Affinity-based probes (AfBPs) provide a powerful tool for large-scale chemoproteomic studies of drug–target interactions. The development of high-quality probes capable of recapitulating genuine drug–target engagement, however, could be challenging. “Minimalist” photo-crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine-containing photo-crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of AfBPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug–target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC-based chemoproteomic studies have led to the discovery of a novel off-target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up-regulation of endogenous DNMT1 expression under normoxia conditions.
Bibliographical noteFunding Information:
Funding was provided by National Medical Research Council (CBRG/0038/2013), Singapore, KIST (2E26632/2E26110, CAP-16-02-KIST) and the Bio & Medical Technology Development Program of the National Research Foundation (NRF-2016M3A9B6902060) from Ministry of Science, Korea.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
- affinity-based probes
- bioorthogonal chemistry
- live-cell imaging
ASJC Scopus subject areas