De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

Abstract

The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 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 αvβ6 and αvβ8 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 αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with β subunits

“…Data were fitted globally using previously developed equations 26 (Supplementary Equation S28), with the maximum FP value in the absence of antibody and the minimum FP value as shared parameters, and affinities for each titrator as individual parameters. The α5β1 minibinder used in this experiment was obtained using a method similar to that described in 9 .…”

“…Monoclonal antibodies, peptidomimetics, and small molecule antagonists against RGDbinding integrins have been continuously developed to address the role of integrins in cellular processes [16][17][18] . However, the similar ligand binding sites among RGD-binding integrin pairs, such as αVβ3 and αVβ5 5 and αVβ6 and αVβ8 [8][9][10] , pose a significant challenge to development of antibodies that selectively block binding of small, RGD-like ligands. This emphasizes the need to urgently develop molecules specific to RGD-binding integrin subtypes, enabling the discrimination of individual integrins and inhibiting ligand binding at the cellular level.…”

“…There are 24 known integrin heterodimer pairs formed by 18 α subunits and 8 β subunits. Eight are RGD-binding integrins that interact with the Arg-Gly-Asp (RGD) motif in extracellular ligands, thereby regulating diverse pathological processes 4–9 ;10 . αVβ1, αVβ3, αVβ5, and α5β1, expressed on endothelial cells and fibroblasts, bind to fibronectins, exhibiting overlapping functions in cell spreading and migration 11,12 ; αVβ6 and αVβ8 promote TGF-β activation subsequent to binding to the RGD-like motifs in prodomain 13 ; αIIbβ3 on platelets binds to fibrinogen, playing a critical role in hemostasis 14 ; and α8β1 binds to nephronectin in the extracellular matrix and regulates kidney development 15 .…”

Glutamine‐derived peptides: Current progress and future directions

“…The construction of TRAF6 inhibitory peptides from scratch has been achieved using computational peptide backbone algorithms and targeted complementary protein fragments (Swanson et al, 2022). Using the developed algorithm, it was possible to design ultra-stable RGD (Arg-Gly-Asp) mini-proteins and succeed in designing highly selective αvβ6 and αvβ6 inhibitors (Roy et al, 2023). The DL model allowed for the characterization of the sequence of melanin-binding peptides and was used to successfully predict their cell penetration and cytotoxicity, enabling the rapid screening of binding peptides for the sustained delivery of ophthalmic drugs (Hsueh et al, 2023).…”