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Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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couo and novo c methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and Novobiocin antibiotic biosynthesis
Biochemistry, 2005Co-Authors: Michelle Pacholec, Christopher T WalshAbstract:During the biosynthesis of the streptomycete aminocoumarin antibiotics Novobiocin and the dimeric coumermycin A1, the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the speci...
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characterization of the aminocoumarin ligase siml from the simocyclinone pathway and tandem incubation with novm p n from the Novobiocin pathway
Biochemistry, 2005Co-Authors: Michelle Pacholec, Caren Freel L Meyers, Markus Oberthür, Daniel Kahne, Christopher T WalshAbstract:Simocyclinone D8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics Novobiocin, clorobiocin, and coumermycin A1, 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.
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mass spectrometric characterization of a three enzyme tandem reaction for assembly and modification of the Novobiocin skeleton
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Caren Freel L Meyers, Michelle Pacholec, Christopher T Walsh, Julie A LearyAbstract: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.
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initial characterization of novobiocic acid noviosyl transferase activity of novm in biosynthesis of the antibiotic Novobiocin
Biochemistry, 2003Co-Authors: Caren Freel L Meyers, Markus Oberthür, Daniel Kahne, John W Anderson, Christopher T WalshAbstract:The aminocoumarin class of antibiotics, exemplified by Novobiocin, is composed of tripartite l-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar component interacts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa l-noviosyl transferase from Streptomyces spheroides into Escherichia coli, expressed it in soluble form, and purified it to homogeneity as a C-terminal His(8) fusion protein. The aglycone novobiocic acid, obtained from selective degradation of Novobiocin, and TDP-l-noviose, obtained by an 11-step chemical synthesis from l-rhamnose, were shown to be robust substrates for NovM to produce the desmethyldescarbamoyl Novobiocin intermediate with a k(cat) of >300 min(-1). NovM displays activity with variant coumarin aglycones, suggesting it may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovM shows no activity with and is inhibited by TDP-l-rhamnose (K(i) = 83.5 +/- 5.5 microM), the sugar donor that most closely structurally resembles the natural substrate TDP-l-noviose. The NovM reaction products generated during the course of this work will serve as substrates for subsequent analysis of the NovP and NovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibiotic activity.
Francisco Soriano - One of the best experts on this subject based on the ideXlab platform.
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comparative efficacy of Novobiocin and amoxicillin in experimental sepsis caused by β lactam susceptible and highly resistant pneumococci
International Journal of Antimicrobial Agents, 2010Co-Authors: Violeta Rodriguezcerrato, Gema Del Prado, Lorena Huelves, Plínio Naves, Vicente Ruiz, Ernesto García, Carmen Ponte, Francisco SorianoAbstract:Therapeutic alternatives are needed against infections caused by highly multidrug-resistant Streptococcus pneumoniae. Novobiocin, an old antibiotic, was tested in vitro and in a murine sepsis model against one amoxicillin-susceptible and three amoxicillin-resistant strains [minimum inhibitory concentrations (MICs) 8-64 mg/L]. Novobiocin MICs for all strains were 0.25-0.5 mg/L. In sepsis, Novobiocin and amoxicillin were evaluated at 25, 50, 100 and 200 mg/kg given at 1, 5, 24 and 48 h post bacterial challenge. The most effective regimens in animals infected with the amoxicillin-susceptible strain were 200 mg/kg Novobiocin and 25 mg/kg amoxicillin, achieving 100% survival and undetectable organisms in the peritoneum. Among mice infected with amoxicillin-resistant S. pneumoniae, 200 mg/kg Novobiocin gave the highest protection (90-100% survivors), followed by 200mg/kg amoxicillin (60-100%), 100 mg/kg Novobiocin (50-87.5%) and 50 mg/kg amoxicillin (14.3-25%). The killing effect of antibiotics in the peritoneum (mean Deltalog(10) colony-forming units/mL between treated and control mice) was as follows: 200 mg/kg Novobiocin (-6.6)>200 mg/kg amoxicillin (-5.6)>100 mg/kg Novobiocin (-3.7) > 50 mg/kg amoxicillin (-0.7). Total plasma and ultrafiltrate pharmacokinetics of Novobiocin (200 mg/kg, single dose) in non-infected mice showed, respectively, half-lives of 151 min and 215 min, area under the concentration-time curves (AUCs) of 945.0 mgh/L and 136.6 mgh/L and maximal concentrations of 147 mg/L and 18 mg/L. Novobiocin may be a promising agent for therapy of highly beta-lactam-resistant pneumococcal infections.
