Spotlight Cite This: ACS Chem. Biol. 2018, 13, 5−10
impact human health and reproduction. One example of an EDC is bisphenol A (BPA), commonly used in the production of epoxy resins and polycarbonate plastics over the past half century. BPA exhibits estrogen-like properties, and though it is far less potent than the natural hormone, growing concerns about its long-term effects have inspired ban legislation and coaxed some plastic manufacturers to remove it from food and drink packaging. Today, the identities and effects of many EDCs are not fully tractable, so there is a growing need for assays that can both hunt for EDCs and quantify their activity on natural receptors of interest. In a recent study, Furst et al. (ACS Cent. Sci., DOI: 10.1021/ acscentsci.6b00322) built a disposable chip capable of detecting soluble EDCs that bind to the human estrogen receptor (ERα). Specificity was mediated by an ERα-specific monobody, a synthetic fibronectin domain-based protein previously selected for affinity to the ligand-bound form of ERα. When a ligand or EDC-bound receptor is present, it binds the monobody immobilized to the gold electrode surface, and electrochemical detection is achieved through impedance spectroscopy. Since the receptor is not enough to produce a significant signal, the researchers used freeze-dried E. coli expressing the estrogen receptor on the bacterial surface to dramatically increase the mass and in turn the impedance change. The results showed that with a sample volume as low as 10 μL, the chip could detect natural estrogen down to the femtomole level and detect numerous ERα-binding EDCs either diluted into a simple buffer or in the relevant and far more complex solution, infant formula. Jason G. Underwood
We have selected our favorite articles published in ACS Chemical Biology and elsewhere in 2017. We hope you enjoy this year in review, and wish all of our readers, authors, and reviewers a Happy New Year!
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FIGHTING DRUG ABUSE WITH A VACCINE
Harnessing the power of opioids for pain management changed the course of medicine, but the United States has seen a recent spike in overdoses from heroin and prescription pain medicines such as oxycodone. With fatalities from opioids quadrupling since the year 2000, lawmakers and the medical community are actively searching for new avenues to curb this devastating trend. Drugs such as naloxone, which competitively bind to yet do not activate the opioid receptor, have saved lives in emergency situations, but other ways to mitigate overdoses will be vitally important. Kimishima et al. (DOI: 10.1021/acschembio.6b00977) bring the immune system into the fight against opioid overdoses, essentially creating antiopioid vaccines. Their approach is to chemically couple commonly prescribed opioids with tetanus toxin and inject mice with the combination. When opioids are administered to the mice, the opioid-vaccinated mice show reduced ̈ mice, indicating that drug efficacy in pain tests compared to naive the drug is being sequestered by antibodies. Consistent with this interpretation, the opioids also show increased serum half-life, and vaccination also protects the mice from overdose, hinting at the potential for this approach in humans. Jason G. Underwood
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REPAIRING DNA WRAPPED IN HISTONES
SENSING XENOESTROGENS
Chemical and enzymatic damage to the genome can have profound consequences. To keep lesions in check, many proteins serve to monitor and repair the integrity of double-stranded DNA. One diverse set of key enzymes, the DNA glycosylases, recognize abasic or nonstandard base sites and catalyze their excision by a variety of mechanisms. In general, these enzymes work by distorting the double helix to flip out the damaged base. In the case of a eukaryotic genome, the nucleosome architecture fundamentally stabilizes the helix, adding an extra thermodynamic barrier to DNA repair pathways. Now, Olmon and Delaney (DOI: 10.1021/acschembio.6b00921) develop assays to test how nucleosomes affect five different DNA
Reprinted with permission from Furst et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.6b00322. Copyright 2017 American Chemical Society.
