Search

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13 C-labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin-dependent monooxygenase ChaZ catalyses a Baeyer-Villiger oxidation on resomycin C to form a seven-membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the α-pyrone containing pentacyclic intermediate in an NADPH-dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol-type reaction, followed by a spontaneous γ-lactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of biolayer interferometry (BLI) as a tool for rapidly screening natural product sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC- and AT-rich DNA oligonucleotides with known DNA intercalating, groove binding, and covalent binding agents including actinomycin D (1), doxorubicin (2), ethidium bromide (3), propidium iodide (4), Hoechst 33342 (5), and netropsin (6). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (8), actinomycin V (9), and chartreusin (10). This biosensor-based DNA binding screen is a novel, low-cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries. Graphical abstract.

An endophytic actinomycete strain SKH1-2 isolated from Musa (ABB) cv. 'Kluai Sao Kratuep Ho' collected in Suphan Buri province (14° 54' 22.5″ N/100° 04' 50″ E), Thailand, was identified as Streptomyces pseudovenezuelae based on phenotypic and chemotaxonomic characteristics, and 16S rRNA sequence analyses. A chemical investigation led to the isolation of two polyketide molecules from the n-butanol crude extract of the strain SKH1-2 culture broth. The compounds were purified using various chromatographic techniques and identified using spectroscopic methods compared with earlier published data. Compound 1, chartreusin, is known as an anti-Gram (+) bacterial compound and was active against Bacillus subtilis ATCC 6633, Kocuria rhizophila ATCC 9341 and Staphylococcus aureus ATCC 6538p with MIC values of 3.1, 1.6 and 12.5 μg/mL, respectively. Compound 2, lumichrome, did not show activity against all tested microbes. To our knowledge, this is the first report of chartreusin and lumichrome isolated from S. pseudovenezuelae. Taken together, it could be proved that Thai plant species are valuable reservoirs of interesting endophytic actinomycetes producing several interesting biologically active compounds.

Cancer stands in the frontline among leading killers worldwide and the annual mortality rate is expected to reach 16.4 million by 2040. Humans suffer from about 200 different types of cancers and many of them have a small number of approved therapeutic agents. Moreover, several types of major cancers are diagnosed at advanced stages as a result of which the existing therapies have limited efficacy against them and contribute to a dismal prognosis. Therefore, it is essential to develop novel potent anticancer agents to counteract cancer-driven lethality. Natural sources such as bacteria, plants, fungi, and marine microorganisms have been serving as an inexhaustible source of anticancer agents. Notably, over 13,000 natural compounds endowed with different pharmacological properties have been isolated from different bacterial sources. In the present article, we have discussed about the importance of natural products, with special emphasis on bacterial metabolites for cancer therapy. Subsequently, we have comprehensively discussed the various sources, mechanisms of action, toxicity issues, and off-target effects of clinically used anticancer drugs (such as actinomycin D, bleomycin, carfilzomib, doxorubicin, ixabepilone, mitomycin C, pentostatin, rapalogs, and romidepsin) that have been derived from different bacteria. Furthermore, we have also discussed some of the major secondary metabolites (antimycins, chartreusin, elsamicins, geldanamycin, monensin, plicamycin, prodigiosin, rebeccamycin, salinomycin, and salinosporamide) that are currently in the clinical trials or which have demonstrated potent anticancer activity in preclinical models. Besides, we have elaborated on the application of metagenomics in drug discovery and briefly described about anticancer agents (bryostatin 1 and ET-743) identified through the metagenomics approach.

ChaP is a non-heme iron-dependent dioxygenase belonging to the vicinal oxygen chelate (VOC) enzyme superfamily that catalyzes the final α-pyrone ring formation in the biosynthesis of chartreusin. In contrast to other common dioxygenases, for example, 2,3-catechol dioxygenase which uses the dioxygen molecule as the oxidant, ChaP requires the flavin-activated oxygen (O

DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of Biolayer Interferometry (BLI) as a tool for rapidly screening natural products sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC and AT rich DNA oligonucleotides with known DNA intercalating, groove-, and covalent-binding agents including actinomycin D (1), doxorubicin (2), ethidium bromide (3), propidium iodide (4), Hoechst 33342 (5), netropsin (6), and cisplatin (7). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (8), actinomycin V (9), and chartreusin (10). This biosensor-based DNA binding screen is a novel, low cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries.

