Enediyne Antitumor Antibiotic Research Articles

Understanding and exploiting nature's chemical arsenal: the past, present and future of calicheamicin research.

The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology, and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the critical research discoveries relevant to the clinically most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clinical studies are also highlighted.

Release of the Neocarzinostatin Chromophore from the Holoprotein Does Not Require Major Conformational Change of the Tertiary and Secondary Structures Induced by Trifluoroethanol

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.

Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the cagA gene that encodes the C-1027 apoprotein.

C-1027, the most potent member of the enediyne antitumor antibiotic family, is produced by Streptomyces globisporus C-1027 and consists of an apoprotein (encoded by the cagA gene) and a nonpeptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazolinate, a deoxyamino hexose, a beta-amino acid, and an enediyne core. By adopting a strategy for cloning of the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to cagA, we have localized 75 kb of contiguous DNA from S. globisporus. DNA sequence analysis of two regions of the cloned gene cluster revealed two genes, sgcA and sgcB, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the cagA gene resides approximately 14 kb upstream of the sgcAB locus. The involvement of the cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production. These results represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis and provide a starting point for future genetic and biochemical investigations of C-1027 biosynthesis.

The reactivity of the 2-deoxyribonolactone lesion in single-stranded DNA and its implication in reaction mechanisms of DNA damage and repair.

The formal C1'-oxidation product, 2-deoxyribonolactone, is formed as a result of DNA damage induced via a variety of agents, including gamma-radiolysis and the enediyne antitumor antibiotics. This alkaline labile lesion may also be an intermediate during DNA damage induced by copper-phenanthroline. Oligo-nucleotides containing this lesion at a defined site were formed via aerobic photolysis of oligonucleotides containing a photolabile ketone, and were characterized by gel electrophoresis and electrospray mass spectrometry (ESI-MS). Treatment of oligo-nucleotides containing the lesion with secondary amines produces strand breaks consisting of 3'-phosphate termini, and products which migrate more slowly in polyacrylamide gels. MALDI-TOF mass spectrometry analysis indicates that the slower moving products are formal adducts of the beta-elimination product resulting from 2-deoxyribonolactone and one molecule of amine. The addition of beta-mercapto-ethanol to the reaction mixture produces thiol adducts as well. The stability of these adducts suggests that they cannot be the labile species characterized by gel electrophoresis in copper-phenanthroline-mediated strand scission. The characterization of these adducts by mass spectrometry also provides, by analogy, affirmation of proposals regarding the reactivity of nucleophiles with the beta-elimination product of abasic sites. Finally, the effects of this lesion and the various adducts on DNA repair enzymes are unknown, but their facile generation from oligonucleotides containing a photolabile ketone suggests that such issues could be addressed.

Enediyne Biosynthesis and Self-Resistance: A Progress Report

The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, remarkable biological activity, and fascinating mode of action, and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains, and eventually lead to an enediyne combinatorial biosynthesis program. This article reviews the published enediyne biosynthetic labeling and blocked mutant studies and provides a brief account of efforts in the Shen laboratory to elucidate the route to C-1027 biosynthesis and efforts in the Thorson laboratory to elucidate the calicheamicin biosynthetic cascade.

