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Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP- N -acetylglucosamine to yield corresponding midecamycin 2'- O -glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP- N -acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2'- O -glucopyranoside, midecamycin 2'- O -GlcNAc and midecamycin 2'- O -xylopyranoside. In contrast to midecamycin, these midecamycin 2'- O -glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca , we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

The cytochrome P450 enzyme RosC catalyzes a two-step, hydroxylation and alcohol oxidation, oxidation reaction to form the C-20 formyl group in the biosynthesis of a 16-membered macrolide antibiotic rosamicin produced by Micromonospora rosaria IFO13697. RosC is presumed to be involved in the formation of 20-carboxyrosamicin because it has been isolated from the culture broth of M. rosaria. Here, we confirmed that RosC has catalytic activity, with E. coli expressing RosC converting rosamicin into 20-carboxyrosamicin. Therefore, it was revealed that RosC is a multifunctional P450 that catalyzes a three-step oxidation reaction that leads to the formation of the hydroxyl group, formyl group and carboxyl group at C-20 on the macrolactone ring in the rosamicin biosynthetic pathway. Moreover, the cytochrome P450 enzyme TylI, which is involved in formation of the formyl group of a 16-membered macrolide antibiotic tylosin produced by Streptomyces fradiae ATCC 19609, also converted rosamicin into 20-carboxyrosamicin., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The S-adenosyl-l-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2‴-O-methylation of the 6-deoxy-d-allose bound to demethylmacrocin or demethyllactenocin precedes the TylF-catalyzed C3‴-O-methylation of the d-javose (C2‴-O-methylated 6-deoxy-d-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of d-mycinose (C3‴-O-methylated d-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.

Salinipyrones and pacificanones are structurally related polyketides from Salinispora pacifica CNS-237 that are proposed to arise from the same modular polyketide synthase (PKS) assembly line. Genome sequencing revealed a large macrolide PKS gene cluster that codes for the biosynthesis of rosamicin A and a series of new macrolide antibiotics. Mutagenesis experiments unexpectedly correlated salinipyrone and pacificanone biosynthesis to the rosamicin octamodule Spr PKS. Remarkably, this bifurcated polyketide pathway illuminates a series of enzymatic domain- and module-skipping reactions that give rise to natural polyketide product diversity. Our findings enlarge the growing knowledge of polyketide biochemistry and illuminate potential challenges in PKS bioengineering., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A biotransformation approach using microbes as biocatalysts can be an efficient tool for the targeted modification of existing antibiotic chemical scaffolds to create previously uncharacterized therapeutic agents. By employing a recombinant Streptomyces venezuelae strain as a microbial catalyst, a reduced macrolide, 10,11-dihydrorosamicin, was created from rosamicin macrolide. Its chemical structure was spectroscopically elucidated, and the new rosamicin analog showed 2-4-fold higher antibacterial activity against two strains of methicillin-resistant Staphylococcus aureus compared with its parent rosamicin. This kind of biocatalytic approach is able to expand existing antibiotic entities and can also provide more diverse therapeutic resources.

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. The D-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and D-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site PhiC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of "unnatural" natural mycinosyl compounds by various actinomycete strains using the bacteriophage PhiC31 att/int system.

Novel prodrugs of the cytotoxic antibiotic CC-1065 for an antibody-directed enzyme prodrug therapy (ADEPT) were prepared that show an excellent selectivity with a high toxicity of the corresponding drug. In particular, the seco-CBI analogue of CC-1065, 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole, as well as the novel methyl-seco-CBI analogue 1-(1'-chloroethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole, were synthesized and transformed into their galactosides 10 a and 10 b, respectively. These galactosides can be cleaved with beta-D-galactosidase to give the free cytotoxic compounds. They were tested in in vitro cytotoxicity assays by using human bronchial carcinoma cells of line A549 in the presence and in the absence of beta-D-galactosidase. While the seco-CBI prodrugs revealed only modest selectivity, prodrugs of the methyl-seco-CBI analogue bearing an anti orientation of the substituents at the two stereogenic centers of the N-heterocycle displayed an excellent selectivity with an ED(50) quotient of about 750. The cytotoxicity of the corresponding phenol was rather high, with an ED(50) of 1.3 nM. The diastereomer with a syn orientation at the stereogenic centers was much less toxic.

