“Minimalist” Cyclopropene-Containing Photo-Cross-Linkers Suitable for Live-Cell Imaging and Affinity-Based Protein Labeling

Target
identification of bioactive compounds within the native cellular environment
is important in biomedical research and drug discovery, but it has
traditionally been carried out in vitro. Information about how such
molecules interact with their endogenous targets (on and off) is currently
highly limited. An ideal strategy would be one that recapitulates
protein–small molecule interactions in situ (e.g., in living
cells) and at the same time enables enrichment of these complexes
for subsequent proteome-wide target identification. Similarly, small
molecule-based imaging approaches are becoming increasingly available
for in situ monitoring of a variety of proteins including enzymes.
Chemical proteomic strategies for simultaneous bioimaging and target
identification of noncovalent bioactive compounds in live mammalian
cells, however, are currently not available. This is due to a lack
of photoaffinity labels that are minimally modified from their parental
compounds, yet chemically tractable using copper-free bioorthogonal
chemistry. We have herein developed novel minimalist linkers containing
both an alkyl diazirine and a cyclopropene. We have shown chemical
probes (e.g., BD-2) made from such linkers could be used
for simultaneous in situ imaging and covalent labeling of endogenous
BRD-4 (an important epigenetic protein) via a rapid, copper-free,
tetrazine-cyclopropene ligation reaction (k2 > 5 M–1 s–1). The key features
of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting
moiety, are their tunable reactivity and solubility, relative stability,
and synthetic accessibility. BD-2, which is a linker-modified
analogue of (+)-JQ1 (a recently discovered nanomolar protein–protein-interaction
inhibitor of BRD-4), was subsequently used in a cell-based proteome
profiling experiment for large-scale identification of potential off-targets
of (+)-JQ1. Several newly identified targets were subsequently confirmed
by preliminary validation experiments.