Spotlight pubs.acs.org/acschemicalbiology
■
BEST OF SPOTLIGHTS IN 2016 Our editors have selected their favorite articles published in ACS Chemical Biology and elsewhere in 2016. We hope you enjoy these selections. We would like to wish all our readers a Happy New Year!
■
mycoides genome (JCVI-syn1.0) into bacterial cells (Science 2010, 329, 52).
INTERACTIONS FIT FOR NANOBIT
Monitoring protein−protein interactions in vivo can be difficult, but split reporters offer one tool to assess such interactions. Several split reporter strategies take an enzyme or fluorescent protein, expressed in two polypeptide chains that together reconstitute their activity. A relatively low affinity between the complementary reporter parts makes reconstituting the activity dependent upon the interaction of the test proteins fused to the two halves. Among the well-behaved split reporter strategies is luciferase, but here, Dixon et al. (ACS Chemical Biology, DOI: 10.1021/ acschembio.5b00753) take this one step further by introducing NanoBiT featuring the smaller NanoLuc enzyme enabled for complementation-dependent activity. They demonstrate that removing just 13 amino acids from the C-terminus imparts the desired two-piece enzymatic activity and then goes on to optimize both pieces to make a useful, quantitative reporter. Their final peptide is just 11 amino acids, and the study explores the split strategy for probing a variety of biological interactions.
Inspired by the question of how few genes an organism would actually need to survive given a complete nutrient supply and an absence of selective pressure, Hutchison and co-workers at JCVI have recently developed a functioning minimal bacterial genome (Science 2016, 351, aad6253). Their initial attempt to minimize JCVI-syn1.0 by deleting all genes that were putatively nonessential failed, in part because removing genes of proteins with redundant function left the transformed cells without any protein to carry out the necessary function. However, by subjecting JCVI-syn1.0 to multiple rounds of mutagenesis and screening the mutated genome portionwise to determine which genes were essential, nonessential, or simply necessary for robust growth, the team produced JCVI-syn3.0, a synthetic bacterium containing only 473 genes. Most of the retained genes were known to sustain cytosolic metabolism and cell membrane integrity or enable gene preservation or expression, but 79 of the genes could not be placed into a functional category, which opens the door for further genome optimization as the roles of these mystery genes are elucidated. Heidi A. Dahlmann
Jason G. Underwood
■
HYPOTHETICAL MINIMUM GENOME REACHES NEW LOW Life can be complicated, even for the simplest of organisms, bacteria. Depending on their habitat, bacteria may need to scavenge for nutrients, synthesize their own amino acids, and produce antimicrobial defense molecules, in addition to maintaining cell growth and replication. In the absence of complicated demands, however, bacteria trim down their genomes in a “use it or lose it” manner. This has enabled parasitic species that colonize relatively stable niche environments to survive and thrive with astonishingly few genes; for example, Mycoplasma genitalium gets by with only 525 genes, the smallest known genome. The brevity of such genomes has made them a feasible target for synthesis in the laboratory; in 2010, researchers at the J. Craig Venter Institute (JCVI) chemically synthesized and installed a version of the Mycoplasma © 2017 American Chemical Society
■
TROLLING FOR ANTIBIOTIC TARGETS
Published: January 20, 2017 7
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
ACS Chemical Biology
Spotlight
Mindful of these limitations as well as motivated by the ominous increase in antibiotic-resistant pathogenic bacteria, a research team led by Andrew G. Myers of Harvard University has developed a new platform for discovering novel macrolide antibiotics (Nature 2016, 533, 338−345). Macrolides, a class of molecules including erythromycin, are macrocyclic lactones typically consisting of 14- to 16-membered rings. The research team, including first authors Ian B. Seiple and Ziyang Zhang, focused on linking together readily available building blocks to form molecular fragments, which were then convergently assembled into novel macrolide structures. The piece-wise approach allowed the researchers to append various side chains and introduce heteroatom substitutions in a site-specific manner, ultimately resulting in the gram-scale synthesis of over 300 14- and 15-membered ring antibiotic candidates. Several of these macrolides were found to be potent against multiple pathogenic bacteria, including methicillin-resistant Staphylococcusaureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The authors anticipate that their convergent-synthesis platform can be applied to the development of libraries of other classes of antibiotics.