A series of CBI analogues of the duocarmycins and CC-1065 exploring substituent effects within the first indole DNA binding subunit are detailed. Substitution at the indole C5 position led to cytotoxic potency enhancements that are > or =1000-fold, providing simplified analogues containing a single DNA binding subunit that are more potent (IC(50)=2-3 pM) than CBI-TMI, duocarmycin SA, or CC-1065.

The last few years have represented an accelerated accumulation in detailed information about ligand-DNA interactions. A collected view of literature information is essential for advancing our understanding of the principles of ligand-DNA recognition, utilizing this valuable information for construction of a modeling database, and eventually the rational design of DNA-binding ligands possessing desired properties. This review is concentrated on structure-based information on ligand-oligodeoxyribonucleotide (DON) complexes published since 1995, especially focusing on the results obtained from NMR structure elucidation. The discussions emphasize the sequence specific recognition of novel binding motifs or binding modules of ligand molecules rather than specific atomic details. A comprehensive list of DNA binding ligands are discussed in the text and are also summarized in a table. The DNA sequences that are recognized by specific ligand molecules as studied by NMR are annotated in a figure to provide a clear view of target selection. This review also briefly describes NMR methods for characterization and structure elucidation of ligand-DNA complexes.

DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new class of PBD-lexitropsin conjugates is also discussed. Certain functional models of the bleomycins (BLMs) show outstanding DNA cleavage activity comparable with that of and positionally distinct from natural BLM.

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA., (Copyright 2000 Academic Press.)

Duocarmycins have been reported to derive their potent antitumor activity through a sequence-selective minor groove alkylation of N3 adenine in double-stranded DNA. We have used gel mobility shift assays to detect proteins that bind to DNA treated in vitro with duocarmycin SA and identified a protein, named duocarmycin-DNA adduct recognizing protein (DARP), which binds with increased affinity to duocarmycin-damaged DNA. Examination with partially purified DARP revealed that the protein recognized not only the DNA adduct of structurally related drug, CC-1065, but unexpectedly, the protein also recognized the DNA adduct of another chemotype of minor groove binder, anthramycin. These results demonstrate that DARP recognizes the structural alteration of DNA induced by these potent DNA-alkylating drugs, suggesting the possibility that the protein might modulate the antitumor activity of these drugs.

We have recently synthesised a series of compounds based on the simplest functional unit of CC-1065 containing a para substituted phenethyl halide moiety. These compounds alkylate N3 of adenines in a similar fashion to CC-1065, as well as N7 of guanines to a limited extent. In this work we compared the para amino substituted derivative (2) with the published hydroxyl compound (1) in terms of stability, DNA reactivity and pH dependence using gel electrophoresis techniques. The results show that 2 has a shorter lifetime and is at least 2.5 times more reactive with DNA than 1. It is completely hydrolysed between 30 and 60 min in buffer and its reaction with DNA is complete within 5 min. In contrast, only a fraction of 1 is hydrolysed after 60 min and retains reactivity towards DNA even after 3 h. The reactivities of both 1 and 2 with DNA are pH dependent and reaction rates rapidly decrease in the range pH 5.8-8.8. Preliminary molecular modelling studies suggest that the p-amino group on 2 enables the drug to bind to the AT-rich minor groove more effectively, thus stabilising the orientation of the substrate in the groove such that the reactive cyclopropyl ring is located close to the nucleophilic centre N3 of adenine. A possible mechanism of action of these drugs is presented based on these findings.

