On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening

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)T315I kinase 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-ABLT315I tyrosine kinase, with an IC50 value 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.