De novo design of highly selective miniprotein inhibitors of integrins v6 and v8

The RGD (Arg-Gly-Asp)-binding integrins alpha v beta 6 and alpha v beta 8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous alpha v beta 6 and alpha v beta 8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective alpha v beta 6 and alpha v beta 8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the alpha v beta 6 and the alpha v beta 8 integrins. In a lung fibrosis mouse model, the alpha v beta 6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.