Mechanistic studies on the base-catalyzed transformation of neocarzinostatin chromophore: roles of bulged DNA.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom) in a base-catalyzed, radical-mediated reaction. Studies designed to elucidate the detailed mechanism of the base-catalyzed activation of NCS-chrom and to evaluate the roles of bulged DNA in its activation are described. They show that nucleobases in the DNA bulge are not required to form an effective bulge pocket but enhance the binding of the wedge-shaped activated drug molecule. Analysis of solvent deuterium isotope effects on NCS-chrom degradation and DNA cleavage efficiency experiments suggests that the spirolactone biradical 6 is a relatively stable species and that intramolecular quenching of the C2 radical of 6 to form the biologically active cyclospirolactone radical 7a occurs first (pathway a in Scheme 2), leaving the C6 radical to abstract the hydrogen atom from the DNA deoxyribose and to form the cyclospirolactone 8. Binding of the activated drug at the bulge site is required, but not sufficient, for efficient 8 formation, whereas cleavage of bulged DNA is not essential. Efficient generation of 8, but inefficient DNA damage generation, comes mainly from the likely high off-rate of 7a binding. The finding that thymidine 5'-carboxylic acid-ended oligonucleotide fragment can be formed in the reaction suggests that the process of DNA cleavage is rather slow and that sequential oxidations of the target 5'-carbon are possible. Study of the effect of solvent (methanol) concentration on NCS-chrom degradation indicates that bulged DNA acts to assist the intramolecular quenching of the radical at C2 by C8' ' of the naphthoate moiety by excluding solvent from the binding pocket, thus preventing the formation of spirolactones 9, and by blocking radical polymerization. Because in the absence or near absence of solvent methanol 8 formation does not reach even 10% that formed in the presence of bulged DNA, it is possible that the DNA bulge also induces a conformational change in the drug to promote the intramolecular reaction.

Synthesis of the namenamicin A–C disaccharide: towards the total synthesis of namenamicin

Progress towards the total synthesis of namenamicin (1), the only enediyne antitumor antibiotic of marine origin, is reported. Two methods were investigated for the stereoselective introduction of the highly unusual quaternary C-4 center of the A-ring. Spirocyclic [3,3]-sigmatropic rearrangements of the thiocarbonates 6 and 10 gave products 7 and 11, from processes involving approach of the sulfur atom solely to the α-faces of the substrates. The desired stereochemistry of the quaternary center was ultimately obtained by way of an intramolecular Michael addition of a xanthate anion derived from the α,β-unsaturated methyl ester 18. Further functional group manipulations provided an olefin 25 which underwent dihydroxylation to give a mixture of diastereomeric diols 26, allowing access to both potential diastereomers of the natural product. The A ring intermediate 29 was found to be a viable substrate of glycosidation with a glycosyl fluoride of the C ring aminosugar 30, providing the protected A–C disaccharide subunit of 1.

ChemInform Abstract: Pyrenylboronic Acids as a Novel Entry for Photochemical DNA Cleavage: Diradical‐Forming Pyrene‐1,6‐diyldiboronic Acid Mimics the Cleavage Mechanism of Enediyne Antitumor Antibiotics.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Diastereoselective Synthesis of the Monosaccharide Kedarosamine and Incorporation in an Analogue of the Enediyne Kedarcidin Chromophore

Kedarcidin chromophore, as with most enediyne antitumor antibiotics, contains unusual monosaccharide moieties. Synthesis of one of these moieties, the 2,4,6-trideoxy-4-dimethylaminohexose kedarosamine, from d-threonine and incorporation into an analogue of kedarcidin chromophore (1) is described herein. Conversion of d-threonine into allyl ketone 7 and stereoselective reduction by using tetramethylammonium triacetoxyborohydride for intramolecular hydride delivery were key steps in the preparation of kedarosamine. A thioglycoside derivative of kedarosamine (12) was found to be less efficient as a glycosyl donor, whereas a 1-O-acetate (15) gave the desired α-glycoside exclusively in 60−80% yield when treated with borontrifluoride etherate. Use of a Cbz instead of a Fmoc protecting group for the C-4 amino group of kedarosamine was essential for the successful preparation of analogue 1. Finally, dimethylation of the amino group at C-4 of kedarosamine was found to require careful adjustment of the reaction con...