Pathogenic bacterial strains pose a significant public health threat when they are resistant to conventional antibiotics. To uncover new antimicrobials, researchers can go fishing with clever screens of natural product or small molecule libraries, but bridging growth inhibition hits into drugs in the clinic is especially difficult. Identifying the mechanism of action (MOA) for a putative hit is a helpful step, guiding chemical modifications and structure−function studies, but pinning down the molecular target is also a tough task. Here, Lamsa et al. (DOI: 10.1021/acschembio.5b01050) demonstrate a useful system to uncover an antimicrobial compound’s MOA. Their method is based on a prior observation that antibiotic-treated microbes have different cytological characteristics under a fluorescent microscope depending on the target of the antibiotic. Inspired by this observation, the researchers engineer B. subtilis strains harboring inducible destruction constructs aimed at several key cellular pathways including DNA replication and fatty acid biosynthesis. They show that antibiotics of a known cellular target display similar cytological characteristics when compared to engineered strains that destroy that same cellular pathway. They go on to use their system, which they dub rapid inhibition profiling or RIP, to probe the antimicrobial mechanism for nonsteroidal anti-inflammatory drugs.
Heidi A. Dahlmann
■
Jason G. Underwood
■
SYNTHESIS PLATFORM ENABLES DISCOVERY OF MACROLIDE ANTIBIOTICS
FISHING FOR THE NOISE
It is often a daunting task to search for the intracellular target of a small molecule or the binding partner of a protein of interest, but photoaffinity techniques can provide helpful clues. Labeling a molecule of interest with a photoactivatable cross-linker enables a fishing expedition for specific binding partners. Unfortunately, as with other affinity methods, the analysis is complicated by the nonspecific interactions from sticky binding partners. Park et al. (DOI: 10.1021/acschembio.5b00671) take on this complication in a study designed to ask the question, which proteins are nonspecific background in a photoaffinity experiment? In parallel, they study three structurally dissimilar, commonly used photoactivatable linkers. They generate the binding protein inventories for several different mammalian cell lines with each of these linkers and learn along the way that each linker structure has its own set of partners. Their lists are of great utility to the scientific community, as are the useful lessons for improving future fishing expeditions.
Since its discovery in 1949, erythromycin has become a workhorse antibiotic for treating respiratory and skin infections caused by Streptococcus and Staphylococcus bacteria, among others. Because erythromycin is unstable in the digestive tract, limiting its application in oral treatments, pharmaceutical companies developed more effective erythromycin derivatives. These derivatives are generally synthesized by modifying erthryomycin obtained by fermentation, a process which produces the parent compound on a ton scale. Despite the availability of the starting material, erythromycin derivatives can be very challenging to make; for example, the synthesis of solithromycin, an advanced clinical candidate effective against a broad range of Grampositive bacteria, requires 16 consecutive chemical transformations beginning with erythromycin. Morever, development of further erythromycin derivatives is confined to manipulations of the reactive functional groups present in the parent molecule, which can be difficult to target selectively.
Jason G. Underwood 8
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
ACS Chemical Biology
■
■
GENETICALLY RECODED ORGANISM UNDER CONSTRUCTION
Spotlight
CROSS-LINKING THE PATH TO STABILITY
With type 2 diabetes on the rise, potential therapies to treat the epidemic will be of increasing interest to clinicians. One hormone therapy that shows promise is the peptide oxyntomodulin, or OXM, which binds both the glucagon receptor, GCGR, and the glucagon-like peptide receptor, GLP1-R. Unfortunately, applying OXM as a therapeutic has been hampered by its short half-life in the body. Here, Muppidi et al. (DOI: 10.1021/acschembio.5b00787) apply a cross-linking approach that vastly improves the half-life of OXM while maintaining a reasonable affinity for the two receptors of interest. They begin by installing pairs of cysteine residues into OXM one α-helical turn away from one another, guiding the locations by the crystal structure of glucagon and a structural model of OXM bound to GLP1-R. After establishing a new OXM peptide that tolerates the cysteine substitutions, a variety of bifunctional cross-linkers are tested to connect these residues. The result is a class of conformationally constrained OXM analogs displaying increased stability in mice, yet still displaying subnanomolar affinities to both peptide receptors.
