title: Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.
abstract: Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease.
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Rachel Friedman
authors.initials: RF
authors.lastname: Ohana
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California, United States.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kirkland
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California, United States.
authors.forename: Carolyn C
authors.initials: CC
authors.lastname: Woodroofe
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California, United States.
authors.forename: Sergiy
authors.initials: S
authors.lastname: Levin
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California, United States.
authors.forename: H Tetsuo
authors.initials: HT
authors.lastname: Uyeda
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Otto
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Robin
authors.initials: R
authors.lastname: Hurst
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Robers
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Kris
authors.initials: K
authors.lastname: Zimmerman
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Lance P
authors.initials: LP
authors.lastname: Encell
authors.affiliation: Promega Corporation , Madison, Wisconsin, United States.
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
mesh_terms: D000473:Alkanes / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002713:Chlorine / Q000737:chemistry*; D002853:Chromatography, Liquid; D016503:Drug Delivery Systems; D055808:Drug Discovery; D057809:HEK293 Cells; D056572:Histone Deacetylase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D000077337:Vorinostat
chemical_list: D000473:Alkanes; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D002713:Chlorine; D000077337:Vorinostat