Pyrenylboronic acids as a novel entry for photochemical DNA cleavage: diradical-forming pyrene-1,6-diyldiboronic acid mimics the cleavage mechanism of enediyne antitumor antibiotics

Pyren-1-ylboronic acid 4 and pyrene-1,6-diyldiboronic acid 5 are bound to supercoiled double-strand circular DNA plasmid ColE1 (colDNA) and effect its efficient photocleavage under visible light irradiation. Mechanistic studies show that the cleavage occurs according to the radical mechanism. The cleavage activity of monoacid 4 could be suppressed by the addition of D-fructose because of boronic acid–saccharide complexation. Diacid 5 is bound to colDNA more strongly than is monoacid 4 and effects its efficient photocleavage to yield Form III. The results show that diradical-forming diacid 5 is able to cleave DNA at two reaction sites, mimicking the cleavage mechanism of enediyne antitumor antibiotics.

Mechanism of Formation of Novel Covalent Drug·DNA Interstrand Cross-Links and Monoadducts by Enediyne Antitumor Antibiotics

The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4', C1', and C5' positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5'GTTA1T/5'ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin gamma1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin gamma1I was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.

Chromatographic and spectroscopic assignment of thiol induced cycloaromatizations of enediyne in neocarzinostatin

Neocarzinostatin is an enediyne antitumor antibiotic. Upon attack by a thiol, the enediyne nucleus is cycloaromatized into two stable 1:1 thiol adducts. After analyzing products from various thiols, the chromatographic and spectroscopic characters that associate only with the cyclized aromatic moiety from enediyne nucleus were assigned. Based on HPLC analysis we have derived, products from picomole ranges of the drug sample can be detected. Confirming the type of cycloaromatization at nanomole ranges can be achieved by photodiode array UV spectroscopy. Three-dimensional fluorogram presents ten times more sensitive identification. The method provides a sensitive tool for massive screening study in microscale.

Selective cleavages of tRNA Phe with secondary and tertiary structures by enediyne antitumor antibiotics

Some enediyne antitumor antibiotics induce site-selective cleavages for yeast tRNA Phe with three-dimensional structure. Of special interest is the fact that tRNA Phe is specifically cleaved at the anticodon arm regions by C-1027 and esperamicin A l in the presence of Mg 2+ ions. Although neocarzinostatin strongly breaks tRNA Phe at 5′-G Pu steps in the absence of magnesium ions, its cleavage ability is completely lost in the presence of 100 μM Mg 2+ ions. Dynemicin A, which favors an intercalative binding, causes no strand scissions for the RNA with secondary and tertiary structures. This cutting of tRNA Phe may reveal that RNA as well as DNA constitutes a therapeutically relevant target for certain enediyne antitumor antibiotics.

Design of Diradical‐based Hydrogen Abstraction Agents

Why are diradicals used as hydrogen abstraction agents in the enediyne antitumor antibiotics, and what would happen if a “nonnatural” diradical were to be used? The very slow abstraction rates by arene diradicals suggest an answer to the question of the functional significance of diradicals versus monoradical abstraction agents in the natural products, and furthermore, suggest potential strategies for the rational modification of DNA‐cleaving agents by alteration of the diradical “warhead”.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

9,10-Dehydroanthracene: p-Benzyne-Type Biradicals Abstract Hydrogen Unusually Slowly

The 9,10-dehydroanthracene biradical, a model for the p-benzyne-type biradicals implicated in DNA cleavage by the enediyne antitumor antibiotics, was prepared by photodissociation of a propellane in solution. Trapping products characteristic of biradicals, e.g. anthracene-d2, are found. The rates of hydrogen abstraction by the biradical from acetonitrile and isopropyl alcohol are measured directly by flash photolysis/transient absorption spectroscopy, giving second-order rate constants of kMeCN,abstr = (1.1 ± 0.2) × 103 M-1 s-1 and ki-PrOH,abstr = (6.5 ± 0.6) × 103 M-1 s-1 at room temperature, which are 100−200 times lower than the corresponding rate constants for phenyl or 9-anthryl radical. A second decay route for the biradical is found, and assigned, based on thermochemical, kinetic, and trapping arguments, to a retro-Bergman reaction that converts the 9,10-dehydroanthracene biradical into the ring-opened 3,4-benzocyclodeca-3,7,9-triene-1,5-diyne. Although the retro-Bergman reaction is relatively fast...