Hybrid agents 5-11 containing the C-terminus DNA binding domain of bleomycin A2 linked to the CBI analogue of the CC-1065 and duocarmycin DNA alkylation subunits were prepared and evaluated. The agents exhibited little or no enhancement of the DNA alkylation efficiency and in some cases the linkage resulted in diminished properties relative to the simple alkylation subunit itself. Moreover, the DNA alkylation selectivity (5'-AA > 5'-TA) of the resulting agents proved identical to that of simple derivatives of the CBI alkylation subunit, e.g. N-BOC-CBI. Thus, the linkage to the DNA binding domain of bleomycin A2 did not alter this inherent DNA alkylation selectivity to reflect a DNA binding or cleavage selectivity of bleomycin A2, nor did it reflect the greater 5- or 3.5-base-pair AT-rich selectivities observed with CC-1065 or the duocarmycins, respectively. Consistent with these observations, the cytotoxic properties of 5-11 were diminished relative to those of even simple derivatives of the CC-1065/duocarmycin alkylation subunits, e.g. N-BOC-CBI.

A number of indirect observations are summarized that suggest the rate acceleration for the CC-1065 and duocarmycin. DNA alkylation reaction is derived in part from a DNA binding-induced conformational change in the agents which substantially increases their inherent reactivity. This ground-state destabilization of the agent, which we suggest results from a binding-induced twist in the linking N2 amide and requires a rigid extended N2 amide substituent, disrupts the vinylogous amide stabilization and activates the agents for DNA alkylation.

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1H-indol-2-yl]carbonyl)-1H-indol-2-yl] carbonyl]-7-methyl-1,2,8,8a-tetrahydrocyclopropa [c]-pyrrolo-[3,2-e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.

A study of the DNA cleavage efficiency and selectivity of CDPI3-EDTA (4), an affinity cleavage agent based on the structure of CC-1065, is described. The studies with 4 provide direct evidence of AT-rich noncovalent binding coincidental with all DNA alkylation sites observed with (+)- or ent-(-)-CC-1065.

To gain insight into the interactions between transcriptional factor proteins and DNA, the DNA-reactive drugs (+)-CC-1065 and pluramycin were used to target specific protein-DNA complexes. The structural features of the complex between the transcriptional activator Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA were examined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The intercalating drug pluramycin was used to probe the structural details of the interaction between the TATA binding protein (TBP) and the TATA box DNA sequence. TBP, which directs initiation of RNA transcription, exhibits two-fold symmetry and apparently interacts with the TATA box in a symmetrical fashion. However, the interaction results in an asymmetric effect, in that transcription is initiated only in the downstream direction. Using pluramycin as a probe, it was determined that TBP binding to the human myoglobin TATA sequences enhances pluramycin reactivity at a site immediately downstream of the TATA box. The implications on transcriptional control of ternary complexes comprised of transcriptional factors, DNA, and DNA-reactive compounds will be presented.

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Antitumor agent CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups have been synthesized. The extent and the relative rates of DNA cleavage following alkylation by these CPI structures and thermal treatment were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The anticipated preferential covalent binding to adenine sites within the minor groove was confirmed by sequencing determination of selected agents on high-resolution gels. Certain of the synthetic agents, unlike CC-1065, also bind covalently to G sites with weaker intensity. The cytotoxicities of these compounds were also determined against KB cells in vitro. Compounds bearing a bromo or nitro group in the benzene ring and a methylsulfonyl as a leaving group are 10 and 5 times more potent than their unsubstituted counterparts, respectively. Compounds bearing a methylsulfonyl as a leaving group are more potent than those bearing a chlorine.

Although considerable work has focused on characterizing the bonding chemistry and sequence selective alkylation of DNA by cyclopropylpyrroloindole compounds, little is known about the molecular consequence of their N-3-adenine adducts in whole animal systems. We have utilized a transgenic mouse system, harboring a lambda phage shuttle vector, to assess the mutagenic potential of the antitumor compounds CC-1065 and adozelesin and, for the first time, to track the in vivo fate of their unique DNA modifications at the nucleotide level. Mice were inoculated with a single therapeutic dose of these agents and sacrificed at either 18 h, 3 days, or 15 days for extraction and analysis of liver DNA. Mutant frequencies obtained from drug treated and control animals were determined by in vitro packaging of the phage vector from genomic DNA followed by a colorimetric plaque assay to screen for phage in which the accompanying lacI repressor gene had mutated. Although undetectable at 18 h posttreatment, by 72 h a 3-fold increase in mutant frequency was observed in drug treated animals such that sequence analysis of drug induced mutations could be performed and a direct comparison made between in vitro and in vivo DNA alkylation. Base substitution involving guanine or cytosine accounted for 64% of the 41 mutations sequenced from drug treated animals. Only 7 of the mutations occurred at a cyclopropylpyrroloindole alkylation site while 23 occurred 1 to 4 nucleotides from a potentially alkylated adenine.

