A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

While
targeted therapy based on the idea of attenuating the activity
of a preselected, therapeutically relevant protein has become one
of the major trends in modern cancer therapy, no truly specific targeted
drug has been developed and most clinical agents have displayed a
degree of polypharmacology. Therefore, the specificity of anticancer
therapeutics has emerged as a highly important but severely underestimated
issue. Chemical proteomics is a powerful technique combining postgenomic
drug-affinity chromatography with high-end mass spectrometry analysis
and bioinformatic data processing to assemble a target profile of
a desired therapeutic molecule. Due to high demands on the starting
material, however, chemical proteomic studies have been mostly limited
to cancer cell lines. Herein, we report a down-scaling of the technique
to enable the analysis of very low abundance samples, as those obtained
from needle biopsies. By a systematic investigation of several important
parameters in pull-downs with the multikinase inhibitor bosutinib,
the standard experimental protocol was optimized to 100 μg protein
input. At this level, more than 30 well-known targets were detected
per single pull-down replicate with high reproducibility. Moreover,
as presented by the comprehensive target profile obtained from miniaturized
pull-downs with another clinical drug, dasatinib, the optimized protocol
seems to be extendable to other drugs of interest. Sixty distinct
human and murine targets were finally identified for bosutinib and
dasatinib in chemical proteomic experiments utilizing core needle
biopsy samples from xenotransplants derived from patient tumor tissue.
Altogether, the developed methodology proves robust and generic and
holds many promises for the field of personalized health care.