Mode of reversible binding of neocarzinostatin chromophore to DNA: base sequence dependency of binding.

The reversible binding of neocarzinostatin chromophore to polynucleotides was studied in order to understand the molecular basis of its base sequence-specificity in DNA damage production. Studies of the spectroscopic and thermodynamic properties of chromophore-polynucleotide interactions reveal that the binding of the chromophore to poly(dA-dT) is qualitatively and quantitatively different from that to poly(dG-dC) (and poly(dI-dC]. From these and other experiments using double-stranded mixtures of homopolynucleotides, it is proposed that the observed A T specific intercalation might result from differential recognition of minor variations in the B-DNA type structure by the chromophore.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove

Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS), which has DNA-degrading activity comparable to that of the native antibiotic, was found to have a strong affinity for DNA. Binding of chromophore was shown by (1) quenching by DNA of the 440-nm fluorescence and shifting of the emission peak to 420 nm, (2) protection by DNA against spontaneous loss of activity in aqueous solution, and (3) inhibition by DNA of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these three methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence, suggesting spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence seven-fold and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercapto-ethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, suggesting intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.     

Cryospectrokinetic evidence for the mode of reversible binding of neocarzinostatin chromophore to poly(deoxyadenylic-thymidylic acid)

The spectra of neocarzinostatin (NCS) chromophore during its reversible association with poly(dA-dT).poly(dA-dT) [poly(dA-dT)] were recorded (at intervals of 17 ms or more) by a cryospectroscopic method. Examination of the spectral changes of a drug during its interaction with DNA has not been previously reported. Such studies indicate binding of chromophore to poly(dA-dT) is a two-step process in which the spectral properties of the intermediate poly(dA-dT). NCS chromophore species closely resemble those of the final equilibrium species. On the basis of cryokinetic studies (at single wavelengths) carried out at low temperature (2 degrees C), the following proposed mechanism of the DNA-drug (PD) interaction was quantitated: (Formula: see text). In analogy with the other reports on the kinetics of drug-DNA interaction, (PD)I and (PD)II could represent externally bound and intercalated complexes, respectively. However, since the spectra of (PD)I and (PD)II are closely similar, it can also be proposed that (PD)I and (PD)II represent two forms of an intercalated complex. The rate and equilibrium constant for each step were determined by examining the kinetics of the forward and reverse reactions. This was accomplished by determining the polynucleotide concentration dependence of the apparent fast and slow first-order rate constants observed during a double-exponential increase in transmittance (at 330 nm) associated with the binding and the apoprotein-induced dissociation rate constant of the chromophore from poly(dA-dT). The opportunity to use apoprotein, instead of a detergent, to follow the kinetics of the reverse reaction provides a novel approach to these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore

Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.     

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.     

Chemical synthesis and cytotoxicity of dihydroxylated cyclopentenone analogues of neocarzinostatin chromophore

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.     

Sequence-specific binding of luzopeptin to DNA.

We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.     

Stoichiometric uptake of molecular oxygen and consumption of sulfhydryl groups by neocarzinostatin chromophore bound to DNA

In the presence of DNA, and under conditions which resulted in efficient DNA degradation, the reaction of the neocarzinostatin chromophore with sulfhydryl groups was accompanied by a rapid drop in the oxygen tension of the solution. The total extent of oxygen uptake indicated that, consistently, 1 mol of O2 was consumed/mol of chromophore. The rate of oxygen uptake, however, was strongly dependent on the sulfhydryl concentration, and uptake occurred within a few seconds of the sulfhydryl-induced increase in 420-nm fluorescence of the chromophore. Parallel experiments, in which the sulfhydryl concentration of the solution was monitored, showed that approximately 2 mol of sulfhydryl groups were consumed/mol of chromophore, with kinetics similar to those of O2 uptake. Under anaerobic conditions, only 1 mol of sulfhydryl was consumed, but the sulfhydryl-induced fluorescence increase was not inhibited. These results suggest that (i) a reaction with a single sulfhydryl group converts the chromophore to an activated form, (ii) in the presence of DNA this activated chromophore participates in a subsequent reaction which consumes 1 mol of O2 followed by an additional mole of sulfhydryl, and (iii) each chromophore molecule undergoes only one such reaction cycle. In the absence of sulfhydryl groups, the chromophore slowly degraded, giving a product with intense 490-nm fluorescence. This spontaneous degradation reaction, which does not result in DNA damage, was also accompanied by uptake of nearly 1 mol of O2/mol of chromophore.     

Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS), which possesses the full in vitro and in vivo deoxyribonucleic acid (DNA) strand-breaking activities and the ability to inhibit DNA synthesis and growth in HeLa cells of the holoantibiotic, is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, whereas bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of aurodomomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0 degrees C in serum-free medium so as to inhibit subsequent DNA synthesis at 37 degrees C. Such interaction at 0 degrees C is very rapid, reaching 50% completion in about 15 s, and is not found with native NCS or when apo-NCS is added before the chromophore or when serum is included in the preincubation at 0 degrees C. Washing with apo-NCS or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0 degrees C fails to reverse the subsequenct inhibition of DNA synthesis.     

