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PubMed1981

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.

Bacterial Proteins↗
PubMed1994

Enhancement of gustatory nerve fibers to NaCl and formation of ion channels by commercial novobiocin.

Single fibers of the rat chorda tympani nerve were used to study the mechanism of action of the antibiotic novobiocin on salt taste transduction. In the rat, novobiocin selectively enhanced the responses of sodium-specific and amiloride-sensitive chorda tympani nerve fibers (N type) without affecting more broadly responsive cation-sensitive and amiloride-insensitive fibers (E type). In the presence of amiloride, novobiocin was ineffective at enhancing the response of N-type fibers toward sodium chloride. Novobiocin also increased the conductance of bilayers formed from neutral lipids by forming nonrectifying ion channels with low conductance (approximately 7 pS in 110 mM NaCl), long open times (several seconds and longer), and high cation selectivity. Amiloride did not alter either the conductance or kinetics of these novobiocin channels. These observations suggest that even though novobiocin is able to form cation channels in lipid bilayers, and possibly in cell membranes as well, its action on the salt-taste response is through modulation of existing amiloride-sensitive sodium channels.

Amiloride↗
PubMed1987

Novobiocin enhances alkylating agent cytotoxicity and DNA interstrand crosslinks in a murine model.

DNA-DNA crosslinks are the lethal cellular mechanism of bifunctional alkylating agent cytotoxicity. Novobiocin, an inhibitor of DNA topoisomerase II, impairs eukaryotic DNA repair of alkylating agent adducts and may increase the number of adducts and their resultant cytotoxicity in malignant cells. The effect of novobiocin on clonogenic survival and DNA crosslinking due to cisplatin (cDDP) and carmustine (BCNU) was studied. Novobiocin caused synergistic cytotoxicity in Chinese hamster ovary cells exposed to cDDP or BCNU. Novobiocin and cDDP increased the formation of DNA-DNA interstrand crosslinks six-fold greater than cDDP alone. The effect was schedule dependent. Novobiocin and cDDP or BCNU markedly reduced in vivo growth of a murine fibrosarcoma without increased host toxicity. As a modulating agent of cytotoxicity due to DNA-DNA crosslinking, novobiocin may enhance the clinical effectiveness of the alkylating agents in human cancer and offer insight into new therapeutic strategies.

Alkylating Agents↗
PubMed1990

The isolation of salmonellae from poultry environmental samples by several enrichment procedures using plating media with and without novobiocin.

A two-part study was conducted to examine the efficacy of several enrichment-broth techniques and of plating media for detecting salmonellae from poultry environmental samples. The data are reported on pooled samples collected from five poultry houses. The samples were cultured for salmonellae, using up to four different enrichment procedures and employing plating media with and without novobiocin. The primary enrichment-broth procedures were: 1) buffered peptone water preenrichment to Hajna's tetrathionate (TT) broth; and 2) direct inoculation in TT broth. The delayed secondary-enrichment procedure involved prolonged incubation at room temperature and transfer of the primary broths. The plating media consisted of: 1) xylose lysine desoxycholate agar (XLD); 2) xylose lysine desoxycholate agar containing 15 or 20 micrograms per mL of novobiocin (XLDN); 3) brilliant green sulfapyridine agar (BGSP); and 4) brillant green agar containing 20 micrograms per mL of novobiocin (BGN). Of the 94 Salmonella-positive recoveries from the enrichment broths in which complete comparisons could be made, an average of 75% were recovered from the primary enrichment broths and an average of 86% were recovered from the delayed secondary-enrichment broths. Of the 254 Salmonella-positive isolations in which complete comparisons could be made, an average of 65% were isolated on the plating media without novobiocin and an average of 97% were isolated on the plating media containing novobiocin. Overall, the delayed secondary enrichment and enteric plates supplemented with novobiocin significantly improved Salmonella detection from the farm environmental samples.

Animals↗
PubMed1989

Novobiocin, nalidixic acid, etoposide, and 4'-(9-acridinylamino)methanesulfon-m-anisidide effects on G2 and mitotic Chinese hamster ovary cell progression.

Exponentially growing Chinese hamster ovary cells, exposed to inhibitors of topoisomerase II (novobiocin, nalidixic acid, etoposide, and 4'-(9-acridinylamino)methanesulfon-m-anisidide were blocked in progression through G2. The manner of recovery from the novobiocin-induced block, following drug removal, indicated that the blockade was at and before a specific point in G2 (a transition point). The transition point for novobiocin and putative transition points for nalidixic acid and 4'-(9-acridinylamino)methanesulfon-m-anisidide were about 30 min before metaphase. The transition point for nalidixic acid varied with concentration from about 70 min before metaphase, at 1 microgram/ml, to 24 min before metaphase at 15 micrograms/ml and above. The novobiocin- and nalidixic acid-induced G2 block could not be accounted for by cytotoxicity or DNA damage (detected by neutral elution). The novobiocin-induced G2 block could not be attributed to gross RNA synthesis inhibition. Progress beyond metaphase was blocked by novobiocin but not by nalidixic acid, when cells were exposed to drug concentrations which inhibited G2 cell progression. It is suggested that the progression of Chinese hamster ovary cells into but not through mitosis may require topoisomerase II.

