Spotlight pubs.acs.org/acschemicalbiology
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HUMAN STRESS HORMONE BINDS TO BACTERIAL SENSOR KINASE, TRIGGERING A DEFENSE RESPONSE
THREE-WAY SCREEN REVEALS CONTRIBUTIONS OF GUT MICROBE METABOLISM TO DRUG EFFICACY
Reprinted from Cell, 169, Scott et al. Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans, 442−456, DOI: 10.1016/j.cell.2017.03.040. Copyright 2017 with permission form Elsevier.
Gut microbes are well-known to influence host metabolism, immunity, and gut function. Researchers attempting to determine why the efficacy of certain anticancer therapies varies widely among patients have now also demonstrated that gut microbe-mediated metabolism affects drug efficacy (Cell 2017, 169, 442−456). The research team, led by Filipe Cabreiro of University College London, developed a drug−microbe−host high-throughput screening method to reveal previously undiscovered mechanisms for fluoropyrimidine cancer drugs. Fluoropyrimidines such as 5-fluorouracil (5-FU) work by inhibiting thymidylate synthase, an enzyme involved in nucleotide biosynthesis, thus disrupting the rampant DNA replication carried out by cancer cells. Although 5-FU is a first-line treatment for colorectal cancer, it is tolerated very differently among patients in a manner that cannot be completely explained by genetics. Therefore, Cabreiro and co-workers decided to explore the potential role of gut microbes in controlling fluoropyrimidine activity. The research team selected the nematode worm C. elegans as a model host organism because, like humans, the worms maintain symbiotic relationships with gut microbes, and they are furthermore sensitive to fluoropyrimidine-mediated impairment of cell division, which causes easily observable readouts of drug activity on nematode populations. To screen the effects of different gut microbes on fluoropyrimidine activity, the research team first inoculated C. elegans with a library of E. coli mutants, then treated the nematodes with varying concentrations of fluoropyrimidine drugs. Using their three-way screening platform, the research team found that gut microbe-mediated metabolic activation of fluoropyrimidines, carried out by microbial ribonucleotide biosynthesis pathways, was critical for obtaining optimum drug efficacy against the host worms. On the basis of these results, the authors suggest that manipulation of intestinal microbiota may be a powerful way to optimize therapeutic outcomes during disease treatment.
Reprinted with permission from Wright, M. H., et al. J. Am. Chem. Soc., 139, 6152−6159. Copyright 2017 American Chemical Society.
Many of us are familiar to some extent with the unhealthy consequences of stress, including sleep disruption, weight loss or gain, and weakened immunity, to name a few. As if these effects were not bad enough, stress has also been linked to activation of virulence in bacteria that commonly colonize human hosts. Researchers led by Sieber and Wright at the Technical University of Munich have now uncovered another facet of stress-induced effects on the microbiome: a molecular mechanism by which bacteria may sense host stress to defend themselves (J. Am. Chem. Soc. 2017, 139, 6152−6159). Humans release peptide hormones called dynorphins in response to stress and pain. Although dynorphins target endogenous k-opioid receptors, they can also activate quorum sensing and trigger virulence in Pseudomonus aeruginosa, the opportunistic pathogen commonly associated with hospitalacquired infections and various sepsis syndromes. Specifically, dynorphin-A (Dyn-A) exposure induces P. aeruginosa production of procyanin, a redox-active toxin that facilitates colonization in vivo (PloS Pathog., 2007, 3, e35). Sieber and co-workers designed a truncated Dyn-A analog (DYN) that retained the ability to stimulate procyanin production in P. aeruginosa. By incorporating into DYN a photoreactive tag, the research team was able to cross-link DYN to bacterial target proteins. The team discovered that DYN bound ParS, a membrane sensor kinase involved in boosting bacterial defenses in response to cationic antimicrobial peptides. Notably, bacteria with mutant ParS could not respond to DYN treatment and were more easily killed by the peptide. The authors comment that the connection between ParS-mediated signaling and procyanin production is unclear, leaving interesting directions to be explored in further studies.
Heidi A. Dahlmann
Heidi A. Dahlmann
Published: May 19, 2017 © 2017 American Chemical Society
1167
DOI: 10.1021/acschembio.7b00379 ACS Chem. Biol. 2017, 12, 1167−1169
ACS Chemical Biology
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MIMICKING ENZYMATIC NUCLEOSIDE PHOSPHORAMIDATION WITH CHIRAL CATALYST
Spotlight
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.
Reprinted from DiRocco, D. A. et al. Science 2017, 356, 426−430. Reprinted with permission from AAAS.
