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

Abstract

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 (K d 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.

Target identification and mechanism of action in chemical biology and drug discovery

“…64 ). The other is based on a characteristic shift in the chromatographic retention-time profile after a compound binds a protein target 65 . Although the generality of these approaches remains to be determined, their combination with quantitative proteomics is quite promising.…”

Affinity purification in target identification: the specificity challenge

“…Coupled with quantitative mass spectrometry, SPROX measures the rate difference of methionine oxidation of proteins in order to identify specific targets (West et al 2010;Dearmond et al 2011;Strickland et al 2013;Xu et al 2014;Adhikari et al 2015). Finally, with a different concept, Emili and coworkers developed a label-free method based on ligandinduced change of retention time of proteins during chromatographic co-elution (Chan et al 2012). For their intrinsic advantage of using label-free small molecules, these approaches have great potential and nicely complement traditional affinity purification methods.…”

Mass spectrometry approaches to monitor protein–drug interactions

“…In this respect, promising initial results have been obtained with the above mentioned DARTS method [36] and an approach utilizing chromatographic co-elution of test-compound and target protein under non-denaturing conditions (Target identification by chromatographic Co-elution, TCC) [120]. For TCC and related affinity selection mass spectrometry approaches [121] the compound of interest is incubated with cell extract or purified proteins and subjected to non-denaturing liquid chromatography.…”

“…Sufficiently stable drug-protein complexes will be characterized by co-occurrence in collected fractions. In a recent report proof of principle was demonstrated by proteome wide identification of established targets for a number of potent inhibitor compounds including the HDAC inhibitor Trichostatin A and others [120].…”

“…The laborious synthesis of inactive analogs might be avoided by adding a competition-step with the free compound into the experimental work-flow (Fig 3b). In this approach, target proteins are identified by their reduced [120] binding to the affinity matrix in the presence of excess free compound as compared to the vehicle control [81][82][83][84][85][86]. Probe matrices or reactive probes selectively binding to a wide range of proteins of a particular target class can be employed to establish target selectivity of free test compounds in competition-based experiments.…”