The in vitro sensitivity of Plasmodium falciparum to bleomycin, busulfan, camptothecin, CC-1065, cisplatin, daunomycin, distamycin, luzopeptin, mAMSA, mitomycin C, naladixic acid, U71184, VM26, and VP16, or combinations of them was examined. The lethal dose concentration for 50% of parasites for 3 compounds, luzopeptin, CC1065, and U71184, that bind to adenine-thymidine-rich nucleotide sequences, were in the range of 10(-11) M. They were at least 2 orders of magnitude more effective than the other compounds.

(+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol, 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3' adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3' side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5'-AGTTA* or 5'-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5'-AGTTA* the bending magnitude increases with increased temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed.

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.

Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity, [Hurley, L. H., Lee, C.-S., McGovern, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19 degrees, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5'-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered.

Studies on the structural origin of the DNA alkylation selectivity of the antitumor antibiotic (+)-CC-1065 are detailed. The sites of alkylation of double-stranded DNA were examined for simple derivatives of 7-methyl-1,2,8,8a-tetrahydrocycloprop[1,2-c]pyrrolo[3,2-e]indol- 4(5H)-one (CPI), (+)-CC-1065, and agents incorporating the parent 1,2,7,7a-tetrahydrocycloprop[1,2-c]indol-4-one (CI) left-hand subunit. The CI subunit of the agents is a much more reactive alkylating agent than the natural CPI alkylation subunit of CC-1065. Consequently, simple derivatives of CI were found to alkylate double-stranded DNA under milder conditions than were simple derivatives of CPI, and the marked similarities in the CI and CPI DNA alkylation profiles illustrate that CI represents the minimum pharmacophore of CPI. Comparisons of the DNA alkylation profiles of (+)-N-butyloxycarbonyl-CPI, (+)-N-acetyl-CPI, and (+)-CC-1065 revealed distinctions in the CPI and (+)-CC-1065 sites of alkylation, whereas the incorporation of the reactive CI electrophile into an analog of CC-1065 (CI-CDPI2) (CDPI, N3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid) provided an agent that possesses the characteristic CC-1065 DNA alkylation profile (site selectivity and relative site intensity). These observations suggest that the noncovalent binding selectivity of the agents may restrict the number of available DNA alkylation sites and play a productive role in controlling the sequence-selective alkylation by effectively delivering the electrophile to A + T-rich minor groove regions of DNA possessing accessible adenine N-3 alkylation sites. In turn, the noncovalent binding selectivity may be derived from preferential binding within the narrower, sterically more accessible A + T-rich minor groove of double-stranded DNA.

The DNA base pair preferences of the antitumor antibiotic CC-1065 and two analogs of CC-1065 were studied by following the rate of covalent bond formation (N-3 adenine adduct) with DNA oligomers containing the 5'NNTTA* and 5'NNAAA* sequences (N = nucleotide, A* = alkylated adenine). The rate of adduct formation of CC-1065 is greatly affected by DNA base changes at the fourth and fifth positions of the bonding site for the 5'NNAAA sequences, but not the 5'NNTTA sequences. However, an analog of CC-1065 containing the same alkylating moiety as CC-1065, but not the third fused ring system or additional methylene and oxygen substituents, shows similar rates of adduct formation for all sequences. A second analog of CC-1065 containing three fused ring systems, but not the methylene and oxygen substituents of CC-1065, shows rates of adduct formation with the same sequence dependence as CC-1065, but does not distinguish between the sequences to the degree shown by CC-1065. Adduct formation of CC-1065, but not the analogs, competes with a reversibly bound species. Thymine bases to the 3' side of a potentially reactive adenine or a cytosine base at the fifth position from the bonding adenine create reversible binding sites which decrease the rate of adduct formation of CC-1065. The sequence 5'GCGAATT binds CC-1065 only reversibly. This sequence can compete for CC-1065 with covalent bonding sequences if the sites are located in different oligomers, or if the sites are located (overlapped or not overlapped) in the same oligomer. The results of these competitive binding experiments suggest that the transfer of CC-1065 from the reversible binding site to the covalent bonding site with both sites located on a single DNA duplex, not overlapped, occurs through an equilibrium of CC-1065 in solution, not by migration of CC-1065 in the minor groove.

