coumermycin and clorobiocin

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.

Topics: Aminocoumarins; Genes, Bacterial; Molecular Structure; Multigene Family; Novobiocin; Streptomyces

The coumarin group of antibiotics have as their target the bacterial enzyme DNA gyrase. The drugs bind to the B subunit of gyrase and inhibit DNA supercoiling by blocking the ATPase activity. Recent data show that the binding site for the drugs lies within the N-terminal part of the B protein, and individual amino acids involved in coumarin interaction are being identified. The mode of inhibition of the gyrase ATPase reaction by coumarins is unlikely to be simple competitive inhibition, and the drugs may act by stabilizing a conformation of the enzyme with low affinity for ATP.

Topics: Adenosine Triphosphatases; Aminocoumarins; Bacterial Proteins; Binding Sites; Coumarins; DNA Topoisomerases, Type II; DNA, Bacterial; DNA, Superhelical; Escherichia coli; Models, Molecular; Novobiocin; Protein Binding; Protein Structure, Tertiary; Structure-Activity Relationship; Topoisomerase II Inhibitors

Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant to many antibiotics, there is a paucity of effective treatment options and there is no vaccine. In the present study, the efficacies of selected aminocoumarin compounds, DNA gyrase inhibitors that were discovered in the 1950s but are not in clinical use for the treatment of melioidosis were investigated. Clorobiocin and coumermycin were shown to be particularly effective in treating B. pseudomallei infection in vivo. A novel formulation with dl-tryptophan or l-tyrosine was shown to further enhance aminocoumarin potency in vivo. It was demonstrated that coumermycin has superior pharmacokinetic properties compared with novobiocin, and the coumermycin in l-tyrosine formulation can be used as an effective treatment for acute respiratory melioidosis in a murine model. Repurposing of existing approved antibiotics offers new resources in a challenging era of drug development and antimicrobial resistance.

Topics: Aminocoumarins; Animals; Burkholderia pseudomallei; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Drug Therapy, Combination; Female; Melioidosis; Mice; Mice, Inbred BALB C; Moths; Novobiocin; Tryptophan

The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A(1) and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2-2 mg l(-1)) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l(-1) in the presence of 0.6% Q2-5247 and 0.2 mg l(-1) CoCl(2)). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l(-1) on average).

Topics: Aminocoumarins; Azepines; Gene Expression; Genes, Bacterial; Multigene Family; Novobiocin; Streptomyces coelicolor

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are formed by different Streptomyces strains and are potent inhibitors of bacterial gyrase. Their biosynthetic gene clusters have been analyzed in detail by genetic and biochemical investigations. Heterologous expression of these gene clusters by site-specific integration into the genome of the fully sequenced host Streptomyces coelicolor A3(2) readily results in an accumulation of the antibiotics in yields similar to the wildtype strains. In recent years, the aminocoumarins have developed into a model system for the generation of new antibiotics by genetic methods. Prior to heterologous expression in S. coelicolor, cosmids containing the complete biosynthetic clusters can be manipulated in Escherichia coli by lambda RED-mediated recombination, creating single or multiple gene replacements or gene deletions. Thereby, mutant strains are generated which are blocked in the synthesis of certain intermediates or in specific tailoring reactions. For instance, mutasynthetic experiments can subsequently be carried out to generate aminocoumarin antibiotics that contain modified acyl moieties attached to the aminocoumarin core, and chemoenzymatic synthesis can be employed for the acylation of the deoxysugar moiety of structural analogues of the aminocoumarin antibiotics. Metabolic engineering-the combination of gene deletions and foreign gene expression via replicative expression vectors-can be used to generate further structural variants of these antibiotics. These methods can be combined, allowing the generation of a wide variety of new compounds. This chapter may provide general pointers for the use of genetic methods in the generation of new antibiotics.

Topics: Amide Synthases; Aminocoumarins; Models, Genetic; Molecular Structure; Novobiocin

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

Topics: Adenosine Triphosphate; Aminocoumarins; Aminoglycosides; Bacillus subtilis; Bromine; Catalytic Domain; Enterobactin; Mass Spectrometry; Multigene Family; Novobiocin; Peptide Synthases; Phenols; Pyrrolidinones; Substrate Specificity; Sulfhydryl Compounds; Thiazoles

The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway.

Topics: Acyltransferases; Adenosine Monophosphate; Aminocoumarins; Antibiotics, Antineoplastic; Catalysis; Cloning, Molecular; Escherichia coli; Genes, Fungal; Kinetics; Novobiocin; Recombinant Proteins; Streptomyces

The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.