Many pathways in the human body are finely controlled by low concentrations of hormones produced by the endocrine glands. Processes from basal metabolism to fertility are tuned by hormone levels, so disorders that alter the complex endocrine system can have profound consequences. Complicating the situation further, endocrine disrupting compounds (EDCs) found in the environment can produce off-target effects which potentially © 2018 American Chemical Society
Published: January 19, 2018 5
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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. Heidi A. Dahlmann
glycosylases found in bacteria or humans. Though the enzymes all perform well on free duplex DNA, they differ widely in their activity on numerous damaged DNA substrates packaged into nucleosome core particles. The study finds that the size and mechanism of the glycosylase are key parameters, but the damaged base identity and location with respect to the nucleosome also significantly alter the repair kinetics. The assays provide an in vitro platform to test how other enzymatic activities that act on DNA are affected by nucleosomes. Jason G. Underwood
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LSD-BOUND SEROTONIN RECEPTOR CRYSTALLIZED
INHIBITORS KILL THE MESSENGER
Many cancer types share the common thread of dysregulated kinase signaling, and drugs to inhibit specific kinases have already arrived in the clinic. To find new ones, researchers often exploit the subtle differences in kinase ATP-binding sites to design specific mimetic inhibitors. Once catalytic function grinds to a halt, the crippled kinase is often degraded by the proteasome. Here, Field et al. (DOI: 10.1021/acschembio.7b00116) test the specificity of a previously identified JAK3 inhibitor and uncover an additional effect. Growing cells in the presence of the inhibitor significantly reduces the protein levels of both JAK2 and JAK3 after 22 h, while JAK1 and several other control kinase levels remain unaltered. The additional effect was on the mRNA levels of these two kinases. Both mRNAs are specifically reduced by the inhibitor, implying that JAK2/3 kinases regulate the transcription, RNA processing, or degradation of their own messages. Jason G. Underwood
Reprinted from Cell, 168, Wacker, D., et al., Crystal Structure of an LSD-Bound Human Serotonin Receptor, 377−389. Copyright 2017, with permission from Elsevier.
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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 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
RNA: NOW AVAILABLE ON VINYL
Reprinted with permission from George, J. T., et al., Bioconjugate Chem., DOI: 10.1021/acs.bioconjchem.7b00169. Copyright 2017 American Chemical Society.
Many RNAs harbor natural modifications to the bases or ribose, and many flavors of synthetically modified RNAs deliver utility in the research lab or stability in the clinic. In lieu of full synthesis, a new chemical moiety can be inserted into an RNA by in vitro transcription in the presence of a modified nucleoside triphosphate. The 5-position of uracil is a popular place to engineer a 6
DOI: 10.1021/acschembio.8b00008 ACS Chem. Biol. 2018, 13, 5−10
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modification since it is away from the base pairing face, and bacteriophage RNA polymerases are relatively tolerant of extra groups in this position. This approach can add biotins, fluorophores, or other reactive groups within a full length RNA. Recently, George and Srivatsan (Bioconjugate Chem. 2017, DOI: 10.1021/acs.bioconjchem.7b00169) introduced a new reactive moiety to RNA by attaching a vinyl group to the 5-position of uracil. After detailing the synthesis of vinyl-UTP (VUTP), the researchers showed that it is readily incorporated during in vitro transcription. With modified RNAs in hand, they tested two different chemistries that conjugate to the vinyl alkene group. An oxidative Heck reaction was used to deliver fluorophores to the modified uracil by reaction with boronic acid substrates. The coupling reaction with several different fluorogenic substrates was efficient under palladium-EDTA conditions. The second chemistry, an inverse electron demand Diels−Alder (IEDDA) reaction, was also efficient and required no special reaction conditions. In this case, the electron-rich nature of the vinyl group reacts with tetrazines which are electron-deficient. They demonstrated that using modified tetrazines, biotin, or the fluorophore Cy5 can be installed onto vinyl uracils. This study drops a new tool into the RNA toolbox, enabling new ways to add chemical moieties to assist in structural, biochemical, and biophysical studies. Jason G. Underwood
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Spotlight
PRECISION EXCITATION OF FLUOROPHORES EXPANDS IMAGING PALETTE
Reprinted by permission from Macmillan Publishers Ltd.: Nature, Wei, L., et al., 544, 465−470, copyright 2017. Reprinted by permission from Macmillan Publishers Ltd.: Nature, Wei, L., et al., 544, 465−470, copyright 2017.
