Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag
| Autor: | Rachel Friedman Ohana, Keith V. Wood, Robin Hurst, Kristopher Zimmerman, Michael M. Rosenblatt, Sergiy Levin, Thomas Machleidt, Thomas A. Kirkland |
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| Rok vydání: | 2021 |
| Předmět: |
Proteomics
0301 basic medicine Azides Membrane permeability Hydrolases Ultraviolet Rays Dasatinib 01 natural sciences Biochemistry Histone Deacetylases Mass Spectrometry 03 medical and health sciences Low affinity Receptors Adrenergic alpha-2 Hydrocarbons Chlorinated Humans Vorinostat 010405 organic chemistry Chemistry Drug discovery Affinity Labels General Medicine Selective isolation Propranolol 0104 chemical sciences Transmembrane domain Cross-Linking Reagents 030104 developmental biology Membrane Diazomethane Covalent bond Biophysics Molecular Medicine Deconvolution K562 Cells Protein Kinases Chromatography Liquid |
| Zdroj: | ACS Chemical Biology. 16:404-413 |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/acschembio.0c00987 |
| Popis: | Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains. |
| Databáze: | OpenAIRE |
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