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 out method, cysteine-tethered peptides adsorbed onto a gold surface produced changes hi, the relative peak intensitiesof the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kitiase 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 duster region-abelson (BCR-ABL)(T315I) kinase inhibitor, henceforth referred- to as KR135861. KR135861- showed no cytotoxicity and was subsequently confirmed to be superiOr to irriatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABL(T315I) tyrosine kinase, with an IC50 value as low as 1.3 mu M. In in Vitro experiments, KR135861 reduced the viability of both Ba/F3 cells.expressing wild-type BCR-ABL and BCR-ABL7315I, in contrast to imatinib 8 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.