NATURAL PRODUCTS ON THE CLOCK
Many researchers are accustomed to imaging cellular components with a limited palette of fluorescent dyestypically red, green, and blue. Wei Min and colleagues have now reported the development of a refined imaging technique along with the concurrent synthesis of finely tuned dyes that brings the number of colors available for cellular imaging to an amazing 24 hues (Nature 2017, 544, 465−470). The imaging technique begins with the traditional step of labeling target cellular components with fluorescent dyes. The cell sample is then exposed to two precisely defined laser pulses designed to stimulate the spectrally sharp vibration (which only occurs over a much narrower frequency range than that of fluorescence emission) of a specific chemical bond in one of the dyes in the sample, eliminating off-target excitation and significantly reducing background fluorescence. The dyes are excited one at a time to generate a series of images that can be overlaid to view intricate interactions between various cell structures. Because the excitation technique precisely targets specific vibrational frequencies, which are inversely proportional to the square root of masses of each atom or functional group attached to the targeted bond, even dyes with identical molecular structure but containing different isotopes can produce distinct frequencies. Min and co-workers synthesized 14 xanthene analogs with various structures and isotopic patterns, but they predict that dozens more resolvable dyes could be developed. Notably, the new dyes are relatively nontoxic and photostable, allowing the research team to perform proof-of-concept labeling experiments on live cells under normal cell culture conditions as well as in conditions designed to induce cell stress to mimic disease states. The authors expect that the technique could be further improved to allow simultaneous dye stimulation and visualization and predict that due to its sensitivity, resolution, labeling versatility, and biocompatibility, their approach will become widely applied to probing complex biological systems. Heidi A. Dahlmann
Brevicompanines are a family of natural products named from the fungal species that produces them, Penicillium brevicompactum. These products were first described 20 years ago as plant growth regulators, but the overall mechanism of action has remained elusive perhaps due to their inconsistent effects. When lettuce plants were treated with brevicompanines, they displayed increased root growth, while rice plants did not show this effect. Now, de Montaigu et al. (DOI: 10.1021/acschembio.6b00978) search for a mechanism using gene expression analysis in the model plant Arabidopsis thaliana. After showing that brevicompanines impede root growth in this species, the researchers used expression microarrays to hone in on a set of circadian genes that are dysregulated in response to treatment. Further experiments demonstrate that the natural products affect the amplitude of circadian patterns rather than the period length. While the precise molecular target of these fungal products remains unknown, this study indicates a path to explore and unlocks a chemical biology tool to throw a wrench in the circadian clock of a model plant. Jason G. Underwood 7
DOI: 10.1021/acschembio.8b00008 ACS Chem. Biol. 2018, 13, 5−10
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Spotlight
PROGRAMMING RIPPS TO PRODUCE NON-NATURAL PRODUCTS
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) post-translationally modifies serine or threonine residues with an N-acetylglucosamine via an O-glycosidic linkage. Many proteins that are important for brain function are decorated with OGlcNAc, including those involved in synapse formation, learning, and memory. Additionally, studies in rodents support a neuronal role for O-GlcNAc modifications. In the brain, the levels of OGT and the enzyme that removes the modification, OGA, are higher in neuronal cells as compared to non-neuronal cells. Now, Andres et al. (DOI: 10.1021/acschembio.7b00232) investigate the connection between O-GlcNAc and the brain using embryonic stem cell differentiation in a dish. After demonstrating that global levels of this modification oscillate as cells escape pluripotency and become neural precursor cells, the researchers perturb the balance by shutting down the transferase activity with an inhibitor compound. These experiments demonstrate that reducing O-GlcNAc modifications affects the kinetics of neural differentiation, promoting an earlier emergence of neural pathway markers. Jason G. Underwood
Reprinted with permission from Burkhart, B. J., et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.7b00141. Copyright 2017 American Chemical Society.
One strategy for overcoming the obstacles in organic synthesis is to modify biosynthetic pathways to produce molecules of interest. In the latest twist on this idea, Burkhart et al. have developed novel, chimeric, ribosomally synthesized and post-translationally modified peptides (RiPPs) to produce new non-natural products (ACS. Cent. Sci., 2017, DOI: 10.1021/acscentsci.7b00141). Biology provides a range of valuable molecular templates and enzymatic tools for producing natural products and related compounds in high yields, but it is often hard to program in the desired chemistries. In recent decades, researchers have explored biosynthetic pathways such as modified polyketide synthesis and nonribosomal peptide synthesis to produce complex organic products, with limited success. Another potential biosynthetic platform is RiPPs, which are genetically encoded with a leader sequence and a core peptide sequence. After translation, the leader sequence is bound by enzymes, which then modify the core sequence of the peptides in a variety of ways. In nature, RiPPs generate complex chemical functionalities including azoline heterocycles, many types of macrocycles, d-amino acids, as well as a variety of other modifications. In this study, the team rationally engineered hybrid RiPPs to allow for modifications by enzymes from different pathways on the same core peptide. Initially, they modified