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Comparative efficacy of Novobiocin and amoxicillin in experimental sepsis caused by β-lactam-susceptible and highly resistant pneumococci
International Journal of Antimicrobial Agents, 2010Co-Authors: Violeta Rodríguez-cerrato, Gema Del Prado, Lorena Huelves, Plínio Naves, Vicente Ruiz, Ernesto García, Carmen Ponte, Francisco SorianoAbstract:Therapeutic alternatives are needed against infections caused by highly multidrug-resistant . Novobiocin, an old antibiotic, was tested in vitro and in a murine sepsis model against one amoxicillin-susceptible and three amoxicillin-resistant strains [minimum inhibitory concentrations (MICs) 8-64mg/L]. Novobiocin MICs for all strains were 0.25-0.5mg/L. In sepsis, Novobiocin and amoxicillin were evaluated at 25, 50, 100 and 200mg/kg given at 1, 5, 24 and 48h post bacterial challenge. The most effective regimens in animals infected with the amoxicillin-susceptible strain were 200mg/kg Novobiocin and 25mg/kg amoxicillin, achieving 100% survival and undetectable organisms in the peritoneum. Among mice infected with amoxicillin-resistant , 200mg/kg Novobiocin gave the highest protection (90-100% survivors), followed by 200mg/kg amoxicillin (60-100%), 100mg/kg Novobiocin (50-87.5%) and 50mg/kg amoxicillin (14.3-25%). The killing effect of antibiotics in the peritoneum (mean Δlog colony-forming units/mL between treated and control mice) was as follows: 200mg/kg Novobiocin (−6.6)>200mg/kg amoxicillin (−5.6)>100mg/kg Novobiocin (−3.7)>50mg/kg amoxicillin (−0.7). Total plasma and ultrafiltrate pharmacokinetics of Novobiocin (200mg/kg, single dose) in non-infected mice showed, respectively, half-lives of 151min and 215min, area under the concentration-time curves (AUCs) of 945.0mg h/L and 136.6mg h/L and maximal concentrations of 147mg/L and 18mg/L. Novobiocin may be a promising agent for therapy of highly β-lactam-resistant pneumococcal infections.
Caren Freel L Meyers - One of the best experts on this subject based on the ideXlab platform.
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characterization of the aminocoumarin ligase siml from the simocyclinone pathway and tandem incubation with novm p n from the Novobiocin pathway
Biochemistry, 2005Co-Authors: Michelle Pacholec, Caren Freel L Meyers, Markus Oberthür, Daniel Kahne, Christopher T WalshAbstract:Simocyclinone D8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics Novobiocin, clorobiocin, and coumermycin A1, 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.
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mass spectrometric characterization of a three enzyme tandem reaction for assembly and modification of the Novobiocin skeleton
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Caren Freel L Meyers, Michelle Pacholec, Christopher T Walsh, Julie A LearyAbstract: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.