Preliminary results of an attempt to recode an organism’s entire genome are in, as reported by Church and co-workers at Harvard University (Science 2016, 353, 819). The research team hopes to produce a genomically recoded organism with synthetic biological features useful for industrial applications. In order for an organism to grow and survive, its genomic information is transcribed from DNA into RNA, which in turn is translated into proteins. During translation, three-nucleotide segments of the RNA strand, or codons, are matched to complementary tRNA molecules charged with specific amino acids. Among the 64 possible codons, many codons are redundant, meaning that multiple codons exist for specifying the same amino acid or translation termination signal. Church and co-workers used a computer program to scan a simplified E. coli genome for all instances of seven codons with redundant functions and design a genome in which each of these codons was replaced with a synonymous alternative, reducing the number of codons to 57. To test whether the recoded genome would be viable, the research team synthesized it in 87 fragments that were incorporated individually into E. coli strains in which the corresponding segments of genomic DNA were removed. At the time of their report, Church and co-workers had checked 55 segments of recoded genome; so far, 99.5% of all genes and greater than 90% of essential recoded genes supported cell viability, and only 13 lethal recoding modifications were identified. They also developed a pipeline to efficiently identify and overcome these design flaws and demonstrated it on one example. Encouraged by these results, the authors anticipate assembling recoded organisms suitable for biotech applications; the organisms would resist viral infection and horizontal gene transfer and could possibly be engineered so that the seven stripped-out codons could be reassigned to non-natural amino acids.
Jason G. Underwood
■
SYSTEMIC DELIVERY MEETS LOCAL THERAPY
Targeting a drug to a specific tissue in the body is a difficult challenge, but overcoming this challenge can present key advantages in the clinic by reducing the overall systemic dose of a drug. This can reduce potential toxicity or side effects. Antibodies can operate in a targeting capacity, carrying a drug or radioisotope to sites of interest, but this depends upon both a specific antigen and adequate density to achieve therapeutic dosing. Oneto et al. (ACS Cent. Sci. 2016, DOI: 10.1021/ acscentsci.6b00150) recently unveiled a bioorthogonal chemistry approach to concentrate a systemically delivered prodrug to a site of interest before it transforms to a bioactive drug. Their system employed a hydrogel injected at a tumor site and harboring tetrazine moieties which constitute half of the inverse-electron
Heidi A. Dahlmann 9
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
ACS Chemical Biology
Spotlight
co-workers at multiple research institutes in Japan sought to discover microbes’ bacterial species that would be up to the task (Science 2016, 351, 1196). The research team screened 250 PET debris-contaminated samples collected from sediment, soil, wastewater, and sludge from PET-recycling site to see if any samples could degrade low-crystallinity PET film; they found one microbial consortium that formed on the PET film that degraded the PET into carbon dioxide and water. From this consortium, a previously uncharacterized species of genus Ideonella that appeared to adhere to and damage the surface of the PET film substrate was isolated. The bacterium, which the research team proposed to name Ideonella sakaiensis, was found to nearly completely degrade the film after 6 weeks at 30 °C. Upon sequencing the bacterium’s genome and transcriptome, the researchers identified two enzymes, PET hydrolase (PETase) and MHETase, which were determined to catalyze sequential hydrolysis steps that break down PET into simpler substrates. These substrates can be further enzymatically converted to environmentally benign monomers that serve as a carbon source for growing I. sakaiensis. The authors speculate that the PET hydrolase may have evolved from a hydrolytic enzyme that breaks down cutin, a form of natural polyester produced in plants.
