A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins

Abstract

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC₅₀-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay.

Figure 1. Illustration of different binding modes of a linear RGD peptide to different integrin subtypes.

Crystal structures of α5β1 (top), αvβ3 (middle), and αIIbβ3 (bottom) in complex with RGD ligands. Figure adapted from.

Figure 2. Schematic illustration of the enzyme-linked immunosorbent assay (ELISA).

(A) 1. Each well (96-well plate) is coated with an ECM protein (e.g. vitronectin for αvβ3). 2. Uncoated surface is blocked by bovine serum albumin (BSA). 3. ECM protein competes with the tested ligand for binding to the soluble integrin (e.g. αvβ3). 4. Integrin bound to ECM protein is detected by an integrin-specific primary antibody. 5. Secondary antibody, conjugated with a peroxidase (POD), detects bound primary antibody. 6. Peroxidase converts a colorless substrate into a colored substrate (TMB, 3,3′,5,5′-tetrametylbenzidine). (B) The ligand inhibits binding of the coated ECM protein to the integrin. Consequently, steps 3–6 are blocked and no color signal can be detected.