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

Abstract

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.

Current advances of carbene-mediated photoaffinity labeling in medicinal chemistry

“…Since the concept of the "minimalist linker" was rst introduced by Yao et al in 2013, 64 both a diazirine and a clickable moiety have been concurrently incorporated into a single alkyl chain (L 1-3 , Scheme 3). 64,65 This strategy has been demonstrated to be a remarkably efficient method for cmPAL and cell imaging. Very recently, a suite of "minimalist linkers" containing alkyldiazirine units and different bioorthogonal tags have been developed to nd targets of BRD4 inhibitors and obtain better insights into how a tag might affect the overall performance of a probe 66 (New).…”

Development and Leading-Edge Application of Innovative Photoaffinity Labeling

“…To buck-up the missing tag, post-labeling approaches were developed by application of click chemistry. [60][61][62][63] A diol-type diazirine 48 was utilized to introduce an aldehyde by periodate oxidation followed by selective coupling with biotinyl hydrazide to detect trace amounts of labeled product. 64) Preinstalled alkylazide tag on 49 was substantially stable to the photolytic 65) The postlabeling approaches require, however, large excess reagents to complete enough tag on the labeled proteins existing very low concentrations.…”

Affinity purification in target identification: the specificity challenge

“…The authors have demonstrated their superiority to other methods with 12 kinase inhibitors. This strategy has also been further used in live-cell imaging and protein profiling (Li et al 2014c;Su et al 2015). Another approach to reduce the effect of linker addition is to include the linker beforehand in the small molecule; therefore, hit compounds generated from phenotypic screening already have a linker available for tagging or immobilization.…”