Target Identification by Chromatographic Co-elution: Monitoring of Drug-Protein Interactions without Immobilization or Chemical Derivatization

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (Kd range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist. Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (Kd range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist. Drugs often act as protein antagonists (inhibitors) or agonists (activators) through selective physical interactions with targets in disease-relevant pathways, yet many pharmaceuticals and chemical probes from cell-based phenotypic screens currently lack defined cellular targets (1Chan J.N. Nislow C. Emili A. Recent advances and method development for drug target identification.Trends Pharmacol. Sci. 2010; 31: 82-88Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although conventional “target-based” drug discovery pipelines emphasize functional characterization and in vitro inhibition/activation assays (2Schreiber S.L. 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Wang J. Ketela T. Brenner C. Brill J.A. Fernandez G.E. Lorenz T.C. Payne G.S. Ishihara S. Ohya Y. Andrews B. Hughes T.R. Frey B.J. Graham T.R. Andersen R.J. Boone C. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.Cell. 2006; 126: 611-625Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 9Lamb J. Crawford E.D. Peck D. Modell J.W. Blat I.C. Wrobel M.J. Lerner J. Brunet J.P. Subramanian A. Ross K.N. Reich M. Hieronymus H. Wei G. Armstrong S.A. Haggarty S.J. Clemons P.A. Wei R. Carr S.A. Lander E.S. Golub T.R. The Connectivity Map: Using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3509) Google Scholar), such approaches do not pinpoint the direct target(s) bound by a drug. Conversely, biochemical characterization of the protein targets of small molecules has traditionally been accomplished by immobilizing or labeling compounds for use as affinity ligands to probe cell lysates (10Terstappen G.C. Schlüpen C. Raggiaschi R. Gaviraghi G. Target deconvolution strategies in drug discovery.Nat. Rev. Drug Discov. 2007; 6: 891-903Crossref PubMed Scopus (333) Google Scholar, 11Rix U. Superti-Furga G. Target profiling of small molecules by chemical Chem. Biol. 2009; PubMed Scopus Google Scholar), but the of functional can a and is not to screening of diverse compounds. unbiased chemical strategies biochemical with have been developed to identify drug a biochemical drug target by the of a cell by the of a cell A.M. of to identify targets and Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), drug affinity target the proteomic of or to identify targets based on the that the drug-protein is to B. R. M. S. Wang J. J.A. J.A. J. J. J. Target identification drug affinity target Natl. Acad. Sci. U. S. A. 2009; PubMed Scopus Google Scholar). Although approaches do not require chemical or labeling of the compound or an is for biochemical for drug affinity target drug binding not or even target the of in drug Drug Identifying and new for Rev. Drug Discov. 2004; PubMed Scopus Google Scholar), and the of cell-based phenotypic novel label-free chemical proteomic methods are to for the unbiased detection of the physical interactions of bioactive compounds with proteins in biological in a Here, we a applicable drug target identification based on liquid to to the interactions of small molecules and bioactive ligands with nearly cellular proteins in biological The is based on a in the chromatographic of a compound that binding to a protein target. is then used to the protein target(s). We of with diverse TICC used identification by chromatographic protein identification used identification by chromatographic protein identification to compound target and we identify novel targets for one and one We that TICC can be used to identify drug-protein interactions the for compound or protein immobilization or target identification by chromatographic co-elution protein identification monitoring synthetic target identification by chromatographic co-elution protein identification monitoring synthetic of and in and in a from C. H. Ho C.H. S.L. D. S. P. Porter J. Gray C.A. Andersen R.J. G. Nislow C. Andrews B. D. Graham T.R. M. Boone C. molecular yeast mode-of-action analysis of bioactive 2009; PubMed Scopus Google Scholar), from as in of drugs by to from protein in the of S. J.J. S. of affinity for protein 2005; PubMed Scopus Google for and from E. and cell protein by and respectively, as G. H. G. A. J. S. A.P. T. M. M. A. A. B. V. A. P. A. J. S. C. S. R. J.J. G. P. S. G. A.M. 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D. to the of and with PubMed Scopus Google involved in selective protein We performed of protein from the protein on and drug by drug binding with as as of protein and to the of analysis of the fractions the of the to drug of the proteins identified the and target of by detection of of in a a drug performed in demonstrated with with the drug off-target binding by TICC even the drug a yeast with or yeast to a of for A.B. Lopez A. Givoni I.E. Williams D.E. Gray C.A. Porter J. Chua G. Sopko R. Brost R.L. Ho C.H. Wang J. Ketela T. Brenner C. Brill J.A. Fernandez G.E. Lorenz T.C. Payne G.S. Ishihara S. Ohya Y. Andrews B. Hughes T.R. Frey B.J. Graham T.R. Andersen R.J. Boone C. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.Cell. 2006; 126: 611-625Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, C. R. for of the molecular by the and Chem. 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Target identification or validation is essential for drug and of the mechanism of action of drugs and chemical probes unexpected off-target in be the development of generic interaction screening that are and well to with diverse compounds and biological TICC has the to the identification of the physical interaction of bioactive compounds and ligands in an unbiased in cell or biological not proteins involved in a known or The method of interactions in a physiologically relevant the for labeling or immobilization of the protein or Although of proteins has been A.M. of to identify targets and Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, B. R. M. S. Wang J. J.A. J.A. J. J. J. Target identification drug affinity target Natl. Acad. Sci. U. S. A. 2009; PubMed Scopus Google Scholar), with drug and proteomic profiling is Furthermore, the compound is not or to TICC by nearly drug-target association We have that TICC is highly reproducible, and in that one a of that is by a such as an and for detection and for in monitoring is well a for protein for shotgun is and a biological for target(s). target can be protein lysates from or or both compound in or in vitro to for the of the is the ability to is when the compound of a or in the target. target deconvolution depends on drug target from we have the of multiple to the of candidate targets for biochemical to identify targets of compounds such as TICC is to chemical genetic assays for drug target Although in chemical genetic assays can identify a of that be a of direct targets and proteins involved in the or biological as the the proteomic be to identify the targets that interact with the compounds of The methods can a for target and to understanding of biological pathways and by bioactive compounds. the identification of or bound targets is by the of the of the target protein identification and the of drug we in strategies that can such as the use of multiple to putative target the use of to detection of binding to and binding and the use of to identify targets that be with protein TICC for compounds that do not well require the to of biological the of the drug-target the is is to use to The development of more and separations with more protein TICC is applicable to both drug discovery pipelines and the characterization of chemical probes from phenotypic screens and can drug mechanism of and off-target effects in a small to the interaction of such as or even with proteins. analysis is based on target and can be by the of a physiologically relevant drug well TICC can both of candidate and target and affinity that are for understanding the of Furthermore, that TICC be for the characterization of protein as drug Conversely, the of an for drug and require and with and that do not the binding interaction protein and to TICC to to are currently under in the of TICC is currently to to biological and to interactions in the to micromolar of the of We E. and G. for and of the Emili for and of the The a from S. The a from R. yeast and from C. H. J. Y. C. and D. V. and performed the and the and with A. E. the D. V. performed and and proteomic S. performed the and and proteomic J. B. performed the and for P. the proteins for target P. H. B. performed the for J. A. H. performed the proteomic for the and Y. and involved in M. M. involved in C. and target A. E. the TICC method and the from with

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