Amsacrine↗
PubMed1989

Effect of novobiocin on the antitumor activity and tumor cell and bone marrow survivals of three alkylating agents.

Our previous in vitro studies demonstrated marked synergy with alkylating agents when novobiocin was present during and after alkylating agent exposure. To determine whether this effect is observed in vivo, novobiocin was administered daily for 3 days prior to alkylating agent treatment, during alkylating agent treatment, and for 2 days after completion of alkylating agent treatment. When combined with cis-diamminedichloroplatinum(II), 1,3-bis(2-chloroethyl)-1-nitrosourea, or cyclophosphamide, there was significant enhancement of the growth delay of the FSaIIC fibrosarcoma implanted s.c. in C3H mice when compared with alkylating agents alone. In a second assay using ex vivo studies of tumor cells exposed in vivo, single doses of 100 mg/kg of novobiocin followed by cis-diamminedichloroplatinum(II) resulted in a 3- to 4-fold increase in tumor cell killing by cis-diamminedichloroplatinum(II). At a dose of 100 mg/kg of 1,3-bis(2-chloroethyl)-1-nitrosourea there was about a 7-fold increase in tumor cell kill upon addition of novobiocin. Cyclophosphamide showed a dose response effect with novobiocin, reaching 13-fold at a dose of 300 mg/kg of cyclophosphamide. In all cases bone marrow elements were affected less than were neoplastic cells, suggesting that the combination of novobiocin and alkylating agents may be a clinically useful strategy.

Alkylating Agents↗
PubMed1986

Novobiocin induces the in vivo cleavage of active gene sequences in intact cells.

When the topoisomerase II inhibitor, novobiocin, is administered to embryonic chicken red blood cells, it induces the in vivo release of an endogenous nuclease which cleaves specifically within internucleosomal spacer DNA and within nuclease-hypersensitive sites in the active chromatin of intact cells. This in vivo released nuclease activity is induced by novobiocin only in metabolically active immature red blood cells. Little induction occurs in mature erythrocytes and no induction occurs in cells previously treated with 2,4-dinitrophenol. Although novobiocin is required to induce release and/or activation of the nuclease, the activity of the nuclease, once activated, is independent of novobiocin. Analysis of the cleaved DNA in drug-treated immature cells demonstrates that the novobiocin-induced nuclease has an unusual blunt-ended double-stranded mode of cleavage. Because of its special properties and apparent chromatin related function in vivo, the novobiocin-induced nuclease activity offers a novel and useful in vivo and in vitro probe of chromatin structure.

2,4-Dinitrophenol↗
PubMed1966

The international standard for novobiocin.

The National Institute for Medical Research, London, was requested by the WHO Expert Committee on Biological Standardization to arrange a collaborative assay of the International Reference Preparation of Novobiocin in order to define the International Unit of Novobiocin. The collaborative assay showed that the International Reference Preparation was not suitable for use as an international standard, and a second preparation of 400 g of novobiocin acid was obtained in 1962. This second preparation was distributed into ampoules in 100-mg amounts and dried in vacuum. The ampoules were then filled with dry nitrogen and sealed. This material-the proposed international standard for novobiocin-has been assayed biologically in eight laboratories in six countries by means of plate diffusion assays; it has been assayed against the working standard preparation of the US Food and Drug Administration.The variation in the potencies obtained for the second proposed international standard for novobiocin were small and not significant. The material has been established as the International Standard for Novobiocin with a defined potency of 970 International Units per mg, and the International Unit has been defined as the activity contained in 0.001031 mg of the International Standard.

Bacteria↗
PubMed1983

The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin.

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.

Animals↗
PubMedDate not supplied

Liquid chromatographic procedure for the determination of novobiocin residues in bovine milk: interlaboratory study.

