Spotlight pubs.acs.org/acschemicalbiology
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HALTING TUMOR GROWTH BY INHIBITING RAL
ZMAPP: A CURE FOR EBOLA?
Adapted by permission from Macmillan Publishers Ltd: Nature (doi: 10.1038/nature13713), copyright 2014.
The Ras protein is a small GTPase that plays a key role in cell signaling pathways controlling the expression of genes involved in cell growth, differentiation, and survival. Greater than onethird of human tumors contain mutations that activate Ras; thus, inhibitors of Ras activity have been hotly pursued as anticancer therapeutics. However, these efforts have been largely frustrated because small molecules that target the active site of the protein need to compete with its natural ligands, GDP and GTP, which are present in very high (e.g., millimolar) concentrations in cells. In a recently reported study, Dan Theodorescu and coworkers decided to circumvent Ras and instead focus on two of its downstream signaling components, the Ras-like small GTPases RalA and RalB ((2014) Nature (doi: 10.1038/nature13713)). The Ral proteins cycle between inactive (GDP bound) and active forms (GTP bound); active Ral binds to effector proteins, including Ral binding protein 1 (RALBP1), that control regulation of cell adhesion, membrane trafficking, mitochondrial fusion, and transcription. Theodorescu et al. hypothesized that molecules that selectively bind to Ral−GDP might restrict Ral to an inactive state in the cell. Comparison of the available three-dimensional structures of RalA revealed a potentially targetable region adjacent to, but distinct from, the guanine nucleotide binding pocket. Using a structure-based virtual screening approach, they assessed 500 000 small molecules for their ability to bind to this site. Of these compounds, 88 were tested in vitro for their ability to inhibit the binding of RalA-GTP to RALBP1; ultimately, dihydropyranopyrazoles RBC8 and BQU57 were found to significantly block RalA activity as well as bind to RalB (as confirmed by NMR studies). Not only did these two compounds inhibit the anchorage-independent growth of Raldependent lung cancer cell lines in vitro, but they also inhibited the growth of tumor xenografts in mice. The authors note that these first-generation Ral inhibitors have the potential to be further optimized both as leads for drug development and as tools for studying Ral signaling. Heidi A. Dahlmann, Ph.D. © 2014 American Chemical Society
Adapted by permission from Macmillan Publishers Ltd: Nature, advance online publication, 29 August 2014, doi: 10.1038/ nature13777
The outbreak of Ebola that began in the west African country of Guinea in spring 2014 became a major international concern as cases emerged in neighboring countries and among visiting international rescue workers. Ebola virus (EBOV) infections, upon 3−21 days incubation in humans, lead to flu-like symptoms that progress to potentially fatal hemorrhage and multiple organ failure. The current outbreak is menacing in terms of geographical spread, number of cases, and number of fatalites; the particular EBOV strain responsible for the outbreak kills more than half of the people it infects. Unfortunately, there are no licensed vaccines or therapeutics available for managing Ebola, limiting medical care to nonspecific treatment of symptoms as they arise. In August, it was reported that collaboration between academic and industrial researchers from Canadian, Chinese, and American institutions yielded a potential approach for treating Ebola up to 5 days postinfection (2014, Nature, 10.1038/nature13777). The research team explored the optimization of two monoclonal antibody (mAb) cocktails, MB-003 and ZMab, which were previously shown to be effective against EBOV up to 3 days postinfection in nonhuman primates (NHPs). After identifying the c13C6 antibody as the most protective component of MB-003 in studies with EBOV-infected guinea pigs, the team screened the effectiveness of c13C6 coadministered with various combinations of antibodies present in ZMab. The best overall combination of antibodies (called ZMapp) was then tested in EBOV-infected NHPs. Rheses monkeys injected with fatal doses of EBOV began to display symptoms (including fever and leukocytosis) 3 days after infection; a control group of untreated monkeys all died by the eighth day postinfection. However, monkeys treated with ZMapp as late as 5 days postinfection experienced 100% survival and eventual Published: October 17, 2014 2177
dx.doi.org/10.1021/cb500797n | ACS Chem. Biol. 2014, 9, 2177−2179
ACS Chemical Biology
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recovery. The authors hope that these positive results expedite safety testing in humans and facilitate the compassionate use of ZMapp for managing the current Ebola outbreak. Heidi A. Dahlmann, Ph.D.
