title: Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.
abstract: A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (kcat/KM) of purified ABL kinase for the lead peptide (125 s-1μM-1) is similar to that of the starting peptide (103 s-1μM-1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR-ABL providing a readout of BCR-ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR-ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR-ABL kinase reporter in the leukemic cells possessing the BCR-ABL mutation.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Proctor
authors.affiliation: Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA, and North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Imola G
authors.initials: IG
authors.lastname: Zigoneanu
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Qunzhao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.|Department of Chemical Biology and Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Lawrence
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.|Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA, and North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D010447:Peptide Hydrolases