1 nM，Cy5标记的结蛋白-RGD的Kd（9.0 nM）更高。α5β1自行车，例如CT3RGDcT3AYJCT3（J = d-Leu），在αvβ5上表现出优异的选择性（IC50比率α5β1/αvβ5在<0.009和0.039之间），在αvβ3上表现出可接受的选择性（IC50比率α5β1/αvβ3在0.090和0.157之间） 。使用共聚焦显微镜在体外用Cy5标记的肽对脂肪干细胞进行染色，发现在自然环境中，α5β1选择性自行车CT3RGDcT3AWGCT3与整联蛋白具有强结合力，