demand Diels−Alder (IEDDA) reaction. The other half was provided on a prodrug which carries a trans-cyclooctene group. After bioconjugation in vivo, active drug is released through an isomerization event. The IEDDA reaction is advantageous since naturally occurring functional groups are not reactive with either half of the reaction. To test the approach in a therapeutic context, the researchers turned to a mouse xenograft of human soft tissue sarcoma. They synthesized a prodrug version of doxorubicin, an inhibitor of topoisomerase, and reacted it with a hydrogel injected adjacent to the xenograft. The results indicated that the in vivo localized doxorubicin was far more effective than the control, a standard systemic doxorubicin therapy. The median tumor volume was far lower, often undetectable, in the prodrug cohort, and the mice maintained a normal reticulocyte count, a proxy for bone marrow function. These promising data indicate an exciting direction for targeted chemotherapies if a viable prodrug can be identified for the disease of interest. Jason G. Underwood
■
IMPROVING A D-GRADE DRUG
Heidi A. Dahlmann
■ Synthetic polypeptide drugs represent a promising avenue for future human therapies, but this class of drugs encounters several fundamental challenges in the body. For example, many peptides are rendered inactive or completely degraded by in vivo proteases. Additionally, synthetic peptides can provoke an immune response, often targeting the therapeutic for destruction. To evade these obstacles, researchers often turn to alternative backbones or use of d-amino acids instead of the l-amino acids found in natural proteins. Here, Uppalapati et al. (DOI: 10.1021/acschembio.5b01006) use an l to d chirality swap to develop a stable antagonist of the vascular endothelial growth factor A (VEGF-A). In a prior study, the group identified a D-protein with high affinity for VEGF-A, but it showed poor stability. In the present study, they optimize their lead D-protein to have high thermal stability and increased affinity. Characterizing the resulting D-protein shows that it is stable in plasma and does not provoke an immune response.
RIBOSENSING WITH PKR
The innate immune system provides a first line of defense against infection. In humans, an important player in innate immunity is the RNA-activated protein kinase, PKR, which surveys for structured viral or bacterial RNAs and, when activated, can shut down translation. A variety of viral and largely double-stranded RNA substrates have been tested for activation of PKR, and it does not appear to show strong primary sequence specificity. Here, Hull et al. (DOI: 10.1021/acschembio.6b00081) test three small functional RNAs from bacteria for their capacity to activate human PKR, demonstrating that even small RNAs with tertiary structure can bind and turn on its kinase activity. Their tests on natural riboswitches and ribozymes reveal PKR activation at levels similar to a long dsRNA. Footprinting experiments investigate the RNA−protein interactions and show that the double-stranded regions of these natural RNAs are protected when PKR binds. In light of their results, the researchers postulate on how the innate immune system might sort out self from nonself RNAs in vivo.
Jason G. Underwood
■
PET-DEGRADING BACTERIUM IDENTIFIED Polyethylene terephthalate (PET), prized for its durability and flexibility, has become ubiquitous worldwide in applications ranging from packaging to polyester clothing. Although PET is in principle recyclable, in practice most PET disposables end up discarded, and the chemical properties that make PET an ideal material for fabrication become liabilities in landfills or other sites of accumulation in the environment, such as waterways. Recognizing that microbes might be able to assist in breaking down PET into reusable substituents, thus reducing the need for further petroleum-based production of its monomers and serving as a potential environmental remediation strategy, Yoshida and
Jason G. Underwood 10
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
ACS Chemical Biology
■
Spotlight
CELLS LINE UP FOR WEIGH-IN
Wang et al. (DOI: 10.1021/acschembio.6b00243) extend on these observations with a chemical biology approach aimed at identifying which particular acetyl-lysines are Sirt6 substrates. They begin by site-specifically installing lysines carrying an alkene group into recombinant histone H3. This affords a handle for dye labeling, but the handle disappears when Sirt6 deacetylates that particular residue. Their in vitro studies in nucleosomes and followup work in cells extend the known repertoire of lysine substrates. Jason G. Underwood
■
In the quest for developing new antibacterial or anticancer compounds, researchers benchmark their progress by determining the effects of their drug candidates on a general population of cellsfor example, by calculating the concentration of drug that kills 50% of cells. Population studies like these do not take into account the effect of rogue cells such as rapidly proliferating cancer cells or slowly growing bacterial cells, both of which may contribute to the development of drug resistance in a patient. Thus, technologies that allow researchers to quickly and comprehensively track the growth of each cell in a sample would help them to predict the effectiveness of pharmacological treatment. A new device enabling the high-throughput measurement of single-cell growth rates has been developed by a team of researchers led by Scott R. Manalis of MIT (Nature Biotechnol. 2016, 34, 1052−1059). The device contains a narrow channel that sends cells through a serial array of oscillating cantilever sensors, the resonance frequencies of which change in response to the buoyant mass of each cell passing through one at a time, with the cantilevers spaced far enough apart that the cells have time to grow or shrink in response to stimuli in the flow media. Knowing the exact time cells take to get from one cantilever to the next, the research team could track the mass of the exact same cell throughout its passage through the device, with masses being measured so precisely that it was possible to extrapolate the growth rates of each individual cell in a sample in less than half an hour. Using their device, Manalis and co-workers followed the growth rate of various eukaryotic and bacterial cell lines, identifying subpopulations of cells with unusual growth kinetics that could possibly have clinical significance.