Novobiocin is used for the treatment of mastitis in dairy cattle. In 1982, the tolerance was set at 0.1 ppm in milk from dairy animals. A laboratory trial has been completed for a liquid chromatographic procedure that can quantitate novobiocin residues in bovine milk at tolerance level. In this procedure the milk is diluted with buffer, the proteins are precipitated with methanol, and the solution is filtered. Novobiocin is determined after separation of milk components using reversed-phase chromatography with UV detection at 340 nm. The participating laboratories analyzed 2 concentrations of biologically incurred residues as well as control milk and control milk fortified at 0.05, 0.1, and 0.2 ppm. Recoveries of novobiocin reported by the participating laboratories were 89 to 99% at 0.05 ppm; 93 to 101% at 0.1 ppm; and 89 to 100% at 0.2 ppm. Coefficients of variation (CVs) ranged from 2.0 to 6.2%. The average concentrations for the low levels of incurred novobiocin in milk samples were 0.073, 0.072, and 0.081 ppm, with intralaboratory CVs of 3.3, 7.2, and 2.4%, respectively. The samples with high levels of incurred novobiocin averaged 0.139, 0.121, and 0.144 ppm, with CVs of 6.2, 0.7, and 4.7%, respectively.

Animals↗
PubMed1965

BIOCHEMICAL EFFECTS OF NOVOBIOCIN ON STAPHYLOCOCCUS AUREUS.

Wishnow, Rodney M. (Washington University School of Medicine, St. Louis, Mo.), Jack L. Strominger, Claire H. Birge, and Robert H. Threnn. Biochemical effects of novobiocin on Staphylococcus aureus. J. Bacteriol. 89:1117-1123. 1965.-Novobiocin induced accumulation in Staphylococcus aureus of uridine nucleotide precursors of the cell-wall glycopeptide, of a cytidine nucleotide precursor of the teichoic acid in the cell wall, and of a large number of other ultraviolet-absorbing materials. These substances accumulated promptly on addition of the antibiotic at levels near the growth-inhibitory concentration. Isotopic experiments showed that, in addition to inhibiting cell-wall synthesis, novobiocin also inhibited both protein and nucleic acid synthesis. The lack of selectivity in the action of novobiocin is similar to that previously observed with gentian violet, and is quite different from the selective inhibition of cell-wall synthesis observed with penicillin or with d-cycloserine. Although inhibition of cell-wall synthesis is not excluded as one of the primary lethal effects of novobiocin, it is also possible that all of the observed phenomena are secondary to some other primary metabolic lesion.

Anti-Bacterial Agents↗
PubMed1997

Novobiocin inhibits vaccinia virus replication by blocking virus assembly.

Novobiocin inhibits the replication of vaccinia virus in cultured BSC40 cells. All classes of viral proteins were synthesized during synchronous infection in the presence of drug. The onset of DNA replication was delayed slightly, yet the extent of DNA replication in the presence of novobiocin was comparable to that of a control infection. A delay in the temporal transition to late viral protein synthesis was in keeping with the effects on DNA replication. Although the precursor forms of the major viral structural proteins were synthesized normally at late times, the proteolytic processing of these polypeptides was inhibited, which suggested an impediment to virus assembly. Electron microscopy revealed that novobiocin blocked virus morphogenesis at an early stage. Conversion of the concatemeric DNA replication intermediates into hairpin telomeres occurred in the presence of novobiocin, confirming that telomere resolution was not coupled to virus assembly. Novobiocin is the latest addition to a class of antipoxviral agents, which includes rifampin and IMCBH, that arrest morphogenesis.

Animals↗
PubMed1979

Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.

Animals↗
PubMed1994

Novobiocin activates the mating response in yeast through the alpha-pheromone receptor, Ste2p.

In a previous publication we reported the isolation of several yeast mutants sensitive to the coumarin drug novobiocin. Here we characterise the wild-type gene (NBS5), obtained by rescuing a novobiocin-sensitive nbs5 strain to natural novobiocin resistance. We show that nbs5 is allelic to the previously characterised gene sst2, conferring super-sensitivity to the mating pheromones. Although nbs5/sst2 cells of both mating types are super-sensitive to the opposite mating pheromone, only cells of mating-type a are sensitive to novobiocin. We show that the entire effect of the drug is mediated through Ste2p, the alpha-pheromone receptor. Thus, novobiocin is a functional agonist of Ste2p, and may identify a potentially useful interaction between coumarin drugs and the family of G-protein-coupled receptors.

Genetic Complementation Test↗
PubMed2001

Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI.

BACKGROUND: Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. RESULTS: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [3H]beta-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. CONCLUSIONS: This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.

Acylation↗
PubMed2007

Intramammary infections in heifers during early lactation following intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin at the first milking after parturition.

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73.1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61.8% (21/34) were cured following treatment with penicillin-novobiocin and 39.6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52.8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41.4% (12/29) were cured following treatment with penicillin-novobiocin and 23.1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.

Animals↗
PubMed1997

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Binding Sites↗
PubMed2000

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone.

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.

Adenosine Triphosphate↗