Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens

McKinney, David C.; Basarab, Gregory S.; Cocozaki, A.; Foulk, M.A.; Miller, M.D.; Ruvinsky, Anatoly M.; Scott, Clay W.; Thakur, Kumar; Zhao, Liang; Buurman, Ed T.; Nimmagadda, Sridhar

doi:10.1021/acsmedchemlett.5b00205

Cited by 9 publications

(5 citation statements)

References 30 publications

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“…Negamycin used in this study was of synthetic origin and inhibited translation in an E. coli cell-free system with an IC of 2.8 µM (0.69 µg/ml, Fig. 1B), in accordance with previously published values (20,22,26). The compound also induced stop codon readthrough in an E. coli whole-cell miscoding assay (Fig.…”

Section: Media Conditions Significantly Affect Negamycin Activitysupporting

confidence: 90%

“…In an attempt to improve the efficacy of negamycin, several derivatization campaigns were conducted by companies and academic groups, which almost exclusively resulted in a loss of activity ( 19 – 21 ). Only a single recently reported derivative, N6-(3-aminopropyl) negamycin, showed 4-fold improved antibacterial activity ( 22 ). Notably, among the derivatives generated over the years, several were active in ribosomal extracts but failed in whole-cell MIC assays, suggesting uptake issues ( 23 ).…”

Section: Introductionmentioning

confidence: 99%

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The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes

Hörömpöli

Ciglia

Glüsenkamp³

et al. 2021

Antimicrob Agents Chemother

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Negamycin is a natural pseudo-dipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli. Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of DtpD, a proton-dependent oligopeptide transporter. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions point to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multi-faceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

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Section: Media Conditions Significantly Affect Negamycin Activitysupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes

Hörömpöli

Ciglia

Glüsenkamp³

et al. 2021

Antimicrob Agents Chemother

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“…Thus, we propose that AP-Neg facilitates nonsense suppression by stabilizing suppressor tRNA binding at the A-site, analogous to the mechanism proposed by which Neg promotes misreading of sense codons 37,39 . Our structure also rationalizes the improved biochemical and antimicrobial activities of AP-Neg over Neg 45 , since the aminopropyl chain of AP-Neg forms additional contacts with the 16S rRNA. This includes potential hydrogen bonds from the terminal amino group with the 2′ OH of the ribose and non-bridging oxygen of the 3′ phosphate of A968 (Fig.…”

Section: Resultssupporting

confidence: 57%

“…10g-i). Consistently, substitutions of the amino group of AP-Neg generate Neg analogs with reduced inhibitory activity 45 .…”

Section: Resultsmentioning

confidence: 84%

Repurposing tRNAs for nonsense suppression

et al. 2021

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Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

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“…The resulting unique secondary structures can improve the proteolytic stability of these compounds [ 22 ]. An acylhydrazine (hydrazide) was first reported in Ugi reactions back in 1961 [ 23 ] and also some natural products contain this moiety, such as the vitamin B6 antagonist linatine [ 24 ] and the antibiotic negamycin, active against Gram-negative bacteria [ 25 ], among others [ 26 ]. Furthermore, trisubstituted acylhydrazines were found to serve as tertiary amide bioisosters [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

Consecutive hydrazino-Ugi-azide reactions: synthesis of acylhydrazines bearing 1,5-disubstituted tetrazoles

Barreto¹,

Santos²,

Andrade³

2017

Beilstein J. Org. Chem.

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Isocyanide-based multicomponent reactions (IMCRs) allow the construction of relatively complex molecules through a one-pot synthesis. The combination of IMCRs in a consecutive or sequential fashion further extends the complexity of the molecules obtained. Herein, we report the efficient application of this approach to the synthesis of acylhydrazines bearing 1,5-disubstituted tetrazoles. Our strategy was accomplished in only three steps: first, a one-pot hydrazino-Ugi-azide four-component reaction; second a hydrazinolysis and finally an additional hydrazino-Ugi-azide reaction. This sequence provides the title compounds in moderate to excellent yields. The products synthesized herein contain functional groups within their structures that can be easily modified to obtain new acylhydrazino 1,5-disubstituted tetrazoles.

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Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens

Cited by 9 publications

References 30 publications

The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes

The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes

Repurposing tRNAs for nonsense suppression

Consecutive hydrazino-Ugi-azide reactions: synthesis of acylhydrazines bearing 1,5-disubstituted tetrazoles

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