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Spotlight
BUILDING A BETTER VANCOMYCIN
BREATH TEST CONFIRMS TB DRUG METABOLISM
Reprinted with permission from Okano, et al., J. Am. Chem. Soc. 136, 13522−13525, DOI: 10.1021/ja507009a. Copyright 2014 American Chemical Society Adapted by permission from Macmillan Publishers Ltd: Nature Communications, Choi et al., 5, 4989, copyright 2014
Though physicians have used vancomycin as a last resort antibiotic against resistance strains for decades, vancomycinresistant strains of enterococci and Staphylococcus aureus have developed. Now Okano et al. report the synthesis of new vancomycin derivatives that are effective against these strains and have a new additional mechanism of activity that may thwart the development of new resistant strains (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja507009a). Vancomycin kills bacteria by interfering with peptidoglycan synthesis that forms the cell wall. A critical carbonyl group in the structure binds to D-Ala-D-Ala residues, and resistant strains have developed that substitute D-Ala-D-Lac for D-Ala-D-Ala. To combat this strategy, chemists in Dale Boger’s laboratory had previously synthesized modified vancomycin analogs that changed a carbonyl group to a thiocarbonyl or amidine, but lacked the disaccharide, to see if they could restore activity. In this study, Okano et al. synthesized full vancomycin analog structures with these modifications and an additional set that also appended a hydrophobic (4-chlorobiphenyl)methyl (CBP) group. The carbohydrates were added using enzymatic reactions, and all the syntheses were carried out without protecting groups. Many of these analog compounds showed potent activity against antibiotic-resistant and vancomycin-resistant bacteria. From the previous study, the researchers had learned that the thiocarbonyl analog without the carbohydrate did not have antimicrobial activity and could not bind to either the D-Ala-DAla or altered D-Ala-D-Lac target. Adding the carbohydrate portion did not restore activity, but adding the CBP group did, which suggested that this compound acted by a different mechanism. Converting the carbonyl to an amidine group alone within the vancomycin restored activity against vancomycinresistant organisms, and adding the CBP group led to a further boost in activity and also provided an antimicrobial compound that was 10 times more effective against vancomycin-resistant strains than vancomycin-sensitive ones. This study provides useful insights for the modification of other complex antimicrobial natural products. In addition, the CBPvancomycin-amidine is most likely acting through two separate mechanisms, which could delay or prevent the development of resistance to this modified antibiotic. Sarah A. Webb, Ph.D.
Mycobacterium tuberculosis is a globally ubiquitous pathogen responsible for causing the often fatal lung disease tuberculosis (TB). Selection of the proper antibiotic to treat M. tuberculosis infection is very important, because many strains have acquired mutations in enzymes required for metabolic activation of antibacterial prodrugs. Unfortunatley, quick selection of an appropriate antibiotic is difficult because the two most common methods for characterizing M. tuberculosis infections, namely in vitro culture or DNA amplification, are respectively very time-consuming or not [always] informative for determining drug sensitivity. The antibacterial prodrug isoniazid (INH) is metabolically activated by the mycobacterial enzyme KatG to form a hydrazyl radical, which subsequently undergoes beta scission to an acyl radical and a diazene side product. The acyl radical covalently binds NAD+, creating an adduct known to inhibit the biosynthesis of mycolic acid, a major component of the mycobacterial cell wall. It is not clear whether diazene, which could be converted to N2 through oxidation by cellular alkenes or through bimolecular disproportionation, plays a role in INHmediated antibacterial activity. However, the diazene decomposition product N2 has recently been shown to be a valuable marker for detecting INH metabolism in vitro and in vivo ((2014) Nature Communications 5, 4989). The team of researchers led by Graham S. Timmins exposed a known INH-sensitive M. tuberculosisstrain to 15N-labeled INH and used isotope ratio mass spectrometry (IRMS) to quantify the metabolic byproduct 15N2 in the headspace of the culture tubes. In contrast, no enrichment of 5N2 was observed in cultures of bacterial species lacking KatG. Furthermore, the team demonstrated that 15N2 production could be detected in the breath of M. tuberculosis-infected rabbits as soon as 5 min after inhalation of a dose of 15N-labeled INH, whereas no 15N2 enrichment occurred in INH-treated noninfected control rabbits. The authors anticipate that their IRMS-based method could facilitate rapid, point-of-care diagnosis of INH-sensitive TB, and they plan to expand the approach to other drugs. Heidi A. Dahlmann, Ph.D. 2178
dx.doi.org/10.1021/cb500797n | ACS Chem. Biol. 2014, 9, 2177−2179
ACS Chemical Biology
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Spotlight
LOOKING UNDER THE HOOD OF THE MICROBIOME
Reprinted from Cell, 158, Donia et al., A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics, 1402−1414. Copyright 2014, DOI: 10.1016/j.cell.2014.08.032 with permission from Elsevier, Inc.
It has long been known that the human body plays host to legions of bacterial species, but with the introduction of highthroughput DNA sequencing, researchers are teasing apart new details about our microbiome at an impressive rate. Efforts from the Human Microbiome Project and many others have uncovered the landscape of microbes populating various body regions in healthy people. Emerging studies indicate that environmental factors such as diet can have rapid and profound effects in remodeling the composition of the microbiome. In turn, this species composition can influence particular human health conditions. Now, Donia et al. (DOI: 10.1016/ j.cell.2014.08.032) sift through the gigabases of microbiome data with an innovative systematic approach that asks what biosynthetic gene clusters, or BGCs, are expressed and whether the resulting small molecules might have an effect on the microbial community at large. Using the ClusterFinder algorithm on metagenomic data, the group detected 3118 BGCs that are present in the microbiomes of healthy individuals. The mouth and gut displayed the largest number of BGCs with the saccharide class being the most abundant. They observed widespread BGCs corresponding to peptides synthesized by the ribosome and displaying posttranslational modifications. Among these were thiopeptide BGCs in isolates of every human body site assayed. They went on to focus on these since thiopeptides show potent antibiotic activity against Gram-positive bacteria. Using a large-scale culture of a vaginal isolate, the thiopeptide was purified for structure determination. They name the compound lactocillin and demonstrate that it has strong activity against known pathogens. This study illustrates that we have just begun to scratch the surface of knowledge on the microbiome and that the species in and on our bodies are capable of producing small molecules that alter their microbiome neighbors and the human host. Jason G. Underwood, Ph.D.
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dx.doi.org/10.1021/cb500797n | ACS Chem. Biol. 2014, 9, 2177−2179
