Role of the C(5)−C(5a) Exomethylene Group in Bicyclomycin:  Synthesis, Structure, and Biochemical and Biological Properties

Park, Hyeung Geun; Zhang, Zhuming; Zhang, Xiangdong; Widger, William R.; Kohn, Harold

doi:10.1021/jo961004i

Cited by 18 publications

(48 citation statements)

References 24 publications

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“…2 The use of analogues acting as irreversible inactivators has proven to be a useful approach for the identification of substrate-binding sites in enzymes (30). A recent structure-activity relationship study of Bcm (31)(32)(33) has identified 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm) as a potential reductive amination probe that warranted further study (34). FD-Bcm has Rho inhibitory properties comparable with Bcm.…”

Section: Bicyclomycin (Bcm)mentioning

confidence: 99%

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Riba¹,

Gaskell²,

Cho

et al. 1998

Journal of Biological Chemistry

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The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH 4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH 4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys 181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174 -184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys 181 . These findings provide the first structural information concerning the site of Bcm binding in Rho.Bicyclomycin (Bcm) 1 is a commercial antibiotic of novel structure (1-8). The primary site of action in Escherichia coli is unique and has been identified as the transcription termination factor, Rho (9). Rho is composed of six identical 47-kDa proteins of 419 amino acids (10) and exists in a planar, hexagonal (11-13) arrangement of proposed C 6 (14) or D 3 (15) geometry. Rho-dependent transcription termination sites occur at the ends of transcription units, at regulatory points before or between genes, and within genes (16). Transcription termination begins with the recognition and binding of Rho to discrete regions (rut sites) within the newly synthesized RNA chains (17). Binding is governed by a general requirement for a cytosine-rich sequence, and it extends across all six subunits of the functional protein, encompassing approximately 80 nucleotides (17)(18)(19)(20). Rho then extends its interaction toward the 3Ј end of the RNA polymerase elongation complex in a process coupled to ATP hydrolysis. The Rho-mediated release of RNA from the transcription bubble results from a destabilization of the complex, believed to be caused, in part, by the ATPase-dependent 5Ј 3 3Ј RNA-DNA helicase action of the protein (21).Details of the Bcm-Rho interaction have emerged in recent investigations (9,(22)(23)(24). Chemical studies have shown that nucleophilic amino acids readily react with Bcm, consistent with sever...

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Section: Bicyclomycin (Bcm)mentioning

confidence: 99%

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Riba¹,

Gaskell²,

Cho

et al. 1998

Journal of Biological Chemistry

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“…In the first, we determined the structureactivity relationship (SAR) for bicyclomycin inhibition of rho [119][120][121][122][123][124][125][126][127][128][129][130][131]. In the second, we identified key amino acid residues in rho necessary for rho binding [74].…”

Section: Preliminary Studies On the Identi-fication Of The Bicyclomycmentioning

confidence: 99%

Section: The C(5)-c(5a) Exomethylene Groupmentioning

confidence: 99%

“…More than 75 C(5)-C(5a) exomethylene-modified bicyclomycin derivatives were prepared [125][126][127][128][129][130][131]. The compounds can be grouped into three categories (Figure 9): C(5)-unsaturated bicyclomycins A [125,126], C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycins B [125,[127][128][129][130], and C(5)-modified norbicyclomycin C [131]. Among the C(5)-C(5a) exomethylene-modified bicyclomycins prepared were C(5a)-methyl-substituted bicyclomycins [126] (43)(44)(45)(46) and C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycins [125,130] (47)(48)(49)(50)(51)(52).…”

Section: The C(5)-c(5a) Exomethylene Groupmentioning

confidence: 99%

“…The compounds can be grouped into three categories (Figure 9): C(5)-unsaturated bicyclomycins A [125,126], C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycins B [125,[127][128][129][130], and C(5)-modified norbicyclomycin C [131]. Among the C(5)-C(5a) exomethylene-modified bicyclomycins prepared were C(5a)-methyl-substituted bicyclomycins [126] (43)(44)(45)(46) and C(5a)-substituted C(5)-C(5a)-dihydrobicyclomycins [125,130] (47)(48)(49)(50)(51)(52). This required new methods for the extension and the modification of the C(5)-C(5a) exomethylene unit in 1 [125][126][127][128].…”

Section: The C(5)-c(5a) Exomethylene Groupmentioning

confidence: 99%

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The Molecular Basis for the Mode of Action of Bicyclomycin

Kohn

Widger

2005

CDTID

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Bicyclomycin (1) is a clinically useful antibiotic exhibiting activity against a broad spectrum of Gram-negative bacteria and against the Gram-positive bacterium, Micrococcus luteus. Bicyclomycin has been used to treat diarrhea in humans and bacterial diarrhea in calves and pigs and is marketed by Fujisawa (Osaka, Japan) under the trade name Bicozamycin. The structure of 1 is unique among antibiotics, and our studies document that its mechanism of action is novel. Early mechanistic proposals suggested that 1 reacted with nucleophiles (e.g., a protein sulfhydryl group) necessary for the remodeling the peptidoglycan assembly within the bacterial cell wall. We, however, showed that 1 targeted the rho transcription termination factor in Escherichia coli. The rho protein is integral to the expression of many gene products in E. coli and other Gram-negative bacteria, and without rho the cell losses viability. Rho is a member of the RecA-type ATPase class of enzymes that use nucleotide contacts to couple oligonucleotide translocation to ATP hydrolysis. Bicyclomycin is the only known selective inhibitor of rho. In this article, we integrate the evidence obtained from bicyclomycin structure-activity studies, site-directed mutagenesis investigations, bicyclomycin affinity labels, and biochemical and biophysical measurements with recent X-ray crystallographic images of the bicyclomycin-rho complex to define the rho antibiotic binding site and to document the pathway for rho inhibition by 1. Together, the structural and functional studies demonstrate how 1, a modest rho inhibitor, can disrupt the rho molecular machinery thereby leading to a catastrophic effect caused by the untimely overproduction of proteins not normally expressed constitutively, thus leading to a toxic effect on the cells.

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Convenient Synthesis of (E)‐5‐Aryl(halo)methylenebicyclo‐ [2.2.2]oct‐2‐enes and ‐[2.2.1]hept‐2‐enes via Lewis Acid‐ Promoted Carbohalogenation of Cyclic 2,6‐Enynols

Yeh¹,

Lin²,

Kuo³

et al. 2014

Adv Synth Catal

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An efficient synthesis of (E)-5-aryl(halo)methylenebicyclo[2.2.2]oct-2-enes is reported. Lewis acid-promoted carbohalogenation of 4-(3-arylprop-2-ynyl)-cyclohex-2-enols in dichloromethane proceeds rapidly to afford the exo-methylenebridged bicycles in good yields. This method also provides an easy access to (E)-5-aryl(halo)methyl-A C H T U N G T R E N N U N G enebicyclo[2.2.1]hept-2-enes from the five-membered ring 2,6-enynols. The reactions are procedurally simple and high yielding, producing the aryl(halo)methylene-bridged bicycles in minutes under air and mild conditions.

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Role of the C(5)−C(5a) Exomethylene Group in Bicyclomycin: Synthesis, Structure, and Biochemical and Biological Properties

Cited by 18 publications

References 24 publications

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

The Molecular Basis for the Mode of Action of Bicyclomycin

Convenient Synthesis of (E)‐5‐Aryl(halo)methylenebicyclo‐ [2.2.2]oct‐2‐enes and ‐[2.2.1]hept‐2‐enes via Lewis Acid‐ Promoted Carbohalogenation of Cyclic 2,6‐Enynols

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