Abstract

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.