| Reference | [1]. Curr Drug Targets Infect Disord. 2005 Sep;5(3):273-95. doi: 10.2174/1568005054880136.<br />
The molecular basis for the mode of action of bicyclomycin.<br />
Kohn H(1), Widger W.<br />
Author information: (1)Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA. [email protected]<br />
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.<br />
DOI: 10.2174/1568005054880136 PMID: 16181146 [Indexed for MEDLINE]<br />
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[2]. Sci Rep. 2019 Dec 27;9(1):20226. doi: 10.1038/s41598-019-56747-7.<br />
Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches.<br />
Witwinowski J(1)(2), Moutiez M(1), Coupet M(1), Correia I(3), Belin P(1), Ruzzini A(1)(4), Saulnier C(1), Caraty L(1), Favry E(1)(5), Seguin J(1)(6), Lautru S(1), Lequin O(3), Gondry M(1), Pernodet JL(1), Darbon E(7).<br />
Author information: (1)Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France. (2)Unit Evolutionary Biology of the Microbial Cell, Department of Microbiology, Institut Pasteur, Paris, France. (3)Sorbonne Université, École Normale Supérieure, PSL University, CNRS, Laboratoire des Biomolécules, Paris, France. (4)Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. (5)Frédéric Joliot Institute for Life Sciences, CEA, SPI, Saclay, France. (6)CEA, DEN, Centre de Marcoule, Bagnols-sur-Cèze, France. (7)Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France. [email protected].<br />
The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.<br />
DOI: 10.1038/s41598-019-56747-7 PMCID: PMC6934819 PMID: 31882990 [Indexed for MEDLINE]<br />
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[3]. Biochemistry. 2018 Jan 9;57(1):61-65. doi: 10.1021/acs.biochem.7b00943. Epub 2017 Nov 7.<br />
Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin.<br />
Patteson JB(1), Cai W(1), Johnson RA(1), Santa Maria KC(1), Li B(1).<br />
Author information: (1)Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.<br />
Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.<br />
DOI: 10.1021/acs.biochem.7b00943 PMCID: PMC5760335 PMID: 29053243 [Indexed for MEDLINE]<br />
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[4]. J Antibiot (Tokyo). 1976 Feb;29(2):155-68. doi: 10.7164/antibiotics.29.155.<br />
Mechanism of action of bicyclomycin.<br />
Tanaka N, Iseki M, Miyoshi T, Aoki H, Imanaka H.<br />
Microscopic examination of cultures of Escherichia coli exposed to bicyclomycin revelaed elongated or spheroplast-like cells. At the lethal level, bicyclomycin was shown to inhibit the synthesis of RNA and protein in the growing cells of E. coli 15 THU, whereas DNA and lipid synthesis were not significantly affected. However, the antibiotic did not block RNA and protein synthesis in vitro. Bicyclomycin was observed to inhibit the synthesis of envelope proteins more markedly than that of cytoplasmic proteins. The synthesis of two major envelope proteins was more sensetive to bicyclomycin than that of the other envelope proteins. One (peak I), which was inhibited to the greatest extent, seemed to be identical with a bound form of lipoprotein, and the other (peak V) with a free form of lipoprotein. Bicyclomycin exhibited inhibitory effects on the exclusive biosynthesis of the lipoprotein in histidine-starved cells of E. coli 15 THU. The biosynthesis of the bound form of lipoprotein was more profoundly inhibited by bicyclomycin than that of the free form. These results indicate that the primary action of bicyclomycin may be due to the interference with the biosynthesis of lipoprotein, and its assembly to peptidoglycan.<br />
DOI: 10.7164/antibiotics.29.155 PMID: 776917 [Indexed for MEDLINE]<br />
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[5]. J Med Chem. 1985 Jun;28(6):733-40. doi: 10.1021/jm00383a008.<br />
Synthesis and antimicrobial evaluation of bicyclomycin analogues.<br />
Williams RM, Armstrong RW, Dung JS.<br />
The synthesis and antimicrobial evaluation of novel bicyclomycin analogues are described. The series of analogues were prepared from the basic 8,10-diaza-2-oxabicyclo[4.2.2]decane-7,9-dione, 7,9-diaza-2-oxabicyclo[3.2.2]nonane-6,8-dione 8,10-diaza-5-methylene-2-oxabicyclo[4.2.2]decane-7,9-dione and 7,9-diaza-4-methylene-2-oxabicyclo[3.2.2]nonane-6,8-dione nuclei. For compounds where R1 = p-methoxybenzyl, deprotection of the lipophilic amides with ceric ammonium nitrate affords the corresponding lipophobic free amides. The basic bicyclic nucleus of bicyclomycin (8h, R1 = R2 = R3 = R4 = H) has been synthesized for the first time as well as increasingly more complex congeners bearing the C-6 OH, 5-methylene; C-1'-C-3' trihydroxyisobutyl group. In general, it has been found that the bicyclic nucleus of bicyclomycin is devoid of antimicrobial activity, the entire structure of bicyclomycin being generally obligate for activity. In one instance, the racemic analogue 10c (R1 = CH2Ph, R2 = OH, R3 = H) showed interesting antimicrobial activity against several Gram-positive organisms; the minimum inhibitory concentrations were of the same order of magnitude as bicyclomycin displays toward Gram-negative organisms. Totally synthetic (+/-)-bicyclomycin was half as active as the natural antibiotic. The design, synthesis, and antimicrobial activity (and/or lack thereof) of bicyclomycin and the analogues are discussed in the context of a proposed chemical mechanism of action.<br />
DOI: 10.1021/jm00383a008 PMID: 4009595 [Indexed for MEDLINE]
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