Oxidative rearrangements play key roles in introducing structural complexity and biological activities of natural products biosynthesized by type II polyketide synthases (PKSs). Chartreusin (1) is a potent antitumor polyketide that contains a unique rearranged pentacyclic aromatic bilactone aglycone derived from a type II PKS. Herein, we report an unprecedented dioxygenase, ChaP, that catalyzes the final α-pyrone ring formation in 1 biosynthesis using flavin-activated oxygen as an oxidant. The X-ray crystal structures of ChaP and two homologues, docking studies, and site-directed mutagenesis provided insights into the molecular basis of the oxidative rearrangement that involves two successive C-C bond cleavage steps followed by lactonization. ChaP is the first example of a dioxygenase that requires a flavin-activated oxygen as a substrate despite lacking flavin binding sites, and represents a new class in the vicinal oxygen chelate enzyme superfamily.

Solution phase and solid-sate electrochemical techniques centered in the voltammetry of microparticles approach are applied for testing the cytotoxic activity of anticancer drugs. The possibility of electrochemical generation of reactive oxygen species (ROS) is exploited for evaluating their contribution to cellular damage. The described methodology is applied to the case of chartreusin (Ch) whose electrochemistry in non-aqueous solutions and in the solid state in contact with aqueous electrolytes is described in the absence (experimental data were confirmed by theoretical calculations) and in the presence of double-stranded DNA (dsDNA). In parallel, scanning electrochemical microscopy (SECM) examination of dsDNA fibers was developed. Electrochemical data suggest that Ch-induced dsDNA interaction can operate by a two pathways: via intercalation and by mechanisms related with ROS generation. Although reduced Ch forms electrochemically generated can act as radical scavengers blocking the ROS generation chain, this property is interrupted upon binding to dsDNA.

DNA-intercalating polyketide glycosides are important leads for cancer therapeutics, yet their use is often limited by their low solubility and challenging synthetic protocols. To overcome these limitations, we employed 1,4'-bipiperidine-1'-carbamate residues as sugar surrogates in daunorubicin and chartreusin, yielding water-soluble derivatives and prodrugs with dramatically improved antiproliferative activities.

RATIONALE The fate of drug delivery systems (DDSs) in vivo is a widely discussed question. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an important tool to localize proteins and small compounds in many different tissues. This basic study was performed as an aid to obtain spatial information on DDSs in the future. METHODS LDI and MALDI-TOF MS was used to investigate five drug molecules, i.e. madurahydroxylactone (MHL), tetrakis(4-hydroxyphenyl)porphyrin (THP), chartreusin (Chart), amphotericin B (AmB) and retinoic acid (RA). The drug molecules were analyzed in terms of their efficiency to act as matrix for different homopolymers and the block copolymer poly(ethylene glycol)-block-poly(e-caprolactone) (PEG-b-PCL). The block copolymer was further utilized as a DDS to encapsulate the drug molecules previously investigated as matrices. The obtained DDSs were investigated by MALDI. RESULTS The spectra obtained with the drugs Chart, MHL, THP and AmB did not reach the quality of the standard matrix RA. Nonetheless, they showed surprisingly good results as matrices for different homopolymers and the block copolymer. However, only the DDSs containing THP as the drug provided spectra where the drug and the block copolymer were detected. CONCLUSIONS These results form the foundation for obtaining mass-related information about the localization of DDSs and drugs in tissues. Copyright © 2013 John Wiley & Sons, Ltd.

Natural products of the benzonaphthopyranone class, such as chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of chartreusin derivatives. Pathway engineering of the chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromochartreusin led to the first crystal structure of a chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new chartreusins with fine-tuned properties as promising candidates for further development as therapeutics.

The molecular strucrures of chartarin and its isomers have been optimized by use of PM3 MO method.These molecules have been calculated to be planar.The calculated results for chartarin well agree with those for the chartarin part of the X-ray-determined structure of elsamicin A.CNDO/S calculations of chartarinand the observed spectrum.Further,PM3 and COSMO calculations show that both in the gas phase and in solvents,chartarin is the most stable.

