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 hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvβ6 small molecule inhibitors were characterized 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. After washout, the αvβ6 cell surface repopulation 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 NHBE cells if present continuously. These observations suggest αvβ6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation.