Abstract C158: A chemoproteomics strategy for target identification and lead compound optimization using chloroalkane derivatized compounds
Authors: Ford Michael , Jones Richard , Amunugama Ravi , Daniels Danette , Ohana Rachel , Levin Sergiy , Kirkland Thomas , Urh Marjeta , Wood Keith 
Published: May 2016

The Identification and validation of drug targets is an industry wide challenge. There is a very clear and urgent need for technologies that can identify the interaction partners of small molecules. Chemical proteomics is one technology that has attracted attention as a solution to the drug target identification problem. Here we present a new approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules to isolate their respective protein partners. In general derivatization of small molecules with the CA moiety does not impact their cell permeability and has limited impact on potency, allowing for phenotypic assays of the derivatized compound to be performed. The retention of cell permeability also allows for the capture process to be performed from live cells treated with the CA-compound. This process is also compatible with competition assays, which can be used to evaluate and compare other compounds exhibiting a similar mode of action.

Here we present a study using Dasatinib-CA, a modified kinase inhibitor and potent anti-cancer drug which binds to a broad range of kinases. First we performed target enrichment experiments by incubating K562 cells with the modified Dasatinib (Dasatinib-CA). Cells were lysed and the Dasatinib-CA, together with the bound targets, was rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute putative interacting proteins. Secondly using the same assay format we evaluated the relative target affinities of Dasatinib-CA versus competing molecules. Competition assays were performed by incubating multiple mixtures of Dasatinib analogues and Dasatinib-CA at varying relative concentrations.

Eluted proteins were processed using SDS-PAGE and in-gel digestion. For target identification experiments peptides were analyzed using label free mass spectrometry. For the competition assays digested material was labeled with Tandem Mass Tags (TMT) 10plex reagents. Peptides were analyzed using nanoscale LC-MS/MS coupled with a Q Exactive mass spectrometer (Thermo). Protein identification and quantitation was performed with MaxQuant (MaxQuant.org) and data validation and visualization was performed using Perseus (Perseus-framework.org).

Using this approach we identified over 30 kinases, including known membrane associated and membrane protein targets. This work presented here highlights a new method for chemical proteomics and demonstrates utility of the platform to enable target identification and to evaluate competitor molecules.

Citation Format: Michael Ford, Richard Jones, Ravi Amunugama, Danette Daniels, Rachel Ohana, Sergiy Levin, Thomas Kirkland, Marjeta Urh, Keith Wood. A chemoproteomics strategy for target identification and lead compound optimization using chloroalkane derivatized compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C158.