Abstract Phenotypic screening has become increasingly common to the drug discovery workflow. Yet, productive outcomes remain hindered by the challenging task of identifying the underlying cellular targets mediating the desired phenotype. Current approaches rely on linking the bioactive compound to a surface or an affinity handle allowing selective capture of interacting proteins for identification by mass spectrometry. These methods can be constrained by ineffective capture of low affinity and low abundance proteins. In addition, for these methods to provide meaningful results, it is critical that the chemical derivatization of the bioactive compound would not disrupt the binding interactions with the cellular targets. Consequently, there is preference for methods compatible with living cells because they allow verification of the pharmacological activity of the modified compound. We have developed such a method based on a novel chloroalkane capture tag that minimally affects compound potency and cell permeability. Following binding with the tagged compound in live cells, the cells are lysed and the chloroalkylated compound, together with the bound targets, is rapidly captured onto immobilized HaloTag protein. The in-situ target engagement combined with rapid covalent capture allows for isolation of difficult targets with low affinity and/or low abundance. The putative targets identified by mass spectrometry are then validated for direct binding relationship with the bioactive compound by bioluminescence energy transfer. We tested this target capture/target-validation workflow using the interaction of histone deacetylases (HDACs) class I/IIb and the inhibitor SAHA (Vorinostat). RESULTS: Treatment of K562 cells with SAHA or SAHA-chloroalkane revealed high cellular potency against HDAC class I/IIb with minimal impact of the chloroalkane modification on the drug potency. Using the SAHA-chloroalkane to selectively enrich for SAHA targets from K-562 cells, we identified and validated all the known targets of SAHA (i.e., HDAC 1, 2, 3, 6, 8 and 10) regardless of their abundance or affinity (nM to low μM). In addition, we also identified and validated two previously undescribed targets of SAHA (CPPED1 and ADO), both metalloenzymes. Because SAHA binds to HDACs by chelating to a bound zinc ion, it may interact by a similar mechanism with other metalloenzymes. The discovery of ADO may also provide insight to a potential novel mode of action for SAHA in neurodegenerative diseases. Other experiments showed that replacing the chloroalkyl group in the SAHA-chloroalkane with biotin significantly reduced drug potency and consequently enrichment efficiency (only HDAC6 was isolated), further demonstrating the advantages provided by the relatively inert chloroalkane tag. Taken together these results indicate that our workflow can reveal the direct binding relationships between bioactive compounds and their cellular targets. Citation Format: Rachel Friedman Ohana, Thomas Kirkland, Carolyn Woodroofe, Sergiy Levin, Robin Hurst, Paul Otto, Tetsuo Uyeda, Michael Ford, Richard Jones, Danette Daniels, Marjeta Urh, Keith Wood. Decoding phenotypic drug screening targets using a novel chloroalkane capture tag. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2446. doi:10.1158/1538-7445.AM2015-2446