Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag

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.