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PubMed2003

Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation.

The left ends of the biosynthetic gene clusters of novobiocin ( nov), clorobiocin ( clo) and coumermycin A(1) ( cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A(1) does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-alpha-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.

Bacterial Proteins↗
PubMed1985

Novobiocin blocks the Drosophila heat shock response.

In the studies reported here we show that the antibiotic novobiocin, an in vitro inhibitor of topoisomerase II, blocks the Drosophila heat shock response. If novobiocin is added prior to induction, there is no detectable expression of the Drosophila heat shock genes. Moreover, analysis of the chromatin organization of the 87A7 heat shock locus indicates that the antibiotic prevents the structural alterations which normally accompany heat induction. When novobiocin is added after induction, transcription appears to be rapidly turned off, and the chromatin organization of the 87A7 locus is "fixed" in an "active" configuration. Novobiocin also prevents the re-establishment of the pre-induced 87A7 chromatin organization which occurs during recovery from heat shock. We have also presented data suggesting that this antibiotic blocks transcription at 25 degrees C. These findings raise the possibility that topoisomerase II may be required in eukaryotes for both gene activation and deactivation.

Autoradiography↗
PubMed1987

Stimulation of a Chlamydomonas chloroplast promoter by novobiocin in situ and in E. coli implies regulation by torsional stress in the chloroplast DNA.

We have characterized regulation of a complex Chlamydomonas reinhardtii chloroplast (PA) whose activity is stimulated by the DNA gyrase inhibitor novobiocin, both in the alga itself and in a heterologous Escherichia coli plasmid system. Since novobiocin is known to reduce torsional stress in E. coli DNA, we interpret our results to mean that PA is regulated by torsional stress in the chloroplast DNA. In E. coli, where we could readily manipulate PA, we found that this regulation depends on sequences upstream of PA. These sequences contain at least two different kinds of silencing elements that inhibit PA in the absence of novobiocin. Novobiocin stimulates PA only when the promoter-distal silencing element is present.

Base Sequence↗
PubMed1994

Determination of plasma novobiocin levels by a reversed-phase high-performance liquid chromatographic assay.

A simple and specific reversed-phase high-performance liquid chromatographic (HPLC) assay for the determination of novobiocin levels in human plasma has been developed. The sample preparation was performed by deproteinization with methanol. Prednisone was used as an internal standard. Both novobiocin and prednisone were separated on a C8 column with a gradient elution of acidic water (pH 3.0)-methanol. The recovery of novobiocin from plasma was nearly complete. The linear range was 5-1000 microM in 0.5 ml of plasma with a minimum limit of determination of 2.25 fmol of novobiocin at 254 nm. The method has been implemented and validated in an ongoing clinical trial.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Should novobiocin be clinically re-evaluated?

The current novobiocin spectrum of antimicrobial activity was evaluated by testing 585 staphylococci (393 oxacillin-resistant) and 779 other bacterial strains. Novobiocin inhibited 98.5% of all staphylococcal isolates (MIC 90, 0.25 micrograms/ml) and a significant number of other Gram-positive species at less than or equal to 1 microgram/ml. Pathogenic Neisseria, Haemophilus influenzae, and Branhamella catarrhalis were also very susceptible to novobiocin, e.g., MIC 100, less than or equal to 1 microgram/ml. These results indicate that novobiocin may have a role in contemporary chemotherapy of oxacillin-resistant staphylococcal and other infections.

Bacterial Infections↗
PubMed2005

Novobiocin induces apoptosis-like cell death in topoisomerase II over-expressing arsenite resistant Leishmania donovani.

