Spotlight pubs.acs.org/acschemicalbiology
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RAMPING UP PROTEIN ANALYSIS WITH CLEAVABLE FLUORESCENT ANTIBODIES
that the platform could be extended to analyzing nucleic acids or metabolites as well, opening up a new avenue for studying cell systems. Heidi A. Dahlmann
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LSD-BOUND SEROTONIN RECEPTOR CRYSTALLIZED
Reprinted with permission from Wiley-VCH from Angew. Chem., Mondal, M. et al., 2017, 56, 2636.
Cells are swarming with complex mixtures of proteins, the relative quantities and localizations of which continually change as cells respond to stimuli and maintain themselves. Being able to pinpoint and quantify proteins is critical to understanding cellular contributions to tissue structure, function, and development, but tools for tracking their spatiotemporal distribution are limited. For example, mass spectrometry on cell extracts can be used to quantify hundreds of different proteins at a time, but information on intracellular localization and expression variation in a population is lost. On the other hand, fluorescence microscopy is a powerful means for simultaneously locating and quantifying proteins in intact cells, but the number of different proteins that can be analyzed in a single experiment is relatively low. A promising solution to this dilemma was recently reported by Jia Guo and co-workers, who developed a detection platform that combines the potential to visualize and quantify scores of proteins in a sample of intact cells (Angew. Chem. 2017, 56, 2636−2639). Their highly multiplexed single-cell in situ analysis protocol involves staining proteins in cells with antibodies labeled with cleavable fluorophores; after one round of visualization, the fluorophores are cleaved so that the same sample of cells can be treated with a new batch of fluorophore-labeled antibodies targeting a batch of different proteins. The research team optimized the antibody-fluorophore linkers so that they could be cleaved with a reducing agent under mild conditions, enabling multiple cycles of staining to be performed with little to no damage to the cells being studied. The authors anticipate © 2017 American Chemical Society
Reprinted from Cell 2017, 168, Wacker, D. et al., Crystal Structure of an LSD-Bound Human Serotonin Receptor, 377− 389. Copyright 2017, with permission from Elsevier.
Few hallucinogenic compounds are as potent as lysergic acid diethylamide (LSD), the psychoactive properties of which were infamously discovered in 1943 when it was accidentally ingested by the chemist who first synthesized the compound in 1938. By the 1960s LSD had become a popular recreational drug, notable not only for the profoundness but also the duration of its psychological effects. The longevity of an LSD “trip” had been attributed to the remarkable slowness with which LSD dissociates from its molecular targets, a phenomenon that may be explained with the help of a recently reported crystal structure of LSD bound to human serotonin receptor 5-HT2BR (Cell 2017, 168, 377−389). When Bryan L. Roth and co-workers examined their crystal structure, they noticed that LSD’s diethylamide moietythe functional group which enables the ergoline derivative to cross the blood−brain barrierwas rotated into a specific orientation that had never been observed in crystals of LSD alone, forming Published: March 17, 2017 583
DOI: 10.1021/acschembio.7b00207 ACS Chem. Biol. 2017, 12, 583−585
ACS Chemical Biology
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contacts that were essential to LSD’s ability to stabilize the receptor and affect its function. The crystal structure also revealed that an extracellular loop (EL2) of the receptor formed a “lid” that presumably hinders LSD’s dissociation from the binding site. Upon mutating the lid to be more flexible, the research team demonstrated that LSD was able to move in and out of the binding site more quickly than in wild-type 5-HT2BR, which subsequently altered time-sensitive interactions of 5-HT2BR with the signaling protein β-arrestin but not with another signaling protein, Gq. The authors note that LSD’s unusual effect on signaling kinetics may be critical to its in vivo hallucinogenic activity.
sulfimide conjugation product. The alkyne or azide side chain can then function as a synthetic handle for a “click” reaction with other molecules of interest. In a series of proof-of-principle experiments, the research team demonstrated ReACT’s utility for tagging model protein substrates with payloads such as biotin or fluorophores, creating antibody−drug conjugates, and identifying functional methionines in cells (Science 2017, 355, 597−602). Heidi A. Dahlmann
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Heidi A. Dahlmann
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TURMERIC WITH A GRAIN OF SALT
NEW TAKE ON SULFUR-LINKED BIOCONJUGATION
Reprinted with permission from Nelson et al. J. Med. Chem., DOI: 10.1021/acs.jmedchem.6b00975. Copyright 2017 American Chemical Society.
