AbstractThe constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant.