A Suite of “Minimalist” Photo‐Crosslinkers for Live‐Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors

Abstract

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.

A Vinyl Sulfone‐Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

“…As shown in Figure 3D,strong dansyl fluorescence signals were detected in DNS-pE2-treated live MCF-7 cells,most of which was retained after cell fixation and permealization (compare panels 1&2), indicating successful Tu rned-ON fluorescence via covalent target labeling.H ighly colocalized rhodamine fluorescence was obtained on cells further clicked with Rh-N 3 (Supporting Information , Figure S5). Finally,tounequivocally confirm the highly selective PHGDH labeling profile by DNS-pE2 at ap roteome-wide scale,i nsitu proteome labeling followed by large-scale PD/ quantitative mass spectrometry (stable isotope labeling with amino acids in cell culture,orSILAC [6,20] )was done on MCF-7, COS-7 and HepG2 cells ( Figure 3F;S upporting Information; Figure S5);selective PHGDH labeling by the probe was observed in all three cell lines,a t1to 3 mm probe concentration. Immunofluorescence (IF) by using anti-PHGDH was used to further confirm the Tu rned-ON fluorescence from DNS-pE2-treated cells was indeed from the PHGDH-bound probe (compare panels 1&2 in Figure 3E).…”

“…Immunofluorescence (IF) by using anti-PHGDH was used to further confirm the Tu rned-ON fluorescence from DNS-pE2-treated cells was indeed from the PHGDH-bound probe (compare panels 1&2 in Figure 3E). Finally,tounequivocally confirm the highly selective PHGDH labeling profile by DNS-pE2 at ap roteome-wide scale,i nsitu proteome labeling followed by large-scale PD/ quantitative mass spectrometry (stable isotope labeling with amino acids in cell culture,orSILAC [6,20] )was done on MCF-7, COS-7 and HepG2 cells ( Figure 3F;S upporting Information; Figure S5);selective PHGDH labeling by the probe was observed in all three cell lines,a t1to 3 mm probe concentration. As shown in Figure 3F (SILACr atio plot), of the > 1000 proteins identified, PHGDH was the only hit that exhibited as ignificant increase in SILACr atios (H/L ratio > 5).…”

Natural Products and Chemical Biology Tools: Alternatives to Target Epigenetic Mechanisms in Cancers

“…investigated the influence of a photo‐crosslinking group for living cell imaging. The authors reported APEX1 as an off‐target of GW841819X and showed the interaction to be responsible of DNMT1 up‐regulation . This approach has the potential to be be the starting point of a new indirect targeting of DNA methylation.…”

A Photoaffinity Displacement Assay and Probes to Study the Cyclin‐Dependent Kinase Family

“…They have been used for profiling changes in protein expression levels to investigate the biological origin of phenotypic changes, [1] and in the determination of inhibitor selectivity profiles. [2][3][4] Photoaffinity probes typically consist of three functionalities ( Figure 1A): the selectivity function, which binds to the target of interest;the photoreactive group, which will crosslink to bound protein;and the bioorthogonal handle for downstream workflows. [5,6] Thed esign of the probe,i ncluding choice of photoreactive group,p rofoundly impacts the likelihood of successful protein capture.T here have been few studies to systematically compare the efficiency of crosslinking of commonly used photoreactive groups and there is still no consensus on the ideal photoreactive group and attachment strategy.…”