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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

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.

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.

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.

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.

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 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.

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.

The apparent aqueous solubility of the water-insoluble cytotoxic agent, chartreusin, was increased at neutral pH in the presence of hydroxybenzoates. Water molecules play an important role in the chartreusin conformation. Studies included solubility and spectral examinations. The weakest and strongest interactants with chartreusin were sodium benzoate and sodium trihydroxybenzoate, respectively, while the effect of mono- and dihydroxybenzoates was intermediate. A plane-to-plane orientation of chartreusin and the ligand molecules brought together by electrostatic and hydrophobic interactions is postulated. The dramatic chartreusin aqueous solubility increase relative to its aglycone, chartarin, under similar conditions was best rationalized by micellization.

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.

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.

The in vitro activity of elsamicin A (ELS) was investigated compared with that of doxorubicin (DX) on two sensitive breast cancer cell lines: one estrogen receptor-positive (ER+, MCF7) and one estrogen receptor-negative (ER-, MDA-MB-231) line, and on a DX-resistant subline (MCF7DX). The activity of the two drugs was also investigated on 19 clinical breast cancer specimens from untreated patients. The drugs were tested at pharamcologically relevant concentrations, as calculated from the area under the curve for a 3 h exposure to the lethal dose producing 10% mortality (LD10) in mice, and at 10- and 100-fold concentrations. In DX-sensitive lines, a greater inhibition of RNA and DNA precursor incorporation, as well as of cell proliferation, was caused by ELS than by DX. Moreover, the antiproliferative effect was 10-fold higher in the ER+ MCF7 than in the ER- MDA-MB-231 cell line (IC50: 0.25 versus 0.21 micrograms/ml). ELS was cross-resistant to DX in the MCF7DX subline. In clinical specimens, effects on DNA precursor incorporation were more often observed for ELS than for DX at the same drug concentrations. The in vitro sensitivity to ELS was more pronounced for ER+ than for ER- tumors: minimal inhibiting concentrations of the drug were 0.1 and 3.5 micrograms/ml, respectively, in the two groups. If confirmed in a larger series of human breast tumors, these in vitro results would indicate a promising role for ELS in clinical treatment, mainly of ER+ breast cancer patients.