Topics: Amino Acid Sequence; Aminocoumarins; Chromatography, High Pressure Liquid; Coumarins; DNA Primers; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Novobiocin; Polymerase Chain Reaction; Pyrroles; Restriction Mapping; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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.

Topics: Amide Synthases; Aminocoumarins; Anti-Bacterial Agents; Carboxylic Acids; Coumarins; Gene Expression Regulation, Bacterial; Glycosides; Novobiocin; Streptomyces antibioticus; Substrate Specificity

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A(1) are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL, novL, and couL, respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and (1)H-NMR spectroscopy.

Topics: Amide Synthases; Aminocoumarins; Animals; Anti-Bacterial Agents; Coumarins; Gas Chromatography-Mass Spectrometry; Gram-Positive Bacteria; Humans; Mutation; Novobiocin; Plasmids; Substrate Specificity; Up-Regulation

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects. The combination of full scan mass spectrometry (MS), parent ion scan MS, product ion scan MS, and in-source collision-induced fragmentation prior to product ion scans (pseudo-MS(3) scan), using characteristic fragmentation of the central aminocoumarin unit, was employed for the detection and structural interpretation of expected and new intermediates. We were able to show the applicability of this methodology to the three culture extracts, where the main intermediates could be found, and to demonstrate its use for interpretation of secondary metabolite biosynthesis. Some new compounds were discovered, including bis-carbamoylated novobiocin, hydroxylated clorobiocin, and several structurally and not yet fully elucidated coumermycin derivatives or precursors.

Topics: Aminocoumarins; Cell Extracts; Coumarins; Enzyme Inhibitors; Mass Spectrometry; Novobiocin; Signal Transduction; Streptomyces

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A(1), and clorobiocin were investigated. All three clusters contained a gyrB(R) resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A(1) clusters were found to contain an additional, similar gene, named parY(R). Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB(R) and likewise of parY(R) in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A(1), suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB(R) or parY(R) and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A(1) cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A(1), suggesting that these genes may be involved in antibiotic transport.

Topics: Aminocoumarins; Anti-Bacterial Agents; Blotting, Southern; Coumarins; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Bacterial; In Situ Hybridization; Microbial Sensitivity Tests; Multigene Family; Novobiocin; Phylogeny; Plasmids; Streptomyces

The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety. The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies. CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase. In the present study, the enzyme CloR was overexpressed in Escherichia coli, purified, and identified as a bifunctional non-heme iron oxygenase, which converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to 3DMA-4HB by two consecutive oxidative decarboxylation steps. In 18O2 labeling experiments we showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product 3DMA-4HB during the second reaction step. CloR does not show sequence similarity to known oxygenases. It apparently presents a novel member of the diverse family of the non-heme iron (II) and alpha-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an alpha-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature.

Topics: Aminocoumarins; Bacterial Proteins; Benzoates; Coumarins; Gene Expression Regulation, Bacterial; Heme; Iron; Novobiocin; Oxygenases; Parabens; Streptomyces

The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.

Topics: Amino Acid Sequence; Aminocoumarins; Cloning, Molecular; Coumarins; Kinetics; Mixed Function Oxygenases; Molecular Sequence Data; Multienzyme Complexes; Novobiocin; Sequence Homology, Amino Acid; Streptomyces; Substrate Specificity

The biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin was cloned by screening of a cosmid library of Streptomyces roseochromogenes DS 12.976 with two heterologous probes from the novobiocin biosynthetic gene cluster. Sequence analysis revealed 27 ORFs with striking similarity to the biosynthetic gene clusters of novobiocin and coumermycin A(1). Inactivation of a putative aldolase gene, cloR, by in-frame deletion led to the abolishment of the production of clorobiocin. Feeding of the mutant with 3-dimethylallyl-4-hydroxybenzoic acid (Ring A of clorobiocin) restored clorobiocin production. Here, it is suggested that the formation of Ring A of clorobiocin may proceed via a retro-aldol reaction catalysed by CloR, i.e. by a mechanism different from the previously elucidated benzoic acid biosynthetic pathway in Streptomyces maritimus. A comparison of the gene clusters for clorobiocin, novobiocin and coumermycin A(1) showed that the structural differences between the three antibiotics were reflected remarkably well by differences in the organization of their respective biosynthetic gene clusters.