Chapter 4 deals with the origin and roles of reactive oxygen species (ROS) and local oxidative stress associated to the imbalance between ROS production and the action of antioxidant defence systems. Membrane phospholipid peroxidation initiated by the attack of a hydroxyl radicals is explained, as well as the generation of malondialdehyde and its consequences. The mechanisms of DNA strand scission, oxidation of DNA bases and nuclear proteins, as well as the defense mechanisms that counteract this damage, are discussed in detail. Anthracyclines are studied as promoters of ROS with the consequent induction of apoptosis, although they are also included in Section 6 of Chapter 7 because of their activity as topoisomerase II poisons. We also discuss their association with chelating agents such as dexrazoxane to prevent the induced cardiotoxicity and how the anthracycline-induced ROS may generate formaldehyde and anthracycline/formaldehyde adducts. The mechanisms that explain how chartreusin (NSC 5159), elsamicin A, the bleomycins (BLMs) and the enediyne antibiotics generate radical species that cause single strand scission of DNA are explained. Radiotherapy is based on the generation of hydroxyl radicals from the homolytic fragmentation of water molecules upon local application of ionizing radiation, and is less efficient in hypoxic cells because in the absence of oxygen biomolecule radicals are repaired by glutathione. We study several hypoxic radiosensitizers that increase the efficiency of radiotherapy, and we also discuss how the normal tissues surrounding the tumor may be protected against the radiation damage with radioprotective compounds. Finally, we study the photodynamic therapy (PDT) of cancer by using photosensitizers (PS) able to absorb harmless visible light energy and transfer it to other molecules in their vicinity.

Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.

The marine Streptomycete isolates B5342 and B5525 forms, beside large amounts of the antitumor antibiotic chartreusin (1a), two chartreusin monoacetates as trace components. Whereas the chemical shift of the acetate methyl in 1c is in the normal range, the methyl group of the 2"-acetate residue in 1b and also in the tetra- and pentaacetates shows an extreme upfield shift. The structures of the monoacetates were confirmed by extensive NMR experiments, the anisotropic shift is explained by semi-empirical calculations.

The aim of this study was to analyze the pharmacokinetics and pharmacodynamics (PK/PD) of 6- O-(3-ethoxypropionyl)-3',4'- O-exo-benzylidene-chartreusin (IST-622) and its metabolites, and to develop limited sampling models (LSM). Based on the data from 18 patients with breast cancer who were treated orally with 280 or 525 mg/m(2) of IST-622 once daily after breakfast for five consecutive days, we analyzed the relationship between the area under the plasma concentration versus time curve (AUC) and toxicities using a sigmoid E-max model and logistic regression. Plasma concentrations of IST-622 and its metabolites, 3',4'- O-exo-benzylidene-chartreusin (A-132) and 3"-demethyl-3',4'- O-exo-benzylidene-chartreusin (A-132M), were measured at 1, 2, 4, 8 and 24 h after administration on day 1. The AUC was calculated using the trapezoidal method. We also developed a LSM using stepwise linear regression analysis. IST-622 was detected in very few patients, and its concentration was very low and could be disregarded. It was suggested that meals promoted absorption of IST-622. AUCs of A-132 plus A-132M showed a better correlation with the rates of decrease and nadir counts of leukocytes, neutrophils and platelets than the AUC of each metabolite separately. Patients with the sum of AUCs more than 70 microg.h/ml showed severe myelotoxicities. Moreover, logistic regression analysis showed that grade 4 myelotoxicities would be seen in 30% of patients at an AUC of 65 microg.h/ml. We also developed an unbiased and precise LSM: AUCSUB0-24h/SUB=CSUB8h/SUBx17.6-0.95, where C(8h) denotes the sum of plasma concentrations of A-132 and A-132M. Myelotoxicities showed a good correlation with AUC(0-24h), and based on the results, it was decided that the target AUC was 65 microg.h/ml. The LSM was very convenient for estimating AUC(0-24h) and sufficiently accurate. These results show the possibility of predicting toxicities and dose adaptation for interpatient variability using LSM.

Unraveling the sequence of reactions that transforms aromatic polyketide backbones into much more elaborate structures, for example the chartreusin bislactone, is challenging, but in this issue of Chemistry & Biology, Xu and coworkers [1] show that sequencing the gene clusters can provide valuable clues.

