Determining the True Selectivity Profile of αv Integrin Ligands Using Radioligand Binding: Applying an Old Solution to a New Problem

The arginyl-glycinyl-aspartic acid (RGD) integrin subfamily contains five members that partner with the alpha v subunit: alpha v beta 1, alpha v beta 3, alpha v beta 5, alpha v beta 6, and alpha v beta 8. Within the alpha v integrins, the epithelially restricted alpha v beta 6 has been identified as playing a key role in the activation of transforming growth factor that is hypothesized to be pivotal in the development of idiopathic pulmonary fibrosis (IPF). As part of a drug discovery program to identify a selective alpha v beta 6 RGD mimetic for IPF, cell adhesion and radioligand binding assays were investigated to screen compounds to determine affinity and v integrin selectivity. In this study, a pan-alpha v radioligand was characterized against all the v integrins and used to determine accurate selectivity profiles for literature and novel RGD ligands, as well as enable an early readout on alpha v beta 6 dissociation kinetics. It has been shown that while cell adhesion offers a high throughput and reliable format for ranking compounds, there are downsides to this format when comparing selectivity across v integrins. By accurately defining the relationship between these assay formats, a medicinal chemistry effort has identified novel, high-affinity, and selective alpha v beta 6 RGD mimetics with slow dissociation kinetics, with the potential to be developed into clinical candidates for IPF.