Maduropeptin: an antitumor chromoprotein with selective protease activity and DNA cleaving properties.

Maduropeptin (MDP) is a recently isolated antitumor antibiotic, consisting of an enediyne-containing chromophore embedded in a highly acidic protein. This holoantibiotic damages duplex DNA in vitro, producing a mixture of single- and double-strand breaks at selected sites. The chromophore, isolated as the methanol adduct from the protein-containing holoantibiotic, exhibits the same selectivity and cleavage chemistry as the chromoprotein complex. Preliminary evidence suggests that the primary DNA breaks involve 4'-H abstraction from the deoxyribose sugars at the cleavage sites. Unlike most other enediyne antitumor antibiotics, DNA strand scission is not bioreductively induced by MDP or the methanol adduct of the chromophore. This was also observed for the C1027 chromophore. DNA cleavage is inhibited in the presence of certain cations (Ca2+, Mg2+) as was observed with the kedarcidin chromophore. 1H NMR spectroscopy studies on the methanol adduct of the maduropeptin chromophore in the presence of calcium chloride provide clues regarding its activation and give insight as to the regions of the chromophore important for DNA binding. Our results suggest that the solvent artifact of the chromophore may in essence be a prodrug and it regenerates the parent chromophore as in the holoantibiotic prior to cleaving DNA. As with kedarcidin and neocarzinostatin, maduropeptin exhibits a high affinity for histones, in vitro, cleaving them to low molecular mass peptides. Histone H1, the most opposite in net charge, is cleaved most readily. This latter activity may serve to disrupt the chromatin superstructure in vivo, prior to exposing the DNA to the chromophore.

RNA Cleavage by C-1027 Chromophore, an Enediyne Antitumor Antibiotic: High Selectivity to an Anticodon Arm

This study demonstrates unique reactivity of the C-1027 chromophore toward a tRNA phe. In the presence of Mg 2+ ions where the tRNA phe attains a stable three-dimensional structure, the enediyne chromophore exhibits high cleavage selectivity to the anticodon arm. The present reactivity of the C-1027 chromophore is useful in development of new chemical probes for mapping of tertiary RNA structure. Considering the paucity of RNA repair mechanisms, RNA may be also an important biological target for certain enediyne antibiotics.

Induction of Apoptosis by Enediyne Antitumor Antibiotic C1027 in HL-60 Human Promyelocytic Leukemia Cells

C1027, a new macromolecular antitumor antibiotic with an enediyne chromophore, displays extremely potent cytotoxicity against cancer cells. HL-60 human promyelocytic leukemia cells treated with C1027 (0.1-10 nM) for 2 hours resulted in morphological changes, including Hoechst 33342-stained condensed nuclei, condensation of nuclear chromatin, and nuclear fragmentation. Agarose gel electrophoresis of C1027-treated HL-60 cells showed a typical ladder-like pattern of DNA fragments. In addition, the apoptotic DNA peak of propidium iodide-stained nuclei was revealed by flow cytometry. Treatment of HL-60 cells with C1027 (5 nM) induced apoptosis in up to 79% of the cells. The results suggest that C1027 may exert antitumor activity by triggering apoptosis.

Diastereoselective synthesis of methyl α-kedarosaminide, a carbohydrate moiety of the enediyne antitumor antibiotic kedarcidin chromophore

Methyl α-kedarosaminide ( 9), a carbohydrate moiety of the enediyne antitumor antibiotic kedarcidin chromophore, was synthesised from d-threonine. Stereoselective reduction of the allyl ketone 5 derived from d-threonine was a key step in the synthesis, which was achieved by using Me 4NBH(OAc) 3 for intramolecular hydride delivery.