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C 2 with unit cell dimensions, a =18.482(4), b =8.749(3), c =43.906(2) A, β =94.87(2)°, and the structure was refined to R -factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F o >4 σ ( F o )) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132–DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.

The antitumor effects of 6-O-(3-ethoxypropionyl)-3′,4′-O-exo-benzylidenechartreusin (IST-622), a new synthetic derivative of chartreusin (CT), were investigated. Following oral administration, IST-622 showed marked antitumor effects against various mouse tumors such as P388 and L 1210 leukemias, B 16 melanoma, Lewis lung carcinoma, Colon 26 and Colon 38 adenocarcinomas, and M5076 reticulum-cell sarcoma. The best antitumor effects were obtained by five intermittent treatments given every 4 days. In addition, IST-622 showed a significant growth-inhibitory effect against two human tumor xenografts, a large-cell lung cancer (Lu-116) and a gastric adenocarcinoma (St-4), among the seven lines tested. IST-622, which was rapidly metabolized into 3′,4′-O-exo-benzylidenechartreusin (A-132) and not into CT in vivo or in culture medium, exhibited remarkable growth-inhibitory activity against P388 leukemia in vitro, its 50% growth-inhibitory concentration (IC50) being over 20-fold lower than that of CT. IST-622 showed an in vivo antitumor effect superior to that of authentic A-132, possibly resulting from a higher absorption ratio of IST-622 through the gastrointestinal tract. IST-622 is now under clinical phase I study in Japan.

Elsamicin (EM) is a recently discovered antitumour agent that is structurally related to several other compounds displaying anticancer activities, including chartreusin (CT), chrysomycin V (CV) and M (CM), gilvocarcin V (GV) and ravidomycin (RM). The biochemical events resulting in cytotoxicity for most of these compounds have not been clearly elucidated. There is some evidence that GV and CT bind to DNA and that GV is photosensitive, causing DNA damage. Therefore, we investigated the effects of these chemicals on DNA in cells and on pBR322 plasmid DNA. Using alkaline elution techniques, we found that all these compounds induced, to a different extent, DNA breakage in the human lung adenocarcinoma A549 cell line. In addition, all either bound to or intercalated into DNA, as indicated by their ability to alter the electrophoretic migration of DNA in agarose gels. Using the P4 unknotting assay, EM, CT, CV, CM, GV and RM were found to be potent inhibitors of the catalytic activity of topoisomerase II (topo II). Their potencies were compared with the known topo II inhibitors teniposide (VM-26) and doxorubicin (DX). EM was the most potent, with an ic 50 of 0.4 μmol/l followed in order by CV, GV, and CT. VM-26 was the least potent with an ic 50 of 15 μmol/l. It was concluded from these results that EM, GV, CV, CM and CT are capable of inhibiting topo II and that EM is the most potent inhibitor of topo II yet discovered.

The structure of chrymutasin A, B and C are determined from spectral studies, incorporation studies of 13C-labelled sodium acetates and an aglycone derivative.

We report guanine-specific recognition and selective cleavage of DNA by the antitumor antibiotic elsamicin A equipped with an amino sugar and compare these results with cleavage by chartarin and chartreusin antibiotics. The preferential cutting sites of DNA strand scission with elsamicin A are on the bases adjacent to the 3'-side of guanine residues such as 5'-GN sites, in particular 5'-GG sites. The present results also indicate that (1) the aglycon portion binds intercalatively to the 3'-side of guanine in host DNA, (2) the guanine 2-amino group has an important effect on selective DNA binding of elsamicin A, and (3) the amino sugar residue of elsamicin A facilitates the drug binding into the minor groove of B-DNA. In addition, we found that an acetylation of the amino group on the elsamicin A sugar portion plays an interesting switch function for the activity of elsamicin A. The biological implication of this switch has also been discussed.

Many anticancer drugs in clinical use interact with DNA through intercalation. The intercalation process starts with the transfer of the intercalating molecule from an aqueous environment to the hydrophobic space between two adjacent DNA base pairs. DNA intercalators are less sequence selective than minor groove binding agents, and, in contrast with them, show a preference for G–C region s. This selectivity is mainly due to complementary hydrophobic or electrostatic interactions, which are due to substituents attached to the chromophore within the major or minor grooves. Intoplicine is an intercalating compound that can be considered as structurally related to the ellipticines. It behaves as a dual topoisomerase I and II poison at cleavage sites different to those of other known topoisomerase inhibitors. Chartreusin and elsamicin A are structurally related antitum or antibiotics that were isolated from Strep- tomyces chartreusis and from an unidentified actino mycete strain, respectively. Identical loops of DNA having different numbers of twists are topoisomers, that is, molecules with the same formula but different topologies, and their interconversion requires the breaking of DNA strands.