Leishmaniasis affects millions of people worldwide every year. Lack of effective vaccination, co-infection with other dreaded diseases like AIDS and generation of drug resistant strains demand immediate attention into this neglected area of research. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which shows a 13-fold increase in resistance compared with wild type. Here we report that the arsenite resistant strain of L. donovani promastigotes shows cross-resistance to novobiocin, a catalytic inhibitor of topoisomerase II, with IC50 value of 320 microg ml-1 as compared with 242 microg ml-1 for wild type L. donovani. Leishmanicidal action of novobiocin induces dose- and time-dependent increase in cell death. Treatment with IC50 of novobiocin caused morphological and biochemical changes which lead to induction of cell death exhibiting characteristic features of metazoan apoptosis. Phosphatidylserine externalization, cytochrome C release to cytoplasm, activation of caspases, oligonucleosomal DNA fragmentation and in situ labelling of condensed and fragmented nuclei in both wild type and arsenite resistant L. donovani promastigotes strongly suggest the apoptosis-like mode of cell death. Cross-resistance to novobiocin in arsenite resistant strain has been correlated to over-expression of topoisomerase II and substantiated by differential inhibition of enzyme activity in wild type and arsenite resistant L. donovani.

Animals↗
PubMed2005

Characterization of the aminocoumarin ligase SimL from the simocyclinone pathway and tandem incubation with NovM,P,N from the novobiocin pathway.

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.

Amide Synthases↗
PubMed2005

Hsp90 inhibitors identified from a library of novobiocin analogues.

Novobiocin is a C-terminal inhibitor of the Hsp90 protein folding machinery, which is responsible for the conformational maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity ( approximately 700 muM), very little attention has been paid toward the development of novobiocin analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological activity of each evaluated by Western blot analysis of Hsp90 client proteins. A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to cause the degradation of several Hsp90 client proteins in both breast and prostate cancer cell lines. In the presence of 1 muM A4, several Hsp90 client proteins were degraded, including AKT, Her2, Hif-1alpha, and the androgen receptor.

Cell Line, Tumor↗
PubMed2006

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Binding Sites↗
PubMed2004

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton.

The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

Kinetics↗
PubMed1983

ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.

Nuclear novobiocin binding proteins (NBPs) from a set of mouse L cells have been extensively purified by affinity chromatography on novobiocin-Sepharose columns. The NBPs, specifically eluted with 100 micrograms of novobiocin per ml, exhibited equivalent DNA topoisomerase activities (measured as ATP-dependent relaxation or catenation of phi X174 replicative-form I DNA substrate) when extracted from equal numbers of wild-type (WT-4) mouse L cells growing logarithmically at 34 degrees C or at 38.5 degrees C, from ts A1S9 cells similarly cultivated at the low, permissive temperature or from revertant ts+ AR cells in exponential growth at either temperature. The NBPs isolated from similar numbers of ts A1S9 cells grown to midlogarithmic phase and then incubated for 24 hr at 38.5 degrees C (the nonpermissive temperature) showed no topoisomerase II activity. Preliminary NaDodSO4/polyacrylamide gel electrophoretic analysis of enzymatically active material revealed that the NBPs of WT-4 and ts+ AR cells grown at 34 degrees C comprised three major polypeptides of 76,000, 74,000, and 30,000 daltons and a number of larger molecular mass components present in trace amounts. The NBP of ts A1S9 cells grown at the permissive temperature was similar, except that the 30,000-kilodalton polypeptide was not detected. Such enzymatically active NBPs from WT-4 and ts+ AR cells were unaffected by 100 micrograms of novobiocin per ml, whereas the analogous preparation from ts A1S9 cells was totally inhibited. On the basis of these and other considerations, it is postulated that the ts A1S9 locus of mouse L cells encodes a temperature-sensitive polypeptide that is required for normal DNA topoisomerase II activity.

Animals↗
PubMed1996

Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.

Aminocoumarins↗
PubMed1982

Specificity and completeness of inhibition of DNA repair by novobiocin and aphidicolin.

