Drug target deconvolution in cancer cells using liquid chromatography, fluorescence polarization and mass spectrometry

Abstract

Drugs are important in curing diseases, and in the latter decade, research in the use of drugs to cure cancer has grown exponentially. There are thousands of drug candidates that go through screening processes each year, but only a handful reach the clinical stage, and in the end get patented for use in treatment of diseases. One of the major difficulties today is to know if the drug binds to the desired target. Identifying the target is termed drug target deconvolution. Existing drug target deconvolution strategies have limitations, hence there is a need for a new/complementary methodology, with a high degree of throughput, versaitility and reliability. We propose a method which combines liquid chromatography (LC) in three orthogonal dimensions together with fluorescence polarization (FP) and mass spectrometry (MS) to identify target proteins in complex mixtures. Results show that FP applied on fluorecein-tagged drugs is a robust, sensitive and spesific technique to identify protein-drug binding in both standards and complex samples. Study of commercially available LC columns – SEC, HIC and mixed mode IEX under non-denaturating conditions, in three dimensions indicated a 90 % orthogonality between each dimension and a total peak capacity of ~3000. Combining LC, FP and LC-MS has shown that target identification of small molecule inhibitor XAV939 in a protein standard mixture is possible. Preliminary results indicate that target identification in SW480 colon cancer cells using the same techniques is viable. Current work indicate that it is possible to measure protein-drug binding with FP on non-tagged Wnt-inhibitors, both in protein standard samples and complex cell lysates.