Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

Proc Natl Acad Sci U S A. 2020 Nov 24;117(47):29684-29690. doi: 10.1073/pnas.2007213117. Epub 2020 Nov 12.


Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.

Keywords: metastasis; peptide mimetics; proline-rich motif; protein–protein interactions; small molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / metabolism
  • Cell Adhesion Molecules / metabolism*
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • DNA-Binding Proteins / metabolism*
  • Female
  • Humans
  • Jurkat Cells
  • Microfilament Proteins / metabolism*
  • Phosphoproteins / metabolism*
  • Proline / metabolism
  • Protein Binding / drug effects
  • Protein Interaction Domains and Motifs / drug effects*
  • Small Molecule Libraries / pharmacology*
  • Zebrafish


  • Cell Adhesion Molecules
  • DNA-Binding Proteins
  • ENA-VASP proteins
  • Microfilament Proteins
  • Phosphoproteins
  • Small Molecule Libraries
  • vasodilator-stimulated phosphoprotein
  • Proline