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG).d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5'AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a general shape-search docking algorithm, potential DNA minor groove binding compounds were selected from a subset from the Cambridge Crystallographic Database consisting of almost 10,000 molecules. The crystal structure of the DNA dodecamer as observed in the d(CGCGAATTCGCG)2.netropsin complex served as the target receptor. Surprisingly, the highest scoring compound turned out to be CC-1065, a potent anti-tumour agent. Netropsin itself was number 6 on the list of highest scoring compounds. A number of the top-10 scoring compounds may serve as a source of inspiration for further drug design.

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The authors have carried out some studies on MOM dry syrup in the field of pediatrics and the results were as follows. 1. After oral administration of MOM at 20 mg/kg, the serum level peaked at 30 minutes and MOM could still be detected in the serum at up to 4 hours. The half-life was 0.67 hour. The urinary recovery rate of MOM was 1.43% up to 6 hours after this oral administration. 2. The clinical efficacy rates with MOM continuous treatment at a daily dose of 19.4 to 50.0 mg/kg (almost about 30 mg/kg) were 85.7% for upper respiratory tract infections, 66.7% for lower respiratory tract infections and 95.7% for Mycoplasma pneumonia. 3. It was easy to administer MOM orally even to the younger children. Although MOM was administered for 4 to 21 days, no side effects were observed locally or systemically except for transient slight diarrhea. These results confirmed the usefulness of MOM in the treatment of acute respiratory tract infections in children.

The concept of residues of antibiotics used as feed additives or veterinary drugs in food producing animals is analysed, and implications on human public health are discussed. The examples of Tylosin and Penicillin are developed to illustrate the both notions of "high risk residue" and "toxicodisponibility" of residues. The "high risk residue" may be an active metabolite different by its chemical structure and by its pharmacological properties from the original drug administered. Slight modifications of the molecule, as the rupture of the beta lactam ring of the Penicillin, occuring in vivo, lead to a metabolite, e.g. penicilloyl group, that has lost all antibiotic activity but possesses allergenic potential. Toxicity of the residue, compared with that of the original drug, can then be modified or increased. On the other hand, such an active metabolite having a definite chemical structure, even if different from the original compound, can be present in the organism, either free or bound to serum or tissues proteins. Moreover, it is shown here, that in the case of a covalent binding of the drug or its metabolite (e.g. penicilloyl group) to serum albumin, the residues are mostly masked inside the tertiary structure of the albumin molecule, and are not accessible to antibodies. These different forms have then an effect upon the biodisponibility, the "toxicodisponibility", of the residues for the human consumer of animal products where they are present. These forms are only accessible with more and more specific and sensitive analytical methods which relates also the qualitative and quantitative notions of residue to the technological degree used for investigation, determination and identification. As to cooking techniques, they can lead to a thermodegradation of the residue or, on the opposite, to an unmasking of the residue present as a protein conjugate, e.g. penicilloyl-protein conjugate in milk.

A macrolide antibiotic turimycin, is a fermentation product of Streptomyces hygroscopicus. It is active in vitro against a range of mycoplasma species and gram-positive bacteria. The antimicrobial activity of the agent was determined in serum, urine, feces and extracts of different organs. In rats, dogs and pigs the serum half life of turimycin ranged from 30 to 120 min. The absorption rate from the intestine was about 30%. After a single iv injection of turimycin into rats, the original substance and its metabolites were excreted preferably by the bile. After oral administration of turimycin to pigs (250 mg/kg daily for 14 days) and to flow (650 mg/kg daily for 10 days) antimicrobial activity was detected in fatty tissue and bile up to the 14th posttreatment day. The pharmacokinetic properties of turimycin are similar to those of corresponding macrolide antibiotics, including tylosin.

