In This Issue Cite This: ACS Chem. Biol. 2019, 14, 566−566
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DEVELOPING A SPLIT PEROXIDASE
breast cancer drug, together with a cholesterol-modified BODIPY FRET pair, colloid particles can be measured using FRET intensity. The researchers use the assay to demonstrate that increased serum concentrations necessitate higher concentrations of the drug in order to form the initial colloid. Additionally, if fulvestrant colloids are first stabilized by treating with polysorbate 80, their stability is enhanced when placed in high serum conditions or injected into mice.
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■ Reporter proteins such as GFP are handy for visualizing biomolecules in their cellular environment, and splitting some reporters into two parts can give helpful interaction information as well. The split versions do not have reporter activity on their own but instead display activity when their halves are tethered together by two interacting proteins. In this issue, Han et al. (DOI: 10.1021/acschembio.8b00919) take apart the engineered reporter enzyme, APEX2, to generate a new dimerization detector, split APEX (sAPEX). After determining an initial cut site to divide APEX into two, yeast display is used in an evolution strategy for improving the peroxidase activity of the split reporter. The optimized version is then used in cellular assays where reconstitution of sAPEX generates local biotinylation. The authors show the utility of sAPEX for detecting protein− protein interactions between mitochondria and the endoplasmic reticulum. In addition, sAPEX halves fused to sequencespecific RNA binding proteins can be readily detected when bound adjacent to one another on an engineered RNA scaffold.
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THE STRESS YOU SEQ
Extensive molecular and genetic studies from yeast to mammals have shown that three stresses, heat shock, oxidative stress, and unfolded protein response, are each coupled to compensatory transcriptional responses. Since these responses are essentially aimed at repairing a broken cell, pharmacologic strategies to induce such a repair program are of key interest for disease therapeutics. However, developing a screening method for easily deconvoluting the stress responses from one another remains a challenge. Now, Grandjean et al. (DOI: 10.1021/acschembio.9b00134) present a medium-throughput strategy for measuring the transcriptional changes induced by various stress responses. For the proof-of-concept, the assay involves growing tissue culture cells in 96-well plates and adding compounds that induce a particular stress. The RNA is subsequently isolated, and 10−20 transcripts known to be associated with a particular stress pathway are then quantified via high-throughput RNA sequencing (RNaseq). After demonstrating the robustness of this targeted RNaseq strategy with several known stressors, the researchers go on to show its utility in classifying the selectivity of stress activating compounds identified in a previous highthroughput screen.
COLLOIDAL AGGREGATES: FRUSTRATION TURNED FRIEND?
Aggregation of small molecules into self-assembled colloids can lead to misleading results in a variety of high-throughput screening assays. However, while this property of sequestering a lead compound into tiny particles may complicate the discovery process, it may hold promise for keeping a drug-rich formulation stable in vivo. With this idea in mind, Ganesh et al. (DOI: 10.1021/ acschembio.9b00032) develop a flexible fluorescent assay for monitoring colloid formation and stability that is compatible with a range of serum conditions. By formulating fulvestrant, a © 2019 American Chemical Society
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Published: April 19, 2019 566
DOI: 10.1021/acschembio.9b00261 ACS Chem. Biol. 2019, 14, 566−566
