Abstract C224: Identification of drug targets via their intracellular interactions. (2013)

Abstract Phenotypic-based screening of small molecule libraries is a significant trend in drug discovery. However, deciphering the intracellular interactions between proteins and small molecules that mediate phenotypic outcomes remains a major challenge. Current approaches rely on linking the small molecule to a surface or affinity tag (e.g., biotin), allowing selective capture of the corresponding protein target for identification by mass spectrometry. Verifying that the linkage method does not disrupt the pharmacological activity is important for ensuring maintenance of the binding interaction with the intracellular targets. Consequently, although target capture is commonly done from cell lysates, methods compatible with living cells are preferable. We have developed such a method based on a novel chloroalkane tag that minimally affects compound potency and cell permeability. This allows phenotypic outcomes to be recapitulated by the modified compound, increasing confidence in the biological relevance of the captured proteins. The tagged compound is allowed to bind at equilibrium to intracellular targets, then cells are lysed and the chloroalkane bound to its protein targets is quickly captured onto magnetic particles containing immobilized HaloTag. The rapid isolation method minimizes complex collapse, preserves low affinity interactions, and in combination with low background adsorption facilitates target identification by mass spectrometry. Engagement of identified targets by compounds in living cells can be verified using bioluminescence energy transfer (BRET). NanoLuc luciferase fused to the target protein serves as a BRET donor to the compound derivatized with fluorophore adducts. As a model to test this combined target capture/target validation work flow we selected the interaction of histone deacetylase (HDAC's) and Vorinostat (SAHA) a broad- HDAC inhibitor. This family of deacetylases exhibits a range of cellular abundance and affinity to SAHA (0.001-1µM), and has members localized in the nucleus or cytoplasm. Results: Treatment of K562 cells with SAHA or a derivative having the chloroalkane tag revealed high cellular potency against HDAC class I/IIb with IC50 of 0.1µM and 0.2µM respectively. These results indicate minimal impact of the chloroalkane modification on the drug potency. For target identification, the SAHA chloroalkane derivative was allowed to bind at equilibrium to intracellular targets followed by rapid capture onto immobilized HaloTag. By this method we were able to identify all the direct interactors of SAHA (i.e., HDAC 1,2,3,6 and 8) regardless of their subcellular localization, abundance, or affinity. The intracellular BRET approach was next used to validate the set of identified HDAC's and to rank order their relative binding affinity to SAHA. Our results suggested that these approaches used in combination provide reliable identification of targets in living cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C224. Citation Format: Rachel Friedman Ohana, Matt Robers, Thomas A. Kirkland, Carolyn C. Woodroofe, Chad Zimprich, Tetsuo H. Uyeda, Paul Otto, Sergiy Levin, Keith Wood. Identification of drug targets via their intracellular interactions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C224.