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A New Nucleoside Antibiotic Chokes Bacterial RNA Polymerase Eric P. Trautman†,‡ and Jason M. Crawford*,†,‡,§ †
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States § Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, United States ‡
he recent discovery of the first nucleoside analogue inhibitor of bacterial RNA polymerase (RNAP) introduces a new weapon in the arms race against drug-resistant bacteria.1 The spread of multidrug resistance among bacteria has outpaced the introduction of new antibiotics into the clinic. Previous last-line-of-defense therapies, such as vancomycin, are losing efficacy as the prevalence of drug-resistant isolates increases with antibiotic use. As a result, the number of deaths caused by bacteria is on the rise, with at least 23000 people dying from drug-resistant infections each year in the United States alone. While antibiotic resistance has been rising, research into new antibiotics has slowed for a multitude of reasons. Because of the impending heath crisis, the discovery of new antibiotics, particularly those with novel mechanisms of action, is key to combating drug-resistant infections. Maffioli et al. describe the characterization of pseudouridimycin, the first nucleoside analogue antibiotic that targets bacterial RNAP in Gram-positive and Gram-negative organisms.1 The antibiotic was identified from two Streptomyces producers included within a 3000-member actinobacterial extract library. This library was examined for in vitro bacterial and viral RNAP inhibitory activities with a focus on discovering selective bacterial inhibitors. While other antibiotics, such as rifampin, target bacterial RNAP, this molecule targets the active site and directly competes with nucleotide substrates, as assessed by cocrystal structures of the molecule and RNAP, examination of resistance mutations, and interpretation of the results from a series of associated in vitro biochemical assays. The authors show, as a result of the differential mechanisms of action, that antibiotic-resistant bacteria, such as strains of methicillinresistant Staphylococcus aureus (MRSA), are susceptible to this new antibiotic and the rate of spontaneous development of resistance to pseudouridimycin is an order of magnitude lower than that of rifampin under the conditions of their experiments. This lower mutation rate is likely due to the similarity between the substrate and inhibitor, which reduces the number of possible mutations that could retain function while attenuating the affinity of the protein for the inhibitor. Additionally, as the target is a central enzyme, pseudouridimycin is expected to have broad-spectrum efficacy. Pseudouridimycin shows promise as a potential small molecule drug lead. Nucleoside analogue inhibitors have been recognized as excellent drug candidates and have found enormous success in the treatment of viral diseases such as hepatitis and HIV. Harvoni, a cure for hepatitis C, was the second highest grossing drug in 2016 and is a combination therapy of two drugs, one being a nucleoside analogue. The first HIV treatment to be introduced was also a nucleoside analogue, azidothymidine, and nucleoside analogues are still a major component of
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© XXXX American Chemical Society
combination therapy in treating HIV to this day. Mechanistically, these successful molecules inhibit viral polymerases or reverse transcriptases, while having a lower level of off-target inhibition of human RNAPs. Similarly, pseudouridimycin inhibits bacterial RNAP while having a lower potency against human RNAP. Maffioli et al.1 show that in in vitro assays, the potency of pseudouridimycin is approximately 6−60 times higher for bacterial cell lines than for human cells. Mouse model experiments extend these promising results one step further, showing that pseudouridimycin can successfully treat a Streptococcus pyogenes infection. While the biosynthetic pathway for pseudouridimycin has yet to be reported, its structure suggests pathways similar or analogous to those of established natural products, as outlined in Figure 1. However, until the gene cluster is reported for this pathway, the identities of the possible substrates and enzymes
Figure 1. Possible proposed outline for pseudouridimycin biosynthesis, which awaits further experimental investigation. Received: July 18, 2017
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DOI: 10.1021/acs.biochem.7b00680 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Chiriac, A. I., Facchetti, G., Kaltofen, P., Sahl, H. G., Deho, G., Donadio, S., and Ebright, R. H. (2017) Antibacterial NucleosideAnalog Inhibitor of Bacterial RNA Polymerase. Cell 169, 1240−1248. (2) Mihali, T. K., Kellmann, R., Muenchhoff, J., Barrow, K. D., and Neilan, B. A. (2008) Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis. Appl. Environ. Microbiol. 74, 716−722. (3) Miranda-CasoLuengo, R., Coulson, G. B., Miranda-CasoLuengo, A., Vazquez-Boland, J. A., Hondalus, M. K., and Meijer, W. G. (2012) The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi. Infect. Immun. 80, 4106−4114. (4) Walsh, C. T., and Zhang, W. J. (2011) Chemical Logic and Enzymatic Machinery for Biological Assembly of Peptidyl Nucleoside Antibiotics. ACS Chem. Biol. 6, 1000−1007. (5) Yang, Z. Y., Chi, X. L., Funabashi, M., Baba, S., Nonaka, K., Pahari, P., Unrine, J., Jacobsen, J. M., Elliott, G. I., Rohr, J., and Van Lanen, S. G. (2011) Characterization of LipL as a Non-heme, Fe(II)Dependent Alpha-Ketoglutarate:UMP Dioxygenase that Generates Uridine-5 ′-aldehyde during A-90289 Biosynthesis. J. Biol. Chem. 286, 7885−7892.