Solution NMR structure investigation for releasing mechanism of neocarzinostatin chromophore from the holoprotein

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.     

Competition between anaerobic covalent linkage of neocarzinostatin chromophore to deoxyribose in DNA and oxygen-dependent strand breakage and base release

Treatment of poly(dA-dT) X poly(dA-dT) with the nonprotein chromophore of neocarzinostatin in the presence of sulfhydryls resulted in both direct and alkali-dependent base release, indicative of DNA sugar oxidation. Covalent chromophore-DNA adducts were also formed. Under anaerobic conditions, base release was strongly inhibited; however, adduct formation was not inhibited and in some cases was markedly enhanced. In the presence of dithiothreitol, anoxia increased adduct formation by a factor of 2, and a particularly stable adduct species was formed, which was recovered from nuclease digests of the treated DNA as a highly fluorescent compound with structure chromophore-d(TpApT). Acid hydrolysis of chromophore-d(TpApT) released free adenine base and both 3'dTMP and 5'dTMP, leaving a compound that contained only chromophore and the deoxyadenosine sugar. These results conclusively confirm that the chromophore forms a covalent adduct with deoxyribose in DNA. Thus, even in the absence of oxygen, activation of the chromophore by sulfhydryls results in the formation of a species capable of reacting with deoxyribose. Several other adduct species were also formed, some of which were nonfluorescent and relatively hydrophilic, but all of which were produced in increased amounts under anoxia. This inverse relation between sugar oxidation and adduct formation suggests that the two lesions share a common precursor. In the presence of other thiols, the effects of anoxia were somewhat different. With glutathione, anoxia markedly enhanced adduct formation, but the total adduct formed was considerably less than with dithiothreitol. With 2-mercaptoethanol, anoxia had no effect on total adduct formation, but the distribution of adduct species was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of phycobilisome core-membrane linker, LCM, by autocatalytic chromophore binding to ApcE

The core-membrane linker, LCM, connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane. Genes coding for the apoprotein, ApcE, from Nostoc sp. PCC 7120 and for a C-terminally truncated fragment ApcE(1-240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli. Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins. If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker LCM, demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE. The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling. The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-alpha84 lyases, indicates that ApcE constitutes a third type of bilin:biliprotein lyase.     

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.     

Activation of neocarzinostatin chromophore and formation of nascent DNA damage do not require molecular oxygen.

Thiol-activated neocarzinostatin chromophore abstracts tritium from the 5', but not from the 1' or 2' positions of deoxyribose in DNA and incorporates it into a stable, non-exchangeable form. The abstracted tritium remains covalently associated with the chromophore or its degradation product after treatment with acid or alkali, respectively. Drug activation and the consequent hydrogen abstraction reaction, presumably generating a carbon-centered radical at C-5', do not require molecular oxygen but have a dose-dependent relation with thiol. Under aerobic conditions, where base release and DNA strand breaks with nucleoside 5'-aldehyde at the 5'-ends are produced, hydrogen abstraction from C-5' parallels these parameters of DNA damage. It is possible to formulate a reaction scheme in which the carbon- centered radical at C-5' is an intermediate in the formation of the various DNA damage products found under both aerobic and anaerobic conditions.     

Induced formation of a DNA bulge structure by a molecular wedge ligand-postactivated neocarzinostatin chromophore

Our previous structure elucidation of the complexes of DNA and postactivated neocarzinostatin chromophore (NCS-chrom) compounds revealed two distinctly different binding modes of this antitumor molecule. A thorough understanding of these results will provide the molecular basis for the binding and DNA chain cleavage properties of NCS-chrom. NCSi-gb is one of the postactivated mimics of NCS-chrom which is formed under thiol-free conditions and is able to bind to DNA. This report describes the structure refinement of the NCSi-gb-bulge-DNA complex [Stassinopoulos, A., Jie, J., Gao, X., and Goldberg, I. H. (1996) Science 272, 1943-1946] and the NMR characterization of the free bulge-DNA and free NCSi-gb. These results reveal that the formation of the complex involves conformational changes in both the DNA and the ligand molecule. Of mechanistic importance for the NCS-chrom-DNA interaction, the two ring systems of the drug are brought closer to each other in the complex. This conformation correlates well with the previously observed marked enhancement of the formation of a DNA bulge cleaving species in the presence of bulge-DNA sequences, due to the promotion of the intramolecular radical quenching of the activated NCS-chrom. Interestingly, the binding of NCSi-gb promotes the formation of a bulge binding pocket; this was not found in the unbound DNA. NCS-chrom is unique among the enediyne antibiotics in its ability to undergo two different mechanisms of activation to form two different DNA binding and cleaving species. The two corresponding DNA complexes are compared. One, the bulge-DNA binder NCSi-gb, involves the major groove, and the second, the duplex binder NCSi-glu which is generated by glutathione-induced activation, involves the minor groove. Since the two NCS-chrom-related ligand molecules contain some common chemical structural elements, such as the carbohydrate ring, the striking differences in their DNA recognition and chain cleavage specificity provide insights into the fundamental principles of DNA recognition and ligand design.