the peptide leader sequence so that it was recognized by enzymes from two different pathways and optimized the spacing between the recognition and substrate components. The initial combination of enzymes was designed to install a thiazoline, a heterocyclic modification, with other RiPP modifications such as thioether cross-links to create hybrid peptide structures that have not been reported in natural RiPP products. They tested their system by expressing the precursor peptide and the modifying enzymes in E. coli. The team also designed various core peptides that could be modified by the same combinations of enzymes to produce different products. Finally, they demonstrated that they could also use secondary tailoring enzymes that install bioactive groups such as D-alanine subsequent to the primary RiPP modifications. Their overall strategy produces hybrid RiPPs in moderate yields (1 mg/L of culture) and points to the potential of combinatorial biosynthesis with RiPPs. Sarah A. Webb
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SEEING THROUGH THE ONION TEARS
The flavor of onions is a staple in many types of cuisine, but preparing this root vegetable can leave the one holding the knife with a tear in their eye. The irritation associated with chopping onions is caused by an enzymatic reaction that liberates a volatile compound known as lachrymatory factor (LF), an unusual thioaldehyde S-oxide. The onion protein responsible for LF is lachrymatory factor synthase (LFS), but the mechanism by which this enzyme converts (E)-1- propenesulfenic acid into LF remains unknown. In this issue, Silvaroli et al. (DOI: 10.1021/acschembio.7b00336) propose a catalytic mechanism for LFS based upon high resolution crystal structures of the free enzyme and in complex with a substrate analog. The structures show that the protein adopts a topology resembling the helix-grip fold common in another family of plant enzymes, the START (star-related lipid transfer) proteins. Computational substrate docking combined with biochemical data on the essential catalytic amino acids help to build a mechanistic framework for how LFS converts a sulfenic acid into a thioaldehyde S-oxide. Jason G. Underwood
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CUTTING AT THE CRITICAL CUTICLE
POST-TRANSLATIONAL MODIFICATIONS MEET THE BRAIN
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To expand their utility, the group now reports a simpler, general method for synthesizing these compounds in higher yields (ACS. Cent. Sci. 2017, DOI: 10.1021/acscentsci.7b00247). In this new approach, the team centered their strategy around an easily synthesized bisaryl dibromide intermediate that supplied the backbone of the fluorescent dyes. This compound could easily be converted to a diaryl lithium or diaryl Grignard reagent. These electron-rich nucleophiles react twice with an ester or anhydride electrophile to install the pendant aryl group on the dye. This electronically matched reaction resulted in conversion from dibromide to dye in 40 to 60% yield. This series of reactions produced Si-fluoresceins in four fewer steps and in yields 8 times greater than the previous synthetic route. This new strategy also allowed the team to incorporate more fluorine into the dye molecules, a strategy that allows the researchers to lower the pKa and make the dyes fluorescent under a larger range of pH conditions. The researchers then studied the fluorescent properties of various Si-fluoresceins. They used the same synthetic approach to produce a large panel of Si-rhodamines with similar yields and spectral results. This new approach allowed the synthesis of previously inaccessible Si-rhodamines with fully fluorinated pendant aromatic rings. This substitution pushed the spectral properties farther into the red and allows fine-tuning of a lipid membrane stain. These fluorinated fluorophores could also be conjugated by facile reaction with different thiol substrates, resulting in live cell labels for cell nuclei and antibody labels for immunofluorescence with impressive photostability. Overall, these researchers demonstrate a useful synthetic strategy for producing new and improved dyes for bioimaging. Sarah A. Webb
Parasitic nematodes present a significant global health issue for humans and livestock, so exploring new ways to treat affected populations remains an active pursuit. Previously, researchers explored one promising avenue by drugging an enzyme that humans lack, the UDP-galactopyranose mutase (UGM). This enzyme, which isomerizes galactose to galactofuranose (Galf), is crucial for nematode development. Galf-containing glycans are found in the hard exoskeleton or cuticle of the worm, so depletion of UGM or inhibition of its activity led to delayed development or lethality. While the previously optimized small molecule inhibitors worked well against the enzyme in vitro, they ran into barriers in vivo, probably due to rapid inactivation by endogenous enzymes. Now, Winton et al. (DOI: 10.1021/acschembio.7b00487) return to these UGM-inhibiting small molecules with the goal of protecting their potency through further modifications. One carboxylate on their drug scaffold was thought to be a prime target for drug inactivation, so the researchers improve upon this design by replacing it with a surrogate group, N-acylsulfonamide. Experiments in vitro and in C. elegans indicate that the new analogs inhibit UGM. Treatment of worms elicits developmental and phenotypic characteristics that are similar to UGM null or knockdown worms. If they can also function in parasitic worms, these stabilized drug compounds may represent a promising way to get tough on parasitic nematodes by throwing a wrench in the specialized glycoconjugates found in the cuticle. Jason G. Underwood
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BRIGHTER DYES IN BETTER YIELDS
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DEATH BY LIGHT: LIGHT-ACTIVATED CASPASE TRIGGERS PRECISE NEURON ABLATION
Reprinted with permission from Grimm, J. B., et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.7b00247. Copyright 2017 American Chemical Society.
