Conformationally restricted analogs of deoxynegamycin

Raju, B. China; Anandan, Sampath-Kumar; Gu, Shenyan; Herradura, Prudencio; O'Dowd, Hardwin; Kim, Bum; Gomez, Marcela; Hackbarth, C J; Wu, Charlotte; Wang, Wen; Yuan, Zhengqiu; White, Richard; Trias, Joaquim; Patel, Dinesh V.

doi:10.1016/j.bmcl.2004.04.036

Cited by 17 publications

(14 citation statements)

References 28 publications

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“…1D). It is also consistent with structure-activity relationship studies, which show the importance of the hydroxyl group, the stereochemistry at position 3, and both termini of negamycin for its activity (12,13,(17)(18)(19)27). The drug interacts primarily with RNA backbone atoms.…”

supporting

confidence: 86%

Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Schroeder

Blaha

Moore

2007

Antimicrob Agents Chemother

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Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.Negamycin is a translation inhibitor that prevents the release of complete proteins from the ribosome and thus causes an accumulation of polysomes. Negamycin also induces miscoding (1, 15, 23, 26) but does not interfere with peptide initiation or elongation (7,25,28). Because the inhibition of peptide release and miscoding vary independently in negamycin derivatives (27), negamycin may interfere with ribosome function in two distinct ways. Here we report the results of crystallographic experiments which show that negamycin binds to the wall of the peptide exit tunnel of the large ribosomal subunit. This interaction may account for negamycin's inhibition of peptide release.The 50S ribosomal subunits were prepared from wild-type Haloarcula marismortui cells and crystallized as described previously (3). Negamycin was soaked into the crystals following previously described protocols (11,16,20,21). Five millimolar negamycin is approximately five times the MIC of negamycin for this organism. Crystals were equilibrated at least twice with solutions containing 5 mM negamycin dissolved in buffer B (12% [wt/vol] polyethylene glycol 6000, 20% [vol/vol] ethylene glycol, 1.7 M NaCl, 0.5 M NH 4 Cl, 1 mM CdCl 2 , 100 mM potassium acetate, 6.5 mM acetic acid [pH 6.0], 30 mM MgCl 2 or 100 mM SrCl 2 , 5 mM ␤-mercaptoethanol) at 4°C over the course of 1 to 4 h, during which SrCl 2 was included in soak solutions, and over 16 to 24 h, during which MgCl 2 was included in soak solutions.Data collected at the Advanced Light Source (ALS), using beamline 8.2.2, were processed as previously described (3). Unbiased-difference electron density maps were computed for each drug-ribosome complex, using as amplitudes [ԽF o (hkl)Խ drug/ribosome Ϫ ԽF o (hkl)Խ best native ], where the ԽF o (hkl)Խs are measured amplitudes of the (hkl) reflection in the data collected from the two crystals whose structures were being compared, and phases obtained by rigid-body refinement of the native structure (Protein Data Bank code 1S72; 12) into the drug-ribosome data set. Structure refinements were done with CNS software (4) and included rigid-body, energy minimization, and B-factor refinement. In the final rounds of refinement, only those atoms within a 25-to 35-Å-diameter sphere centered on the antibiotic binding site were allowed to vary in position. The coordinates and structure factors for the negamycin complex described here may be found in the Protein Data Bank (code 2QEX; http://www.rcsb.org/pdb/home /home.do).The functional significance of the negamycin binding site identified in the crystal structure was investigated by site-directed mutagenesis and random targeted mutagenesis and by the selection of naturally o...

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confidence: 86%

Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Schroeder

Blaha

Moore

2007

Antimicrob Agents Chemother

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“…Consequently, further clinical studies were not pursued (5). Although the antimicrobial activity and efficacy of negamycin have since been confirmed, analogs exhibiting an improved therapeutic window have yet to be found (6,7). Progress on this front has been hampered, at least in part, by the fact that the molecular mechanisms of negamycin-induced cell growth inhibition have yet to be discerned conclusively.…”

mentioning

confidence: 99%

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome

Olivier

Altman

Noeske

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.egamycin, a natural product originally isolated from cultures of Streptomyces purpeofuscus, exhibits broad-spectrum antibacterial activity against key pathogens for which clinical treatment options are dwindling (1, 2). The chemical structure of negamycin, [2-[(3R, 5R)-3,6-diamino-5-hydroxyhexanoyl]-1-methylhydrazino]acetic acid, and synthetic routes for its synthesis were elucidated in the early 1970s ( Fig. 1A) (3,4). Early toxicity studies in dogs revealed that daily administration of negamycin led to the formation of N-methylhydrazinoacetic acid, an inhibitor of glutamate pyruvate transaminase, an outcome that caused reversible hepatic coma. Consequently, further clinical studies were not pursued (5). Although the antimicrobial activity and efficacy of negamycin have since been confirmed, analogs exhibiting an improved therapeutic window have yet to be found (6, 7). Progress on this front has been hampered, at least in part, by the fact that the molecular mechanisms of negamycin-induced cell growth inhibition have yet to be discerned conclusively.Negamycin decreases the viability of Escherichia coli by preferentially targeting protein synthesis (8). Early mechanistic investigations proposed that negamycin may act by inhibiting translation initiation (8), decreasing translational fidelity during the elongation phase of protein synthesis (9, 10), and disrupting proper translation termination (9,(11)(12)(13). Uncertainties regarding negamycin's inhibition mechanism, its highly polar physicochemical properties, and the lack of streptomycin cross-resistance led som...

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“…The increased efficiency of this key C—N bond-forming step now affords a 35% overall yield of deoxynegamycin analog 28 , a nearly three-fold increase in yield as compared to the C—O to C—N route. 23 …”

Section: Reaction Scopementioning

confidence: 99%

Aerobic Linear Allylic C–H Amination: Overcoming Benzoquinone Inhibition

et al. 2016

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An efficient aerobic linear allylic C—H amination reaction (LAA) is reported under Pd(II)/bis-sulfoxide/Brønsted base catalysis. The reaction operates under preparative, operationally simple conditions (1 equiv. olefin, 1 atm. O2 or air), with reduced Pd(II)/bis-sulfoxide catalyst loadings while providing higher turnovers and product yields than systems employing stoichiometric benzoquinone (BQ) as the terminal oxidant. Palladium(II)/benzoquinone π-acidic interactions have been invoked in various catalytic processes and are often considered beneficial in promoting reductive functionalizations. When such electrophilic activation for functionalization is not needed, however, benzoquinone at high concentrations may compete with crucial ligand (bis-sulfoxide) binding and inhibit catalysis. Kinetic studies reveal an inverse relationship between the reaction rate and the concentration of BQ, suggesting that benzoquinone is acting as a ligand for Pd(II) which results in an inhibitory effect on catalysis.

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Conformationally restricted analogs of deoxynegamycin

Cited by 17 publications

References 28 publications

Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome

Aerobic Linear Allylic C–H Amination: Overcoming Benzoquinone Inhibition

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