are speculative and require investigation. The biosynthesis of nucleoside analogues typically joins together nonribosomal peptide (NRP) biosynthesis and nucleotide metabolism. Pseudouridimycin contains a dipeptide coupled to a 5′amino-pseudouridine. A nonribosomal peptide synthetase could form the dipeptide, starting with guanidinoacetate. Guanidinoacetate is synthesized from glycine and an amidino source by an amidinotransferase in the biosynthesis of the NRP, cylindrospermopsin.2 The second amino acid substrate, N2hydroxyglutamine, could be incorporated in a manner similar to those of other hydroxamate-containing nonribosomal peptides, such as heterobactin.3 Separately, 5′-amino-pseudouridine represents the nucleoside moiety of the new antibiotic. In parallel with the biosynthesis of other nucleoside analogues,4 the initial substrate in the pathway could be pseudouridine monophosphate. Work by Van Lanen and colleagues5 has demonstrated that the 5′ position is oxidized by an αketoglutarate-dependent Fe(II) oxygenase during the biosynthesis of related natural products. This intermediate decomposes to an aldehyde, a substrate for a PLP-dependent transaminase. The two converging units could be coupled by a condensation domain/protein to establish the final natural product. Collectively, the findings described by Maffioli et al.1 are important for several reasons. Pseudouridimycin is the first nucleoside analogue inhibitor of bacterial RNAP. The antibiotic is capable of killing drug-resistant bacteria, specifically clinically relevant isolates of S. aureus and Streptococcus pneumoniae. While the authors could readily identify resistance mutations in the lab, pseudouridimycin, or analogues thereof, could still represent promising molecular probes or drug development leads. This example lends further support to the general notion of increasing investments in the discovery of specialized metabolites already found in nature, especially for the identification and development of drugs with alternative binding sites and new modes of action. Additionally, an understanding of their biosyntheses and modes of action could facilitate large-scale fermentation efforts, the engineering of antibiotic analogues, and/or inspiring biomimetic synthetic efforts to level the playing field in the arms race against multidrug-resistant pathogens.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Eric P. Trautman: 0000-0002-0974-2356 Jason M. Crawford: 0000-0002-7583-1242 Funding
Our work on the discovery and characterization of specialized metabolites is supported by the National Institutes of Health (1DP2-CA186575), the Damon Runyon Cancer Research Foundation (DRR-39-16), the Burroughs Wellcome Foundation (1016720), the Camille and Henry Dreyfus Foundation (TC-17-011), the Searle Scholars Program (13-SSP-210), and Yale University. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Maffioli, S. I., Zhang, Y., Degen, D., Carzaniga, T., Del Gatto, G., Serina, S., Monciardini, P., Mazzetti, C., Guglierame, P., Candiani, G., B
DOI: 10.1021/acs.biochem.7b00680 Biochemistry XXXX, XXX, XXX−XXX
