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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.

Since the discovery of streptomycin, actinomycetes have been a useful source for new antibiotics, but there have been diminishing rates of new finds since the 1960s. The decreasing probability of identifying new active agents led to reduced interest in soil bacteria as a source for new antibiotics. At the same time, actinomycetes remain a promising reservoir for new active molecules. In this work, we present several reporter plasmids encoding visible fluorescent protein genes. These plasmids provide primary information about the action mechanism of antimicrobial agents at an early stage of screening. The reporters and the pipeline described have been optimized and designed to employ citizen scientists without specialized skills or equipment with the aim of essentially crowdsourcing the search for new antibiotic producers in the vast natural reservoir of soil bacteria. The combination of mechanism-based approaches and citizen science has proved its effectiveness in practice, revealing a significant increase in the screening rate. As a proof of concept, two new strains, Streptomyces sp. KB-1 and BV113, were found to produce the antibiotics pikromycin and chartreusin, respectively, demonstrating the efficiency of the pipeline.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 reveal different sensitivities against specific inhibitors and show that this T3 system could be used for the indication of specific inhibitors of DNA synthesis.

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.

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

The interaction of chartreusin with covalently closed circular PM2 phage DNA was studied. The antibiotic caused a single strand scission in the presence of reducing agents, such as dithiothreitol, ascorbic acid or NaBH4. The degree of DNA breakage was dependent upon the drug concentration. The DNA-cleaving activity was enhanced by ferrous ion; but was completely blocked by catalase and partially by superoxide dismutase. The results suggest that reduction, chelate formation and auto-oxidation of the antibiotic, presumably the 5,12-dione moiety, produce free radicals, including O 2 • and •OH, which are capable of inducing DNA strand scission.

The organism Streptomyces chartreusis IMRU 3962 produced a mixture of the antibiotics hydroheptin and chartreusin in fermentation broth. The addition of increasing levels of phosphate resulted in a corresponding increase in the production of both antibiotics, with maximum yields of 400 to 450 micrograms of chartreusin per ml and 80 to 100 micrograms of hydroheptin per ml at 0.45 to 0.55 M phosphate. Chartreusin was invariably produced at a higher ratio; however, a reversal in ratio to favor hydroheptin was attained when 0.03% copper sulfate was added to the medium, particularly at a 0.2 M KH2PO4 level, with antibiotic yields of 125 micrograms of hydroheptin per ml and 40 micrograms of chartreusin per ml.

First isolated in 1953 from a fermentation broth, chartreusin (1) has received renewed interest as a result of substantial antitumor activities recently demonstrated in several murine test systems. Poor water solubility frustrated formulation attempts, and rapid biliary excretion observed in mice made 1 an improbable candidate for clinical development but an excellent candidate for an analogue synthesis program. From a common intermediate, which was prepared from 1, three analogues were synthesized wherein the disaccharide moiety of 1 was systematically replaced with fucose (6), glucose (7), and the disaccharide maltose (8). Each of the three analogues had a cytotoxic potency against cultured L1210 cells which was equal to, or better than, that shown by 1. Based on the structural similarity with the parent, an improved water solubility, and a favorable accessibility through synthesis, maltoside 8 was choe P388 leukemia, 8 showed reproducible activity comparable to chartreusin at similar dose levels. Although 8 caused no observable toxic effects at therapeutic dose levels when given ip, neither 1 nor 8 produced active indications when administered subcutnaeously.

Chartreusin binds cooperatively to poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both the site-exclusion model and the specific site model yield cooperative binding constants of about 5 X 10(5) M-1 and 3 X 10(5) M-1 for the AT and GC polymers, respectively, and the same stoichiometry and intrinsic binding constant for both polymers of 5 nucleotides per binding site and 3.1 X 10(4) M-1. The Scatchard plot for calf thymus DNA is curved in the opposite sense from that of cooperative binding. These binding data did not fit the site-exclusion model with the cooperative binding parameter as a variable nor the specific site, negative-cooperative binding model. The site-exclusion model with a cooperative binding parameter of unity yielded a binding constant of about 4 X 10(4) M-1 and a stoichiometry of about 5 nucleotides per binding site. The same model for transfer and ribosomal RNA yielded binding constants of 5 X 10(3) M-1 and 7 X 10(3) M-1 and stoichiometries of about 13 and 6 nucleotides per binding site, respectively.

1 Die Experimente, die zur Aufstellung der Strukturformel IV fur das Aglykon aus dem Antibiotikum Chartreusin gefuhrt haben, werden beschrieben. 2 STERNBACH, KAISER & GOLDBERG [2] haben seinerzeit bei der energischen, katalytischen Reduktion des Chartreusin-Aglykons eine Reihe von Hydrierungsprodukten erhalten. Ein grosser Teil dieser Hydrierungsprodukte wurde nun in ihrer Konstitution aufgeklart und miteinander chemisch verknupft.

