Development of mirror-image monobodies targeting the oncogenic BCR::ABL1 kinase

Nat Commun. 2024 Dec 23;15(1):10724. doi: 10.1038/s41467-024-54901-y.

Abstract

Mirror-image proteins, composed of D-amino acids, are an attractive therapeutic modality, as they exhibit high metabolic stability and lack immunogenicity. Development of mirror-image binding proteins is achieved through chemical synthesis of D-target proteins, phage display library selection of L-binders and chemical synthesis of (mirror-image) D-binders that consequently bind the physiological L-targets. Monobodies are well-established synthetic (L-)binding proteins and their small size (~90 residues) and lack of endogenous cysteine residues make them particularly accessible to chemical synthesis. Here, we develop monobodies with nanomolar binding affinities against the D-SH2 domain of the leukemic tyrosine kinase BCR::ABL1. Two crystal structures of heterochiral monobody-SH2 complexes reveal targeting of the pY binding pocket by an unconventional binding mode. We then prepare potent D-monobodies by either ligating two chemically synthesized D-peptides or by self-assembly without ligation. Their proper folding and stability are determined and high-affinity binding to the L-target is shown. D-monobodies are protease-resistant, show long-term plasma stability, inhibit BCR::ABL1 kinase activity and bind BCR::ABL1 in cell lysates and permeabilized cells. Hence, we demonstrate that functional D-monobodies can be developed readily. Our work represents an important step towards possible future therapeutic use of D-monobodies when combined with emerging methods to enable cytoplasmic delivery of monobodies.

MeSH terms

  • Crystallography, X-Ray
  • Fusion Proteins, bcr-abl* / genetics
  • Fusion Proteins, bcr-abl* / metabolism
  • Humans
  • Models, Molecular
  • Peptide Library
  • Protein Binding
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-abl / metabolism
  • src Homology Domains

Substances

  • Fusion Proteins, bcr-abl
  • Peptide Library
  • Protein Kinase Inhibitors
  • ABL1 protein, human
  • BCR-ABL1 fusion protein, human
  • Proto-Oncogene Proteins c-abl