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 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.
A catalytic system for stereoselective phosphoramidation, a historically underdeveloped reaction in the field of organic synthesis, has been reported by a research team led by Daniel A. DiRocco at Merck USA (Science 2017, 356, 426−430). The new method greatly facilitates the stereoselective synthesis of nucleoside phosphoramidate prodrugs for treating viral diseases and cancer. Nucleoside analogs comprise nearly half of antiviral and anticancer drugs, and many of these drugs are delivered as phosphoramidates that undergo phosphorylation at their primary hydroxyl groups in vivo to trigger their biological activity. These “ProTides” contain phosphorus stereocenters which affect the drugs’ potency, metabolic stability, and toxicity. The importance of these features spurred the search for a catalytic method for controlling the stereochemistry of the phosphorus center during phosphoramidate synthesis. Enzymes catalyze stereospecific phosphorus−oxygen bond formation by activating nucleophilic hydroxyl groups with a basic residue, activating leaving groups on the phosphorus center with an acidic residue, and stabilizing transition states with an “oxanion hole”a region of the enzyme active site typically consisting of backbone amides or positively charged residues that interact with the negatively charged intermediates formed during phosphorylation transition states. Inspired by these catalytic principles, the Merck team screened chiral activating agents, which would create “pro-R” or “pro-S” phosphoramidating reagents, along with bases that would prime the nucleosides’ primary hydroxyl groups for nucleophilic attack on the phosphorus electrophile, and they measured the impact of these reagents on the percent yield, chemoselectivity, and stereoselectivity of nucleoside phosphorylation reactions. The best-performing chiral activating agent and chiral base were then tethered to each other with a semirigid linker to form a single multimode catalyst. The optimized catalyst was used to selectively phosphoramidate a variety of pharmaceutically relevant nucleoside analogs in high yield, with diastereomeric ratios as high as 99:1 at the newly formed phosphorus stereocenters.
Jason G. Underwood
Heidi A. Dahlmann 1168
DOI: 10.1021/acschembio.7b00379 ACS Chem. Biol. 2017, 12, 1167−1169
ACS Chemical Biology
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Spotlight
SPECIFIC PHOTO-CROSS-LINKERS FOR PROBING PROTEIN INTERACTIONS
Reprinted with permission from Tian, Y., et al. J. Am. Chem. Soc., DOI: 10.1021/jacs.7b02615. Copyright 2017 American Chemical Society.
Photo-cross-linkers provide a powerful way to probe protein− protein interactions. But so far, genetically encoded photo-crosslinkers have been nonspecific, completing C−H insertion reactions based on proximity alone. But now, researchers have developed a series of photo-cross-linkers that are specific for nucleophiles, and that they can site-specifically incorporate into proteins (Tian, Y. et al. J. Am. Chem. Soc. 2017, DOI: 10.1021/ jacs.7b02615). Such moieties could serve as better tools for unraveling the details of protein-based molecular recognition. Last year, this research group had reported a new photoaffinity label derived from 2-aryl-5-carboxytetrazole (ACT), which crosslinks with nucleophiles and is the same size as benzophenone. In this new paper, the team synthesized a series of five analogous cross-linkers attached to the ε-amino group of lysine. To develop an enzyme that could incorporate these amino acids during translation, the team used mutagenesis and several rounds of selection to modify and optimize a pyrrolysyl-tRNA synthetase that could incorporate these residues into proteins at an amber codon site. The researchers initially tested the system in bacteria and expressed a modified glutathione-S-transferase protein in E. coli that swapped Glu-52 with the amino acid cross-linker. These experiments demonstrated that the cross-linker reacted with the closest nucleophile in the dimer, Glu-92. They then examined incorporation in mammalian HEK293T cells. They successfully incorporated the modified amino acid into the SH2 domain of Grb2, which interacts with EGFR as part of the RAS signaling pathway. They removed the phosphotyrosine moiety from EGFR and demonstrated that the photo-cross-linker Grb2-EGFR complexes were attached via the photo-cross-linker. In addition, the capture reaction occurred at a rate that matched the stimulation of the EGFR pathway in these cells. Comparisons of cross-linking efficiency of GST in bacteria showed that one of the ACTK analogs, mPyTK, showed much greater efficiency than its nonspecific counterpart AbK. Overall, these moieties could serve as a powerful chemical tool and one that could simplify the tandem mass spectrometry analysis of protein−protein interactions. Sarah A. Webb 1169
DOI: 10.1021/acschembio.7b00379 ACS Chem. Biol. 2017, 12, 1167−1169