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initial characterization of novobiocic acid noviosyl transferase activity of novm in biosynthesis of the antibiotic Novobiocin
Biochemistry, 2003Co-Authors: Caren Freel L Meyers, Markus Oberthür, Daniel Kahne, John W Anderson, Christopher T WalshAbstract:The aminocoumarin class of antibiotics, exemplified by Novobiocin, is composed of tripartite l-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar component interacts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa l-noviosyl transferase from Streptomyces spheroides into Escherichia coli, expressed it in soluble form, and purified it to homogeneity as a C-terminal His(8) fusion protein. The aglycone novobiocic acid, obtained from selective degradation of Novobiocin, and TDP-l-noviose, obtained by an 11-step chemical synthesis from l-rhamnose, were shown to be robust substrates for NovM to produce the desmethyldescarbamoyl Novobiocin intermediate with a k(cat) of >300 min(-1). NovM displays activity with variant coumarin aglycones, suggesting it may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovM shows no activity with and is inhibited by TDP-l-rhamnose (K(i) = 83.5 +/- 5.5 microM), the sugar donor that most closely structurally resembles the natural substrate TDP-l-noviose. The NovM reaction products generated during the course of this work will serve as substrates for subsequent analysis of the NovP and NovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibiotic activity.
Michelle Pacholec - One of the best experts on this subject based on the ideXlab platform.
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couo and novo c methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and Novobiocin antibiotic biosynthesis
Biochemistry, 2005Co-Authors: Michelle Pacholec, Christopher T WalshAbstract:During the biosynthesis of the streptomycete aminocoumarin antibiotics Novobiocin and the dimeric coumermycin A1, the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the speci...
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characterization of the aminocoumarin ligase siml from the simocyclinone pathway and tandem incubation with novm p n from the Novobiocin pathway
Biochemistry, 2005Co-Authors: Michelle Pacholec, Caren Freel L Meyers, Markus Oberthür, Daniel Kahne, Christopher T WalshAbstract:Simocyclinone D8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics Novobiocin, clorobiocin, and coumermycin A1, 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.
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mass spectrometric characterization of a three enzyme tandem reaction for assembly and modification of the Novobiocin skeleton
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Caren Freel L Meyers, Michelle Pacholec, Christopher T Walsh, Julie A LearyAbstract: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.
Alan C Sartorelli - One of the best experts on this subject based on the ideXlab platform.
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structure activity studies of Novobiocin analogs as modulators of the cytotoxicity of etoposide vp 16
Oncology Research, 2001Co-Authors: Germana Rappa, Krishnamurthy Shyam, Aurelio Lorico, Oystein Fodstad, Alan C SartorelliAbstract:We have previously reported that the antibiotic Novobiocin enhanced the toxicity of the anticancer agent etoposide (VP-16) to several drug-sensitive and -resistant tumor cell lines. The increase in VP-16 cytotoxicity produced by Novobiocin was not due to the combined effects of these agents on topoisomerase II, but to inhibition by Novobiocin of VP-16 efflux, which in turn led to increased accumulation of VP-16 and increased formation of potentially lethal VP-16-stabilized topoisomerase II-DNA covalent complexes. We have now identified Novobiocin analogs that are essentially equivalent to Novobiocin as inhibitors of the activity of topoisomerase II, but that are more potent than Novobiocin (a) as modulators of the cytotoxicity of VP-16 to WEHI-3B leukemia and A549 lung carcinoma cells and (b) in increasing VP-16 accumulation in these cell lines. Thus, removal of the sugar moiety of Novobiocin to form novobiocic acid enhanced the potency of the antibiotic as a modulator of VP-16, whereas the substituted coumarin ring alone (U-7587) was devoid of VP-16 modulatory activity. Modifications of the side chain of Novobiocin significantly influenced modulatory activity, with cyclonovobiocic acid, which was formed from novobiocic acid by acid-catalyzed cycloaddition, being the most active in enhancing the cytotoxicity of VP-16. The increased potency of novobiocic acid and cyclonovobiocic acid as modulators of VP-16 activity was achieved with no change from Novobiocin in the capacity of these analogs to inhibit the catalytic activity of mammalian topoisomerase II, indicating a change in the specificity of these analogs.
