title: On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.
abstract: Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315Ikinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315Ityrosine kinase, with an IC50value as low as 1.3 μM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I, in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity.
authors.affiliation: Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science , Daejeon 34113, Korea.
authors.forename: Young-Lai
authors.initials: YL
authors.lastname: Cho
authors.affiliation: Department of Life Science and Institute of Nano Science and Technology, Hanyang University , Seoul 04763, Korea.
authors.forename: Young-Pil
authors.initials: YP
authors.lastname: Kim
authors.affiliation: Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science , Daejeon 34113, Korea.
authors.forename: Jin Gyeong
authors.identifier: 0000-0003-2106-4014
authors.initials: JG
authors.lastname: Son
authors.affiliation: Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science , Daejeon 34113, Korea.
authors.forename: Miyoung
authors.initials: M
authors.lastname: Son
authors.affiliation: Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science , Daejeon 34113, Korea.|Department of Nanoscience, University of Science and Technology , Daejeon 34113, Korea.
authors.forename: Tae Geol
authors.identifier: 0000-0002-6674-4147
authors.initials: TG
authors.lastname: Lee
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000378:metabolism; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D016044:Fusion Proteins, bcr-abl