Abstract

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesised to be pivotal in the development of fibrosis and other diseases. In this study αvβ6 small molecule inhibitors were characterised in a range of in vitro systems to determine affinity, kinetics and duration of TGFβ inhibition. High αvβ6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvβ6 affinity, slow dissociation) and SC-68448 (low αvβ6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvβ6 in normal human bronchial epithelial (NHBE) cells. Following washout, the αvβ6 cell surface re-population was faster for SC-68448 compared with compound 1. In addition, αvβ6-dependent release of active TGFβ from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFβ was restored, whereas after washout of compound 1 the inhibition of TGFβ activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvβ6 in NHBEs if present continuously. These observations suggest αvβ6 can be degraded following high affinity RGD-binding that sorts the integrin for lysosomal degradation post-internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated following sustained engagement of the RGD-binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation.

Significance Statement The fate of RGD integrins post-ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time-dependent. High affinity ligands induced downregulation via lysosomal degradation likely due to slow dissociation, whilst sustained low affinity ligand engagement were only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.