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

Tobias G Kapp,Stefanie Neubauer,Revital Zarka,Johannes Notni,Ute Reuning,Joachim Spatz,Oleg V Maltsev,Hans-Jürgen Wester,Florian Rechenmacher,Elisabetta A Cavalcanti-Adam,Horst Kessler,Benjamin Geiger,Carlos Mas-Moruno

doi:10.1038/srep39805

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.

Highlights

Structural and signalling responses of cells are tightly regulated by multiple adhesive interactions with the pericellular microenvironment, which promotes the physical networking of neighbouring cells and physical attachment to diverse extracellular matrix (ECM) networks
Eight of the 24 known human integrin heterodimers were shown to bind the RGD-recognition sequence[10,11]. Despite their apparent similarity, these integrins can readily distinguish between different RGD-containing ECM proteins, and respond differently to the interaction with each one of them
We have evaluated a large number of well-known and widely used integrin-targeting molecules using the same standardized competitive ELISA-based test system, by measuring the inhibition (i.e. IC50 values) of integrin binding to immobilized natural ECM ligands

Summary

Introduction

Structural and signalling responses of cells are tightly regulated by multiple adhesive interactions with the pericellular microenvironment, which promotes the physical networking of neighbouring cells and physical attachment to diverse extracellular matrix (ECM) networks. For example fibronectin preferentially binds to α5β1, αvβ[6], αvβ[8] and to αIIbβ[3], with different activities, while integrin αIIbβ[3] is primarily expressed on platelets and binds to specific adhesive proteins, such as fibrinogen/fibrin, prothrombin and plasminogen Despite their narrow specificity, integrin ligands that target αIIbβ[3], should be used for therapeutic purposes with great care, since their excessive systemic administration might cause hemorrhagic disorders. A few years later, we addressed the need of focusing on high affinity ligands toward αvβ[3] while maintaining selectivity over αIIbβ[3 ], by using cyclic RGD and incorporating one d-amino acid The latter modification, based on a process called: “spatial screening”[25,26,27,28], had a drastic impact on the backbone conformation, that changed the selectivity and affinity pattern of the cyclic peptides. The crystal structures of integrin antagonists docked into the αvβ[3] or αIIbβ[3] receptor pocket[32,33,34], explained and corroborated this phenomenon, in retrospect (see Fig. 1 and discussion below)

Methods

Results

Conclusion