The ability to engineer dye molecules with specific absorbance and emission properties, boost brightness, and limit photobleaching is critical for a host of molecular and cellular biology experiments. Many chemical dyes are based on classic fluoresceins or the similar rhodamines, polyaromatic xanthene structures with a set of interesting optical and chemical properties. Such dyes can interconvert between a charged fluorescent form and a lactone form, which is colorless and nonfluorescent, based on pH or polarity, which also makes them useful chemical sensors. Recently, Luke Lavis’s group at Janelia Farm reported the synthesis of fluorosceins and related rhodamines where the xanthene oxygen is replaced with silicon. These dyes absorb at longer wavelengths and with improved brightness, which makes them especially useful for bioimaging. But these initial syntheses were difficult and produced low yields of these compounds.
Smart, A.D., et al., Proc. Natl. Acad. Sci., U.S.A., 114, E8174− E8183. Copyright 2017 National Academy of Sciences, U.S.A.
When it comes to deciphering the role of a component in a complex biological system, it is often easiest to figure out how the component functions within the system by seeing what happens when it is removed from the system. At a molecular level, this can 9
DOI: 10.1021/acschembio.8b00008 ACS Chem. Biol. 2018, 13, 5−10
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be achieved by mutating or knocking down the expression of proteins of interest. At the opposite extreme, surgical ablation (i.e., physical removal) can target specific tissues or organs in an organism. At an intermediate level, it becomes a bit more complicated to target individual cell types within a tissue or organ. Recent approaches involve engineering cells to produce reactive oxygen species upon light activation or injecting organisms with viruses to induce selective cell death. However, these techniques are not readily amenable to knocking down cell activity or killing cells at precise time points or locations. These shortcomings have recently been tackled by James A. Wells and co-workers at the University of California, San Francisco, who hypothesized that selective activation of the caspase-3 protease, which is normally activated during apoptosis, would facilitate targeted cell ablation (Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E8174−E8183). To achieve switchable caspase activation, the research team engineered human caspase-3 to include a light-sensitive protein domain derived from oats (LOV2) in its intersubunit linker. In the dark, the LOV2 domain remains rigid, trapping the caspase−LOV hybrid dimer in an inactive state. Upon light exposure, LOV2 binds to its light-harvesting cofactor flavin mononucleotide and becomes flexible, allowing the caspase domain to achieve its active conformation. To demonstrate the utility of their approach, the research team engineered fruit flies to express caspase−LOV in their retinal, sensory, and motor neurons. By carefully controlling the organisms’ light exposure, the research team could knock out neurons at specific developmental stages, revealing differences in the time-course and extent of caspase-activated degeneration among different neuron types. Heidi A. Dahlmann
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PROFILING O-GLCNAC IN IMMUNITY
Activated CD8+ T cells of the immune system must take on two separate roles when fighting an infection, first as effector cells, which tackle the infection today, and second as longer lived memory cells, which establish immunity for the future. Cellular signaling cascades are remodeled during these phenotypic changes via protein phosphorylation. The post-translational modification, β-D-N-acetylglucosamine (O-GlcNAc), is also elevated in activated CD8+ T cells, but its overall role in the immune response is less clear, owing in part to the technical challenge of detecting and quantifying O-GlcNAcylation targets. Now, Aguilar et al. (DOI: 10.1021/acschembio.7b00869) use a chemoenzymatic approach paired with quantitative proteomics to catalog mouse T-cell proteins harboring the O-GlcNAc modification. Their trick is a galactosyltransferase mutant capable of specifically recognizing and modifying O-GlcNAc with an alkynebearing galactosamine for an affinity handle. Both effector and memory T-cells are profiled to generate the most comprehensive list to date of O-GlcNAc modified proteins in the immune response and the putative cellular pathways that are affected. Jason G. Underwood 10
DOI: 10.1021/acschembio.8b00008 ACS Chem. Biol. 2018, 13, 5−10