Novobiocin and aphidicolin were both potent inhibitors of excision repair of u.v.-induced damage to DNA in human embryonic fibroblasts, and both also inhibited semiconservative DNA replication even more strongly. The mechanism of action of these two drugs is, however, different. Novobiocin inhibited repair replication without accumulating single-strand breaks, but aphidicolin inhibited repair replication with the accumulation of numerous single-strand breaks. Novobiocin appears to inhibit repair at an earlier stage than aphidicolin, which may indicate that DNA topoisomerases play a role in eukaryotic DNA repair. Digestion of DNA by exonuclease III indicated that repair patches in novobiocin-treated cells contained no excess 3'OH termini, whereas up to 40% of the repaired DNA in aphidicolin-treated cells had free 3'OH termini. Therefore, although aphidicolin resulted in the accumulation of single-strand breaks, many of the repair events escaped inhibition and the number of breaks is an underestimate of the true number of repair events.

Antibiotics, Antineoplastic↗
PubMed1985

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision step.

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested (nondividing) human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 micrograms ml-1 nalidixic acid and 1.0 micrograms ml-1 aphidicolin is unchanged when compared with that due to treatment with 1.0 micrograms ml-1 aphidicolin alone, while that for 150 micrograms ml-1 novobiocin + 1.0 micrograms ml-1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 micrograms ml-1 novobiocin inhibited repair synthesis by approximately 60% over the fluence range employed. Nalidixic acid at a concentration of 300 micrograms ml-1 caused no detectable inhibition of repair synthesis. We conclude that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage.

Aphidicolin↗
PubMed1985

Activity of novobiocin against methicillin-resistant Staphylococcus aureus.

As infections due to methicillin-resistant Staphylococcus aureus become increasingly prevalent, newer alternative antibiotics, especially those which are orally administered, will be required. In order to provide an in-vitro basis for selecting alternative antibiotics, we studied the susceptibility of 103 strains of methicillin-resistant Staph. aureus from seven institutions to oral antimicrobial agents. Novobiocin, rifampicin, and trimethoprim-sulphamethoxazole had excellent in-vitro activity against virtually all strains of methicillin-resistant Staph. aureus. The MIC90 and MBC90 of novobiocin against methicillin-resistant Staph. aureus were 0.25 mg/l. Since previously reported achievable serum levels with oral novobiocin are 100 to 200 times its MIC90 against methicillin-resistant Staph. aureus, novobiocin should be evaluated further for combination therapy with rifampicin or trimethoprim-sulphamethoxazole.

Humans↗
PubMed1979

Novobiocin; an inhibitor of the repair of UV-induced but not X-ray-induced damage in mammalian cells.

In addition to inhibiting replicative DNA synthesis in HeLa cells, novobiocin has a severe effect on the cellular response to UV irradiation, reducing the number of breaks made in pre-existing DNA by the excision repair process. The inhibition of UV repair by novobiocin is reflected in enhanced UV-killing of these cells. Rejoining of DNA after X irradiation is not impaired by novobiocin. The recognition and removal of UV damage may require unwinding of the DNA by gyrase, which--in bacteria--is the target for novobiocin.

DNA Repair↗
PubMed1981

DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties.

Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.

Adenosine Triphosphatases↗
PubMed1981

Effect of novobiocin and other DNA gyrase inhibitors on virus replication and DNA synthesis in herpes simplex virus type 1-infected BHK cells.

The four known inhibitors of the bacterial DNA gyrase (nalidixic acid, oxolinic acid, novobiocin and coumermycin A) were investigated with respect to their effect on the growth of uninfected BHK cells and the yield of virus from herpes simplex virus type 1 (HSV-1)-infected BHK cells. High concentrations of nalidixic acid and oxolinic acid (about 10 mM) were needed for 50% inhibition of cellular and viral multiplication with less than fourfold preferential inhibition of virus over cell growth. Novobiocin and coumermycin were effective at lower molar concentrations and the amount needed for 50% inhibition was 10-fold higher for cell growth than for virus yield. At 5 x 10(-4) M, novobiocin inhibited DNA synthesis in uninfected cells to approx. 20% of non-treated controls, while virus DNA in infected cells was almost completely inhibited (approx. 1% of controls). Residual cellular DNA synthesis in infected cells was rather insensitive (approx. 90% of controls) to this concentration of novobiocin.

Aminocoumarins↗