The three-dimensional structures of biological macromolecules have been determined for decades by X-ray crystallography. In this technique, a crystalline sample is bombarded with a beam of X-rays; by analyzing the angle and intensity of the diffracted beams, researchers can construct a map of the electron density in the sample. The better the resolution of the map, the higher the certainty with which the precise locations of individual atoms as well as the nature of their covalent bonds can be determined within the macromolecule. X-ray crystallography is not well-suited for studying proteins that resist crystallization, such as those that are normally found embedded in cell membranes or that are highly dynamic. These limitations have driven the development of cryo-electron microscopy (cryo-EM), in which samples of biomacromolecular complexes are flash-frozen into a thin layer of vitreous (liquidlike) ice. In a manner roughly analgous to light microscopy, the frozen samples are exposed to a beam of electrons, generating two-dimensional projection images of thousands of identical copies of the molecular complex. These images are then computationally combined to create 3D structures. The resolution obtainable by cryo-EM has long lagged behind that of X-ray crystallography, and its application has generally been limited to relatively large (>200 kDa) proteins. However, a team of researchers led by Subramaniam have recently shattered these paradigms, reporting the 1.8-Å-resolution cryoEM structure of glutamate dehydrogenase (GDH), the first cryoEM structure with sub-2-Å resolution, as well as the cryo-EM structure of the 93 kDa isocitrate dehydrogenase (IDH), the first cryo-EM structure of a sub-100 kDa protein (Cell 2016, DOI: 10.1016/j.cell.2016.05.040). The research team attributes their barrier-breaking successes to judicious sample selection, improved detector performance, and correction for beam-induced specimen movement. They anticipate that further hardware and software developments will enable routine cryo-EM on metabolic proteins, many of which are below 150 kDa.
Heidi A. Dahlmann
■
CRYO-EM: IMAGING SMALLER PROTEINS AT HIGHER RESOLUTION
SEARCHING OUT SIRT6 SUBSTRATES
Mammalian sirtuins, or Sirts, are a family of deacetylases responsible for removing lysine acetyl marks on histones. While some Sirts have deacetylase activity toward most histone lysines or even acetyl-peptides, Sirt6 acts only on specific lysines. This observation has been difficult to study in depth because in vivo observations paint a picture of high activity on H3K9 and H3K56, whereas in vitro studies show low activity on purified proteins or peptides. Recent studies indicated that Sirt6 deacetylase activity is improved when the substrate histones are packaged with DNA into nucleosomes.
Heidi A. Dahlmann 11
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
ACS Chemical Biology
■
Spotlight
SMALL MOLECULE TAKES ON MYC
MYC is a transcription factor that regulates the cell cycle and apoptosis, so amplification or mutation can lead to oncogenic transformation. Dysregulation is observed in a variety of cancer types, so a MYC remains an important pharmacological target. Here, Felsenstein et al. (DOI: 10.1021/acschembio.5b00577) take aim at the MYC promoter as a drug target in a new small molecule screen. They design an array displaying 20 000 compounds and then screen for binding of a fluorescent MYC promoter DNA containing the signature G-quadruplex element. A candidate small molecule is identified, and biophysical methods are used to confirm specific binding to the MYC element with low micromolar affinity. Interestingly, the compound does not bind to several other well-known G-guadruplex structures. Studies in cancer cell lines demonstrate a G1 cell cycle arrest in MYC-driven lines harboring the G-guadruplex in the promoter. Gene expression profiling investigates the selectivity of the identified compound, which could inspire new drug candidates based around its benzofuran-containing structure. Jason G. Underwood
12
DOI: 10.1021/acschembio.6b01156 ACS Chem. Biol. 2017, 12, 7−12