In order to overcome the rapid biliary excretion of chartreusin, which diminished its activity when administered iv, a series of 3', 4'-O-substituted derivatives of chartreusin were synthesized. Exo-type of 3', 4'-O-benzylidene-chartreusin was found active both by ip and iv administration. Therefore, this compound was selected for further modification on its 6-phenol to obtain broader spectra and better pharmacokinetic parameters than the original compound. Several 6-O-acyl-3', 4'-O-exo-benzylidene-chartreusins had high antitumor activity against some murine tumors both by iv and po administration.

A new methodology for the rapid regiospecific synthesis of benzonaphthopyranones has been developed, on the basis of a tactical extension of the Hauser–Kraus annulation. The prowess of the methodology has been illustrated by a short synthesis of chartarin ( 22b ) and a facile entry to the chrymutasin scaffold ( 26 ).

The antitumor drug elsamicin A contains a coumarin-related chartarin chromophore that intercalates into DNA. It differs from other related molecules in its disaccharide moiety, which bears an amino sugar. Its binding to DNA was analyzed using isothermal titration calorimetry and UV thermal denaturation, and characterized thermodynamically. For the association of elsamicin A with DNA we found ΔG°=-8.6 kcal mol-1, ΔH=-10.4 kcal mol-1, ΔS=-6.1 cal mol-1 K-1, and K obs=2.8(±0.2)×106 M-1 at 20°C in 18 mM Na+. The contributions to the free energy of binding that lead to the DNA-elsamicin complex are compared with the binding to DNA of chartreusin, another chartarin-containing drug. The results are discussed in terms of the contributions of the disaccharide moieties into the strength of binding. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., This work was supported by Grant SAF-2002-00371 from the Ministry of Science and Technology (Spain) and the European Community, and the support of the Centre de Referencia en Biotecnologia of the Generalitat de Catalunya

Characterization of the thermodynamics of DNA- drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 x 10(5) M(-1) at 20 degrees C, in a solution containing 18 mM Na(+). The DNA-drug interaction was enthalpy driven, with a DeltaH(b) of -7.07 kcal/mol at 20 degrees C. Binding enthalpies were determined by ITC in the 20-35 degrees C range and used to calculate a binding-induced change in heat capacity (DeltaCp) of -391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of DeltaG(obs) into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.

Chrymutasins A, B and C are glycosidic antibiotics produced by a mutant of the chartreusin producer-organism Streptomyces chartreusis. We report here the structure elucidation of these compounds. The sugar moieties involved were determined by comparison with the related chartreusins. The structure of the aglycone, the same in all three compounds, was elucidated by NMR, incorporation studies of labeled compounds and synthesis of derivatives. The chrymutasin aglycone differs from that of chartreusin by a single carbon and an amino group.

Three novel antibiotics, named chrymutasins A, B and C, were isolated from the fermentation products of a mutant strain obtained by NTG (N-methyl-N'-nitro-N-nitrosoguanidine) treatment. The mutant strain produced the chrymutasins, which differed in the aglycone moiety from chartreusin, and related compounds. The production of these compounds needed a characteristically long fermentation period. The antitumor activity of chrymutasin A is better in vivo than that of chartreusin, the cytotoxic activity against cell lines in vitro is equivalent, and the antimicrobial spectrum is narrower.

The structure of chrymutasin A, B and C are determined from spectral studies, incorporation studies of 13C-labelled sodium acetates and an aglycone derivative.