We evaluated acute toxicity and estimated LD50 values of Mb12, one of the main metabolites of MOM, in male and female mice after single oral administration. Observations were continued for 1 week after treatment. The LD50 values were calculated according to Litchfield-Wilcoxon's method. It is concluded that LD50 values of Mb12 were 5,750 mg/kg (4,914.5-6,727.5 mg/kg) in male mice and 4,950 mg/kg (4,194.9-5,841.0 mg/kg) in female mice, respectively.

The pharmacokinetics of rosaramicin was studied in subjects receiving 500 mg of the drug (i) by 1-h intravenous infusion, (ii) in solution orally, or (iii) as tablets orally. After intravenous administration, the rosaramicin levels in serum declined rapidly with t1/2S of 0.27 h for the distribution phase and 3.28 h for the elimination phase. The apparent volume of distribution was 3.78 liter/kg, and the total body clearance was 13.41 ml/min per kg, indicating extensive tissue distribution or metabolism or both. Similar pharmacokinetic data were obtained after oral administration of the drug in solution or tablets and after intravenous dosing. The absolute bioavailability of the drug administered orally, in either tablets or solution, was 32 to 39%. The metabolism and excretion of [14C]rosaramicin administered orally were also evaluated in volunteers. The serum area under the curve (infinity) of unchanged rosaramicin was 19% of that of total radioactivity, indicating extensive metabolism of the drug. About 7.0% of the radioactivity was recovered in the urine, and 86.7% was recovered in the feces. Only a small amount of unchanged rosaramicin was present in the urine (7 to 9% of urinary radioactivity), but none was present in the feces. The major metabolite, 20-bis-ureidorosaramicin, represented 17 to 38% of the radioactivity in the urine and 26 to 29% of the radioactivity in the feces.

Studies were conducted to determine certain kinetic variables for tylosin in the normal dog. These variables were determined by pharmacokinetic analysis, measuring the decrease in serum concentration of tylosin activity in 3 separate experiments. Tylosin, given as a single intravenous dose of 10 mg of tylosin activity/kg of body weight, was calculated to have an elimination half life (t1/2beta) of 54 minutes and was no longer detectable in the serum by assay methods at about 270 minutes. In addition, tylosin appeared to be highly distributed with an apparent volume of distribution of about 1.7 L/kg and a tissue-to-plasma ratio of about 0.7. The body clearance of tylosin was calculated to be about 22 ml/kg minute. A separate multiple-dose intravenous experiment with the same dose per injection was conducted and showed similar results. Also, single intramuscular injection of 10 mg of tylosin activity/kg showed tylosin to peak after injection in the serum at 30 minutes, attaining a concentration of approximately 1.5 mug/ml. This concentration was maintained through 60 minutes and decreased slowly through 540 minutes. Data generated through 17 clinical cases indicated that the minimum inhibitory concentration for tylosin in Pasteurella spp tested was about 6.25 mcs/ml and for hemolytic Staphylococcus spp tested greater than or equal to 0.4 mug/ml. Thus, it would appear that tylosin, given at the recommended dose (6.6 to 11.0 mg tylosin activity/kg of body weight once or twice per day), would produce serum levels insufficient to treat all Pasteurella spp and some hemolytic Staphylococcus spp tested since a serum level of tylosin at slightly higher than 1 mug/ml for about 30 minutes was achieved in this study.

Antibiotics, the activity of which enhanced against Pseudomonas aeruginosa by SP127, were restricted to the basic macrolide antibiotics such as erythromycin, maridomycin and oleandomycin, the neutral macrolide antibiotics such as lankamycin and lankacidin C, vancomycin and enramycin. Synergistic activity of SP127 with the above antibiotics was found against Pseudomonas aeruginosa and several strains of Escherichia coli, but not against Proteus vulgaris and macrolide-resistant Staphylococcus aureus. SP127 had extremely weak proteolytic but no lytic activity. From the isotopic experiments, the action of SP127 was partially attributed to the promotion of antibiotic penetration to cells of Pseudomonas aeruginosa.