Durch Abbau-Reaktionen, NMR.-Spektren und Anwendung der Isorotationsregel wurde die Lokalisierung und die Struktur des aus D-Digitalose und D-Fucose bestehenden Disaccharid-Teiles des Antibiotikums Chartreusin bestimmt. Fur das Chartreusin folgt demnach die vollstandige Strukturformel I.

The aglycone of the complex coumarin antibiotic chartreusin has been shown to be entirely derived via the acetate pathway. The labelled aglycone, derived from feeding diethylmalonate- 14 C, has been degraded and a pathway for its biosynthesis is proposed.

Three DNA restriction fragments designated tyrT, 102-mer and 70-mer, have been used as substrates for footprinting studies using DNase I in the presence of the structurally similar antibiotics chartreusin and elsamicin A, The sequence-selective binding sites of the antibiotics can be mapped in regions which are rich In guanine+cytosine. Chartreusin and elsamicin appear to recognize and bind preferentially to sequences containing a CpG step. Regions containing a TpG step also seem to be a good binding site. The binding of elsamicin to these sites appears to be more concentration-dependent. A comparative analysis is performed of the sizes and locations of the different binding sites, aimed to infer whether the different biological effects of chartreusin and elsamicin A can be correlated to differences in their sequence-selective binding to DNA.

Chartreusin, an antitumor and antibacterial antibiotic, was found to inhibit negatively superhelical DNA-relaxation catalyzed by prokaryotic topoisomerase I and conversion of the superhelical DNA into unit length linear form catalyzed by single-strand-specific S1 nuclease. The inhibitory effect of the agent was due to the binding to DNA causing the alteration of tertiary structure. To characterize the binding specificity, we investigated the protection of DNA against cleavages by various restriction endonucleases. It was evidenced that the binding of the agent is not at random and correlates to the sequence 5'CGC 3' 3'GCG 5' on DNA stretch.

New antitumor antibiotics, elsamicins A and B, were isolated from the culture broth of an unidentified actinomycete strain J907-21 (ATCC 39417). They are structurally related to chartreusin, containing the common aglycone, chartarin, but contain different sugar moieties. Elsamicin A, the major component, has an amino sugar in the molecule which makes the antibiotic much more water-soluble than chartreusin. Elsamicin A exhibits strong inhibitory activity against various murine tumors including leukemia P388, leukemia L1210, and melanoma B16 but elsamicin B which lacks the amino sugar showed only marginal activity. The potency of elsamicin A was 10-30 times more potent than that of chartreusin in terms of minimum effective dose.

First isolated in 1953 from a fermentation broth, chartreusin (1) has received renewed interest as a result of substantial antitumor activities recently demonstrated in several murine test systems. Poor water solubility frustrated formulation attempts, and rapid biliary excretion observed in mice made 1 an improbable candidate for clinical development but an excellent candidate for an analogue synthesis program. From a common intermediate, which was prepared from 1, three analogues were synthesized wherein the disaccharide moiety of 1 was systematically replaced with fucose (6), glucose (7), and the disaccharide maltose (8). Each of the three analogues had a cytotoxic potency against cultured L1210 cells which was equal to, or better than, that shown by 1. Based on the structural similarity with the parent, an improved water solubility, and a favorable accessibility through synthesis, maltoside 8 was choe P388 leukemia, 8 showed reproducible activity comparable to chartreusin at similar dose levels. Although 8 caused no observable toxic effects at therapeutic dose levels when given ip, neither 1 nor 8 produced active indications when administered subcutnaeously.

Publisher Summary This chapter presents a comprehensive review of chartreusin, its chemistry, biosynthesis, biological action and drug potential. Spurred on by its activity in mouse leukemias as well as in solid tumour test systems, the present interest in chartreusin is centered on its potential as a cancer chemotherapy drug. Chartreusin is a biologically active parent structure on which to base an analogue synthesis effort. The structure of the antibiotic is not complex and should be amenable to an analogue synthesis programme. The chemical explorations that lead to total syntheses of the aglycone should prove to be useful in the generation of analogues. Because chartreusin is rapidly and efficiently excreted in the bile, the antibiotic could be used to study the process of biliary excretion. Phenolphthalein has been used in such studies, but conjugation with glucuronic acid is a complicating factor.

Chartreusin has been found to inhibit the aminoacyl transferase I specific binding of aminoacyl tRNA to washed reticulocyte ribosomes. This antibiotic also inhibits polyphenylalanine synthesis and the transferase I specific GTP hydrolysis on these ribosomes. Such inhibition by chartreusin is not reversed by preincubation of various components of the system together or by increasing the concentration of GTP, poly (U), phenylalanyl tRNA, or transferase I. Chartreusin binds strongly to the ribosomes and this binding is probably the key to the mode of inhibition by this antibiotic.