In order to obtain high-producing mutants of the lambdamycin producer Streptomyces glaucoachromogenes IMET 31 118, a new selection technique using the thermophilic phage-host system Tal/Thermoactinomyces vulgaris, was developed. Lambdamycin is a yellow-green pigment antibiotic of the chromoglycoside type with antimicrobial, antiviral, cancerostatic, and ergotropic activity in vitro and in vivo. The physicochemical properties of lambdamycin resemble those of chartreusin. By means of the selection technique developed the determination of the concentration-dependent influence of lambdamycin on bacterial growth inhibition and Tal-phage development in the agar diffusion sphere of mutagen-treated colonies is possible without time and material-consuming pre-tests. A positive correlation exists between the biosynthetic capacity of mutants on solid media and in fermentation liquors. Using this new selection technique, the titres of lambdamycin in fermentation liquors of mutants could be increased by 10 to 20 fold in comparison to the wild type strain.

The isolation and purification of the antibiotic lambdamycin are described. The physicochemical properties of the chromoglycoside lambdamycin resemble those of chartreusin. In this paper we compare the physicochemical properties of chartreusin and antibiotic X 465 with lambdamycin (α D, UV, IR). Lambdamycin is identified with chartreusin.

The compound BMY-28090 (elsamicin A) is a new fermentation product with antitumor properties, which has the same aglycone as chartreusin but contains two novel sugars. To define the disposition of BMY-28090 during phase I trials, HPLC procedures were developed and validated for the quantitation of the drug in human plasma and urine. To 1.0 ml plasma were added 0.5 ml 0.2M phosphate buffer (pH 8.0), 125 ng 1-naphthol (internal standard) in 25 μl MeOH and 5 ml ethyl acetate. After mixing and centrifugation, 4 ml ethyl acetate layer was removed, evaporated to dryness, and the residue was dissolved in 250 μl mobile phase and injected (200 μl). To 1.0 ml urine were added 100 μl MeOH and 1.0 ml 0.5M succinate buffer (pH 4.0). After mixing (30 s) and sonication (1 min), the solution was filtered in an Amicon Centrifree micropartition unit and injected (30 μl). An IBM C-8 column 5-μm and fluorescence detection (excitation at 254 mm, 418 nm emission filter) were used for both analyses. The mobile phases for plasma (2 ml/min) and urine (1.3 ml/min) were H2O/CH3CN (7:3 v/v) and H2O/CH3CN/MeOH (6:3:1 (v/v), respectively, with 1.5 ml 85% H3PO4 and 1.5 ml triethylamine/1. BMY-28090 eluted at 8–10 min and 1-naphthol, at 10–11 min. The standard curves were linear from 1 to 50 ng/ml plasma and from 10 to 1000 ng/ml urine. The within- and between-day precision was

A large number of compounds including actinobolin, adrenochrome, amicetin, anisomycin, aurintricarboxylic acid, blasticidin S, chartreusin, chlortetracycline, cycloheximide, doxycycline, edeine A1, edeine complex, emetine, fusidic acid, gougerotin, GppCH2p, oxytetracycline, pactamycin, polydextran sulphate, puromycin, pyrocatechol violet, sparsomycin and tubulosine have been tested for inhibitory effects on protein synthesis in cultured cells from both mouse fibroblasts (3T6 cells) and chick embryo fibroblasts (CEF). Essentially, similar results were obtained with both cell types with the most effective inhibitors being pactamycin, emetine, tubulosine, anisomycin and cycloheximide and with no significant inhibitory activity being detected with edeine complex, edeine A1, GppCH2p, polydextran sulphate, aurintricarboxylic acid, pyrocatechol violet and adrenochrome. The concentration of pactamycin required to produce 50% inhibition of protein synthesis approximated 5 X 10(-9) M, but for most of the inhibitors it ranged from 5 X 10(-6) M to 5 X 10(4) M. The molecular basis underlying these differences may be related, in addition to their intrinsic inhibitory power, to differences in permeability of the cells towards the various drugs tested. Alternatively, active accumulation of the drugs by the cells may be the variable parameter.

In an enzyme-specific drug screening system nalidixic acid and 3'FTdR, inhibitors of DNA synthesis, both reduce the growth of wild type and temperature-sensitive point mutants of phage T3 with different efficiencies. The wild type shows the strongest sensitivity against the drugs, while an exonuclease mutant is the most insensitive variant. The DNA polymerase mutants exhibit an intermediate degree of inhibition. The anthracycline antibiotics violamycin BI and adriblastin which preferentially inhibit RNA synthesis show the same degree of inhibition for all mutants. This is true also for the RNA synthesis inhibitor lambdamycin, which is identical with chartreusin. The protein synthesis inhibitors chloramphenicol and o-phenanthroline, a chelating agent, impair all mutants to the same extent. Our data confirm the hypothesis that structural variants of essential viral enzymes, when compared with the wild type should sensitivities against specific inhibitors and show that this T3 system could be used for the indication of specific inhibitors of DNA synthesis.

