Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket

Harvey, Colin J. B.; Puglisi, Joseph D.; Pande, Vijay S.; Cane, David E.; Khosla, Chaitan

doi:10.1021/ja304682q

Cited by 59 publications

(58 citation statements)

References 40 publications

(66 reference statements)

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“…The successful introduction of telithromycin to the market at the beginning of this century had prompted many labs to develop novel versions of ketolide compounds to combat the rising tide of multidrug-resistant bacteria (2,6,8,9). Such a version of new compounds has been developed by Kosan Bioscience, namely, the four fluoroketolides which we have studied as new possible antibacterial agents.…”

Section: Discussionmentioning

confidence: 99%

“…1A) into clinical practice in the 1950s (1)(2)(3)(4). However, the widespread usage of macrolides has resulted in the selection of resistant strains, providing a strong incentive for the development of newer macrolide drugs that can overcome such resistance mechanisms (5)(6)(7)(8)(9). This has led to the development of a new generation of macrolides, namely, ketolides, with the most distinguished members being telithromycin, cethromycin, and solithromycin ( Fig.…”

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confidence: 99%

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Insights into the Mode of Action of Novel Fluoroketolides, Potent Inhibitors of Bacterial Protein Synthesis

Krokidis

Márquez

Wilson

et al. 2014

Antimicrob Agents Chemother

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Ketolides, the third generation of expanded-spectrum macrolides, have in the last years become a successful weapon in the endless war against macrolide-resistant pathogens. Ketolides are semisynthetic derivatives of the naturally produced macrolide erythromycin, displaying not only improved activity against some erythromycin-resistant strains but also increased bactericidal activity as well as inhibitory effects at lower drug concentrations. In this study, we present a series of novel ketolides carrying alkyl-aryl side chains at the C-6 position of the lactone ring and, additionally, one or two fluorine atoms attached either directly to the lactone ring at the C-2 position or indirectly via the C-13 position. According to our genetic and biochemical studies, these novel ketolides occupy the known macrolide binding site at the entrance of the ribosomal tunnel and exhibit lower MIC values against wild-type or mutant strains than erythromycin. In most cases, the ketolides display activities comparable to or better than the clinically used ketolide telithromycin. Chemical protection experiments using Escherichia coli ribosomes bearing U2609C or U754A mutations in 23S rRNA suggest that the alkylaryl side chain establishes an interaction with the U2609-A752 base pair, analogous to that observed with telithromycin but unlike the interactions formed by cethromycin. These findings reemphasize the versatility of the alkyl-aryl side chains with respect to species specificity, which will be important for future design of improved antimicrobial agents.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Insights into the Mode of Action of Novel Fluoroketolides, Potent Inhibitors of Bacterial Protein Synthesis

Krokidis

Márquez

Wilson

et al. 2014

Antimicrob Agents Chemother

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“…Tagging natural products with a unique chemical handle enables the visualization, enrichment, quantification, and mode of action study of natural products through bioorthogonal chemistry [1][2][3][4] . One prevalent bioorthogonal reaction is the triazole-forming azide-alkyne [3+2] cycloaddition, often referred to as "click" chemistry 5 .…”

Section: Introductionmentioning

confidence: 99%

De novo biosynthesis of terminal alkyne-labeled natural products

2014

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Abstract:The terminal alkyne is a functionality widely used in organic synthesis, pharmaceutical science, material science, and bioorthogonal chemistry. This functionality is also found in acetylenic natural products, but the underlying biosynthetic pathways for its formation are not well understood. Here we report the characterization of the first carrier proteindependent terminal alkyne biosynthetic machinery in microbes. We further demonstrate that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds in E. coli. These results highlight the prospect for tagging major classes of natural products, including polyketides and polyketide/non-ribosomal peptide hybrids, using biosynthetic pathway engineering.2 Natural products are important small molecules widely used as drugs, pesticides, herbicides, and biological probes. Tagging natural products with a unique chemical handle enables the visualization, enrichment, quantification, and mode of action study of natural products through bioorthogonal chemistry [1][2][3][4] . One prevalent bioorthogonal reaction is the triazole-forming azide-alkyne [3+2] cycloaddition, often referred to as "click" chemistry 5 . This reaction has enabled selective imaging and study of azide-or alkyne-labeled glycans, proteins, nucleic acids and lipids. Despite the success with macromolecules, the labeling of natural products has not been adequately explored. It is often challenging to obtain tagged natural products through total synthesis because of their structural complexity, or through semi-synthesis because of their chemical lability and the limited supply of most natural products. Alternatively, precursor-directed biosynthesis (PDB) may be employed to produce azide-or alkyne-labeled natural products based on the promiscuity of biosynthetic machinery. Mainly used as a tool to introduce structural diversity, PDB has allowed us and other researchers to generate labeled natural products 3,[6][7][8] . However, the coexistence of diffusible precursors and final products 9 with the same chemical handle introduces significant background in the PDB production system, making it incompatible with in situ bioorthogonal chemical transformations. We report here the de novo biosynthesis of alkyne-labeled natural products without the feeding of alkynoic precursors by characterizing and engineering the novel terminal alkyne synthetic machinery that living systems offer.Many acetylenic natural products contain a terminal alkyne functionality, which seems to be crucial for their bioactivity (Fig. 1) 10,11 . Terminal alkynes can be formed by acetylenases, a special family of desaturases that catalyze O 2 -dependent dehydrogenation of C-C bonds in a diiron dependent mechanism 12 . Several membrane-bound acetylenases have recently been 3 identified from plants, insects, fungi, and bacteria 4,10,[13][14][15][16][17] , but all of these reports have been limited to bioinformatics or in vivo studies through mutagenesis or ...

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“…Current methods to measure translation rates detect properties of a translation product reporter, such as luciferase luminescence (Hale 2000), green fluorescent protein (GFP) fluorescence (Nomura et al 2001), chloramphenicol acetyltransferase activity (Shaw 1975;Kim and Swartz 2001;Harvey et al 2012), and 14 C radioactivity (Bayley and Griffiths 1968). The throughput and generality of these methods are limited by the requirement for multiple steps of processing or specialized equipment to obtain a translation signal.…”

Section: Introductionmentioning

confidence: 99%

A simple real-time assay for in vitro translation

Capece

Kornberg

Petrov

et al. 2014

RNA

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A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O 6 -alkylguanine DNA O 6 -alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC 50 ) of three common macrolide antibiotics.

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Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket

Cited by 59 publications

References 40 publications

Insights into the Mode of Action of Novel Fluoroketolides, Potent Inhibitors of Bacterial Protein Synthesis

Insights into the Mode of Action of Novel Fluoroketolides, Potent Inhibitors of Bacterial Protein Synthesis

De novo biosynthesis of terminal alkyne-labeled natural products

A simple real-time assay for in vitro translation

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