The action of a new antibiotic identified as CM 9164 ("Midecamycin") and provisionally called "Midecacin", has been experimented on patients admitted for dental conditions. The research was carried out on an open basis and erythromycin was used as a comparison. After a short review of the chemicophysical properties and toxicological and pharmacological features of Midecamycin, the purpose, materials and methods of the research, which was done on two groups of patients, are presented. The results demonstrated the therapeutics usefulness of the new antibiotic as its activity is certainly on a par if not greater than that of erythromycin whether considered "historically" or from the viewpoint of the comparison group. The tolerance of the antibiotic was clinically better than that of the other substances. The doses used were much lower than those required with erythromycin. On the basis of these results, the use of Midecamycin is therefore recommended in the treatment of Gram-positive infections in the dental field.

By microbial transformation of tylosin (I), the following eight new acyl derivatives were obtained: 3-acetyltylosin (II), 3-propionyltylosin (III), 4"-butyryltylosin (IV), 4"-isovaleryltylosin (V), 3-acetyl-4"-butyryltylosin (VI), 3-acetyl-4"-isovaleryltylosin (VII), 3-propionyl-4"-butyryltylosin (VIII) and 3-propionyl-4"-isovaleryltylosin (IX).

Three different bioassay methods for rokitamycin (TMS-19-Q) were compared to each other in vitro and in vivo. Method I was a paper disc method using Micrococcus luteus ATCC 9341 as the test organism in nutrient agar (pH 8.0), method II was a paper disc method using Streptococcus pyogenes Cook in brain heart infusion agar (pH 7.4) and method III was an agar well method using M. luteus ATCC 9341 in the agar medium provided in the Minimum Requirements for Antibiotic Products of Japan (pH 6.5). Ratios of relative potency of TMS-19-Q and its metabolites calculated from standard curves in human plasma using these bioassay methods, were 1.00:1.13:3.34:0.96 (TMS-19-Q:10"-OH-TMS-19-Q:LM A7:LM V) in method I, 1.00:1.58:0.40 (TMS-19-Q:LM A7:LM V) in method II and 1.00:0.51:0.86:0.114 (TMS-19-Q:10"-OH-TMS-19-Q:LM A7:LM V) in method III. The relative potency obtained from method I, generally used for macrolide antibiotics, greatly contradicted MIC values of TMS-19-Q and its metabolites against clinically isolated 190 strains of Staphylococcus aureus. On the other hand, the relative potency obtained from method III reflected closely MIC values of TMS-19-Q and its metabolites. Therefore, method III should be the most suitable bioassay method to measure the plasma concentration of TMS-19-Q. Plasma concentrations measured by these 3 different methods were compared to each other in vivo, after oral administration of TMS-19-Q at a dose of 1,200 mg to each of fasted healthy volunteers. Values obtained from method I were about 4.3 times higher than those method III.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute toxicity studies on miocamycin (MOM), non-crystalline solid, and its metabolite Mb1 were performed in mice in the previous studies. In the present studies, we evaluated acute toxicity of Mb1 in male and female rats after single oral administration at the maximum physically applicable dose of 5,000 mg/kg. Observations were continued for 1 week after treatment. It is concluded that LD0 values of Mb1 were estimated more than 5,000 mg/kg.

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.

1. The dry syrup of MOM was administered orally to 17 patients mainly with heart diseases at doses of 10 mg/kg and 20 mg/kg. In 17 cases, the serum level was measured and in 4 cases, the urinary excretion rate including the metabolites of MOM. 2. The mean maximal concentrations were 0.54 mcg/ml at 30 minutes for the group of 10 mg/kg treatment and 0.33 mcg/ml at 1 hour for the group of 20 mg/kg treatment. The dose response was not observed obviously in both groups. 3. In each of the cases, the sum of excretion rates of metabolites in the 24-hour urine was about 1%. 4. MOM was administered clinically to 39 cases with respiratory tract infections and the overall efficacy rate was 85%. 5. In this study, 5 strains of S. pyogenes were isolated and the eradication rate was 60%. 6. Although severe side effects were not observed, gastrointestinal abnormalities like diarrhea and vomiting were seen in 3 cases. 7. Any pediatric patient did not refuse taking.