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reversal of etoposide resistance in non p glycoprotein expressing multidrug resistant tumor cell lines by Novobiocin
Cancer Research, 1993Co-Authors: Germana Rappa, Aurelio Lorico, Alan C SartorelliAbstract:Previous reports from this laboratory have demonstrated that Novobiocin produces supraadditive cytotoxicity and increases the formation of drug-stabilized topoisomerase II-DNA covalent complexes in WEHI-3B myelomonocytic leukemia and A549 lung carcinoma cells when combined with etoposide (VP-16). Inhibition of the efflux of VP-16 by Novobiocin is responsible for the increase in VP-16 accumulation, which in turn leads to increased formation of VP-16-stabilized topoisomerase II-DNA covalent complexes and increased cytotoxicity. We now report that Novobiocin synergistically enhanced the sensitivity of the multidrug resistant variants, WEHI-3B/NOVO and A549(VP)28, to VP-16, causing almost complete reversal of the resistance to the epipodophyllotoxin. These two tumor cell variants are resistant to several topoisomerase II-targeted drugs, particularly VP-16, but not to Vinca alkaloids; this finding corresponds to the fact that they do not overexpress the P-glycoprotein. The effects of Novobiocin in these resistant sublines are mediated through the intracellular accumulation of VP-16, resulting in an increase in the formation of lethal VP-16-induced topoisomerase II-DNA covalent complexes. In the P-glycoprotein expressing multidrug resistant HCT116(VM)34 colon carcinoma and L1210/VMDRC0.06 leukemia cell lines, the latter being transfected with the human mdr-1 gene, Novobiocin did not potentiate the cytotoxic activity of VP-16 nor increase the intracellular accumulation of VP-16 and the formation of covalent complexes, whereas their normal counterparts were sensitive to the potentiating activity of Novobiocin when used in combination with VP-16. These results indicate that the action of Novobiocin on the intracellular transport of VP-16 is not directed at the level of the P-glycoprotein, but that the action of Novobiocin is antagonized by the presence of the P-glycoprotein. Since Novobiocin is a clinically available antibiotic, has numerous structural analogues available for comparative studies, and has a relatively low toxicity profile, this drug, as well as structurally related agents, would appear to have significant clinical potential in combination with an epipodophyllotoxin for the treatment of non-P-glycoprotein expressing multidrug resistant tumors.
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Potentiation by Novobiocin of the cytotoxic activity of etoposide (VP-16) and teniposide (VM-26)
International Journal of Cancer, 1992Co-Authors: Germana Rappa, Aurelio Lorico, Alan C SartorelliAbstract:The coumermycin antibiotic Novobiocin, which interacts with the nuclear enzyme topoisomerase II, produced supra-additive toxicity to WEHI-3B D+ leukemia cells at clinically achievable concentrations, when combined with teniposide (VM-26) or etoposide (VP-16). Simultaneous exposure of cells to both agents was required for maximum efficacy of the combination. Novobiocin also produced supra-additive toxicity to A549 human lung carcinoma cells when combined with VM-26 or VP-16. At concentrations above the peak plasma levels achievable in patients, Novobiocin lost its potentiating activity. Exposure of WEHI-3B D+ cells to Novobiocin did not modify the cytotoxicity produced by the topoisomerase II inhibitor m-AMSA, whereas, in contrast, Novobiocin antagonized the cytotoxicity of m-AMSA in A549 cells. Although it has been suggested that inhibitors of the syntheses of DNA and RNA interfere with the cytotoxic activity of the epipodophyllotoxins, maximum potentiation of the cytotoxicities of VP-16 and VM-26 occurred at Novobiocin concentrations that decreased the rates of synthesis of both DNA and RNA in WEHI-3B D+ cells by about 50%. The number of DNA-topoisomerase-ll covalent complexes stabilized by VM-26 in WEHI-3B D+ cells was greatly increased when cells were exposed simultaneously to VM-26 and Novobiocin for 1 hr, but not when cells were treated with m-AMSA and Novobiocin for the same period of time. Novobiocin did not affect the amount of covalent complexes produced by VM-26 in isolated nuclei, suggesting that the potentiating activity of Novobiocin was not due to its direct interaction with the nuclear topoisomerase II enzyme. Our findings suggest that therapeutic levels of Novobiocin may be capable of enhancing the clinical activities of VP-16 and VM-26. © 1992 Wifey-Liss, Inc.