Hydroheptin, a new polyene macrolide antifungal antibiotic, is co-produced with the antibiotic, chartreusin, by a strain of Streptomyces chartreusis designated as IMRU 3962 isolated in our laboratory. The unique water-solubility of this antibiotic at neutrality, revealing in aqueous solution molecular dispersion and an ultraviolet-visible absorption spectrum characteristic of an all-trans heptaene chromophore, clearly distinguishes it from all previously-described and naturally-occurring heptaene macrolides. The isolation and identification of the amino sugar, mycosamine (3-amino-3,6-dideoxy-D-mannose), in acid hydrolysates of hydroheptin and the absence of an aromatic amine upon retrograde alkaline dealdolization of the molecule certainly characterize the antibiotic as a member of the non-aromatic heptaene macrolide group. Chromatographic and countercurrent distribution studies likewise support its novelty. With little or no demonstrable activity against bacteria, hydroheptin as compared to other non-aromatic heptaene macrolides exhibits excellent but somewhat less activity against a wide variety of yeasts and fungi. Likewise, its parenteral toxicity appears to be less than that of other heptaene macrolides.

Examination by 13C nuclear magnetic resonance spectroscopy of chartreusin produced in cultures of Streptomyceschartreusis supplemented with [1-13C] and [2-13C]acetate showed that the 19-carbon aglycone component was derived entirely from acetate. In the spectrum of chartreusin enriched from [1,2-13C]acetate the signals for 16 of the carbon atoms were accompanied by satellites due to spin–spin coupling of intact 13C—13C units. The coupled pairs were matched with the aid of homonuclear single 13C-frequency decoupling. Of the uncoupled carbon atoms, two were derived from methyl groups of acetate and the third came from an acetate carboxyl group. The arrangement of paired and unpaired 13C atoms in chartreusin suggests that the aglycone is derived from a single 22-carbon polyketide chain. Cyclization to a benzpyrene-like intermediate followed by ring cleavage and loss of three carbon atoms provides a plausible route from the polyketide to the substituted isocoumarin structure of the aglycone.

Lambdamycin-producing strains were detected by means of the BIP test method. The isolation technique and the physicochemical and biological properties of lambdamycin, an antibiotic produced by Streptomyces glaucoachromogenes, are described. Lambdamycin is a yellow-green pigment of the chromoglycoside type. Digitalose and fucose are the sugar components. The physicochemical properties of lambdamycin resemble those of chartreusin. However, the known biological activity is different. The antibiotic can be isolated from culture filtrates and from the mycelium by extraction with lower aliphatic alcohols. It can be purified by gel filtration methods. Lambdamycin displays antimicrobial activity, particularly against grampositive bacteria. Strains which produce enzymes inactivating different commercial antibiotics are also inhibited. Moreover, lambdamycin shows antiviral activity, as well as cancerostatic and ergotropic action in vitro and in vivo. The acute LD50 of lambdamycin in mice after 21 days was > 125 mg/kg when administered intraperitoneally.

In order to obtain high-producing mutants of the lambdamyein producer Streptomyces glaucoachromogenes IMET 31 118, a new selection technique using the thermophilic phage-host system Tal/Thermoactinomyces vulgaris, was developed. Lambdamyein is a yellow-green pigment antibiotic of the chromoglycoside type with antimicrobial, antiviral, cancerostatic, and ergotropic activity in vitro and in vivo. The physicochemical properties of lambdamycin resemble those of chartreusin. By means of the selection technique developed the determination of the concentration-dependent influence of lambdamycin on bacterial growth inhibition and Tal-phage development in the agar diffusion sphere of mutagen-treated colonies is possible without time and material-consuming pre-tests. A positive correlation exists between the biosynthetic capacity of mutants on solid media and in fermentation liquors. Using this new selection technique, the titres of lambdamycin in fermentation liquors of mutants could be increased by 10 to 20 fold in comparison to the wild type strain.