Topics: Amino Acid Sequence; Aminocoumarins; Bacterial Proteins; Cloning, Molecular; Coumarins; Fructose-Bisphosphate Aldolase; Genes, Bacterial; Molecular Sequence Data; Multigene Family; Novobiocin; Open Reading Frames; Sequence Analysis, DNA; Streptomyces

Heat shock protein 90 (Hsp90) interacts with and stabilizes several oncogenic protein kinases (e.g., p185(erbB2), p60(v-src), and Raf-1) and is required for the stability and dominant-negative function of mutated p53 protein. Two unrelated antibiotics, geldanamycin and radicicol, bind specifically to an atypical nucleotide-binding pocket of Hsp90, a site that shares homology with the adenosine triphosphate (ATP)-binding domain of bacterial DNA gyrase B. This interaction leads to destabilization of proteins that interact with Hsp90. Since the nucleotide-binding site of gyrase B is targeted by coumarin antibiotics (e.g., novobiocin), we investigated whether these drugs can also interact with Hsp90 and affect its activity.. We used immobilized novobiocin, geldanamycin, or radicicol to isolate either endogenous Hsp90 from cell lysates or Hsp90 deletion fragments translated in vitro. Effects of the coumarin antibiotics novobiocin, chlorobiocin, and coumermycin A1 on several proteins interacting with Hsp90 were assessed in vitro and in vivo.. Hsp90 binding to immobilized novobiocin was competed by soluble coumarins and ATP but not by geldanamycin or radicicol. A carboxy-terminal Hsp90 fragment bound immobilized novobiocin but not immobilized geldanamycin, while a geldanamycin-binding amino-terminal fragment did not bind novobiocin. All three coumarins markedly reduced cellular levels of p185(erbB2), p60(v-src), Raf-1, and mutated p53. Furthermore, novobiocin reduced Raf-1 levels in the spleens of mice treated with the drug.. These coumarin antibiotics, particularly novobiocin, represent a first-generation alternative to other Hsp90-targeting drugs that are not as well tolerated. Novobiocin's unique interaction with Hsp90 identifies an additional site on this protein amenable to pharmacologic interference with small molecules.

Topics: Aminocoumarins; Animals; Antibiotics, Antineoplastic; Blotting, Western; Breast Neoplasms; Carcinoma; Carrier Proteins; Coumarins; DNA Topoisomerases, Type II; Enzyme Inhibitors; Female; Fibroblasts; HSP90 Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Mice; Novobiocin; Oncogene Protein pp60(v-src); Protein Binding; Proto-Oncogene Proteins c-raf; Signal Transduction; Spleen; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.

Topics: Adenylyl Imidodiphosphate; Aminocoumarins; Anti-Bacterial Agents; Binding Sites; Coumarins; Cross-Linking Reagents; DNA Gyrase; DNA Topoisomerases, Type II; Kinetics; Novobiocin; Protein Folding; Topoisomerase II Inhibitors

Novobiocin, coumermycin A1, and clorobiocin, structurally related compounds that antagonize the B subunit of the essential bacterial enzyme DNA gyrase, were compared with 18 of their analogs for the inhibition of Escherichia coli DNA gyrase supertwisting activity in vitro and of bacterial multiplication. This family of compounds has a 4-hydroxy-8-methylcoumarin core substituted in the 7 and 3 positions. Important for enzyme inhibition in vitro is a 7 ether linkage to a 3'-substituted noviose sugar. The 3'-ester-linked 5-methylpyrrole, found in the coumermycin series, conferred at least 10-fold more inhibitory activity than did the similarly linked amide, found in the novobiocin series; lack of the pyrrole and amide results in the loss of inhibitory activity. Of many aryl and alkyl substituents linked as an amide at the 3 position, the 4-hydroxyl-3-(3-methyl-2-butenyl)benzoic acid moiety, found in novobiocin and clorobiocin, and the reduplication of the coumarin-noviose-5-methylpyrrole, found in coumermycin A1, were most effective in gyrase inhibition. In vivo, the ability of these compounds to inhibit the growth of E. coli varied greatly. The enhanced inhibition of gyrase in vitro conferred by a 5-methylpyrrole relative to an amide in the 3'-noviose position was reflected in inhibition of bacterial multiplication. Several substitutions at the 3 position of the coumarin core conferring similar antagonism of gyrase in vitro resulted in substantially different inhibitory activities for E. coli, suggesting that these moieties at the 3 position affect drug access to the intracellular target. This target was shown for isobutyryl PNC-NH2 (PNC-NH2 is 3-amino-4-hydroxy-8-methyl-7-[3-O-(5-methyl-2-pyrrolylcarbonyl)noviosyloxy] coumarin) and confirmed for novobiocin, coumermycin A1, and clorobiocin to be in the B subunit of DNA gyrase.

Topics: Aminocoumarins; Anti-Bacterial Agents; Coumarins; DNA Topoisomerases, Type II; Escherichia coli; Microbial Sensitivity Tests; Novobiocin