Development of Truly Selective αvβ8 Cyclopeptide Ligands for Discriminating Homologous Integrins

Introduction: Aberrant αvβ8 integrin activity is associated with pulmonary and renal fibrosis, as well as tumor progression and immune evasion. Together with the closely related αvβ6 integrin, αvβ8 mediates its biological effects primarily through activation of TGF-β1/β3. The high structural homology between these two αv-integrins represents a major challenge for the development of truly αvβ8-selective ligands. Apart from αvβ8-specific miniproteins, no highly selective, high-affinity small molecules targeting αvβ8 have been described to date. Aim: In this context, we aim to design, synthesize, and characterize novel aminoproline (Amp)-based RGD-cyclopeptides with high affinity and selectivity for αvβ8 over αvβ6 integrin, to further explore and modulate the αvβ8-mediated pathophysiology. Methods: The design of αvβ8-targeted cyclopeptides has been guided by in silico structure-based and NMR conformational studies. Cyclic Amp-peptidomimetics have been synthesized using standard solid- and solution-phase synthetic procedures, followed by flash chromatography or preparative HPLC purification, and structural characterization by LC/MS spectrometry and NMR spectroscopy (K. Bugatti et al., Chem. Eur. J., 2020). A FITC-labelled decapeptide probe (J. Wang et al., Proc. Natl. Acad. Sci. U.S.A., 2017) and a focused library of ligands with known or unknown αvβ8 affinity were prepared and subjected to fluorescence anisotropy-based cell-free assays for evaluating their binding profiles. Results: Starting from in-house cyclic Amp-RGD cyclopeptides that exhibit dual αvβ6/αvβ8 activity, structural modifications were implemented in both the cyclopeptide core and the Nα-Amp side chain to investigate the conformational space of the αvβ8 binding site. The successful setup of a biophysical binding assay has enabled the evaluation of the affinity of the synthesized peptidomimetic integrin ligands. The most promising candidates will subsequently be functionalized with bioactive payloads via suitable spacers. Conclusion: Through an integrated approach combining structure-guided design, synthesis, and biophysical screening, the development of αvβ8-selective Amp-cyclopeptides may provide novel αvβ8-targeted platforms for biomedical and pharmacological applications.