Turmeric is a characteristically orange spice often used in savory South Asian or Middle Eastern cuisine. Beyond the pantry, turmeric extracts have long been purported to possess a wide variety of therapeutic benefits. The extracts mostly contain the natural products curcumin and related curciminoids, and a brief web or literature search uncovers reports of curcumin as medicine for conditions ranging from hangovers to Alzheimer’s disease to cancer. To date, there are thousands of published manuscripts on curcumin, and new ones are unveiled each week, but a healthy slice of skepticism is in order given that many reports label it as a PAINS, or pan assay interference compound. As such, it is difficult to parse which reports are seeing the PAINS effect and which show potential for specificity. Curcumin has also been classified as an IMP, or invalid metabolic panacea, a term for natural products which keep inspiring researchers to chase down further results but fail to deliver as lead compounds. Recently, Nelson et al. (J. Med. Chem. 2017, DOI: 10.1021/ acs.jmedchem.6b00975) penned a perspective article chronicling their deep dive into the curcumin literature aimed at sorting fact from artifact. They began their case by describing how curcumin generically interferes with assays in nine separate publications, making it the archetype for a PAINS. They also reviewed the chemical properties of curcumin, showing that it is an unstable and reactive compound displaying none of the key hallmarks of a promising lead compound such as bioavailability or selectivity. Next, in a series of case studies, they combed through manuscripts representing several reported in vitro activities to better understand putative functional results. Of note, high concentrations of curcumin form aggregates, and many studies did not see effects until these concentrations were reached. Finally, they revisited the clinical trial data for cancer, Alzheimer’s disease, and radiation dermatitis, revealing that the compound cannot even be detected in human serum after oral doses. While this perspective piece does not unilaterally discount turmeric as a medicine, it paints the picture of curcumin skepticism through a chemist’s eye, making the case that the chemical properties of a natural product should always be considered in parallel when evaluating phenotypic observations. It should be noted that this perspective on
From Lin, S., et al., Science 2017, 355, 597. Reprinted with permission from AAAS.
Bioconjugation, the process of covalently attaching a small molecule such as a fluorophore or drug to a biomolecule such as a protein, requires carefully choosing reaction conditions that will allow selective targeting of a specific site on the biomolecule without degrading it or damaging its structure. Nucleophilic functional groups on biomolecules are typically the target sites for bioconjugation reactions; for example, thiol groups of cysteine residues in proteins are commonly chemically modified to generate protein−drug conjugates or to probe the impact of specific side chains on the function of the protein as a whole. Compared with cysteines, methionine residues have been largely ignored by chemical biologists, in part because the sulfide component of methionine is far less nucleophilic than the free thiol of a cysteine. However, a research team led by Christopher J. Chang and F. Dean Toste was interested in performing chemoselective methionine bioconjugation for a couple of important reasons: methionine residues are much rarer than cysteine residues and thus would serve as more selective sites for protein labeling, and methionine-labeling could facilitate emerging research into elucidating the role of methionine oxidation in signaling and protein-binding interactions. In considering how to target methionine in preference to cysteine and other nucleophilic residues, the research team was inspired by its inherent tendency to undergo autoxidation. So, they developed a redox-activated chemical tagging (ReACT) method in which the methionine sulfur attacks an oxidizing oxaziridine reagent with alkyne or azide side chains to form a 584
DOI: 10.1021/acschembio.7b00207 ACS Chem. Biol. 2017, 12, 583−585
ACS Chemical Biology
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curcumin is not without controversy; a letter to the editor presents a counterpoint in ACS Medicinal Chemistry (DOI: 10.1021/acsmedchemlett.7b00051). Jason G. Underwood
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UNRAVELING RNA’S ROLE IN UV DAMAGE
Reprinted from Cell, 168, Williamson, L., et al., UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene, 843−855. Copyright 2017, with permission from Elsevier, Inc. Creative Commons Attribution (CC BY 4.0).
Though biochemists have long known that UV radiation interferes with transcription, they have not had a detailed understanding of the underlying mechanisms and how cells recover. Now, researchers have uncovered one piece of that puzzle (Williamson et al., Cell, 2017, 168, 843−855). They have shown that the coding and noncoding transcript isoform of a single genefor a helicase involved in duplex unwindingacts in opposite ways in the late-stage response to UV exposure. The researchers used global run-on sequencing to quantify the transcription of genes associated with RNA-polymerase II both in the presence and in the absence of UV exposure. UV radiation does not eliminate transcription, but it leads to shorter gene transcripts, an effect that persists for hours. UV radiation also induces alternative last exon (ALE) splicing events, and nearly 80% of those events produced shorter mRNA isoforms. In the screening studies, the gene with the greatest response was ASCC3, which encodes for an enzyme that unwinds helical duplexes of nucleic acids. Transcription of the long isoform (373 kb) and short isoform (25 kb) are both reduced in the first 8 h after radiation. After 24 h, short isoform expression increased, while long isoform expression lagged for another 24 h. Using siRNA targeted to these isoforms, the researchers uncovered their roles in transcription. The protein translated from the long isoform within its associated complex suppresses transcription as part of a later response to UV radiation. The short isoform has the opposite effect and helps cells recover transcriptional activity after UV exposure. Finally, the team showed that these two effects counteract each other, suggesting that these two signals are opposing regulatory signals. In addition, the short isoform carries out its function as a noncoding RNA. These results underscore the complex layers of how UV radiation affects transcription in cells. These experiments help to explain some of the details that occur late in this process, in the maintenance of transcription many hours after UV exposure. Further research will be needed to fully understand exactly how the ASCC complex is assembled and how this noncoding RNA isoform disrupts its activity. Sarah A. Webb
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DOI: 10.1021/acschembio.7b00207 ACS Chem. Biol. 2017, 12, 583−585
