Interaction of the C14-OH Group of Adriamycin with DNA Phosphate as Spectroscopically Evidenced

Abstract

Monitoring the band of the antisymmetric stretching vibration of the backbone PO₂ ^? group in DNA-anthracycline complexes demonstrates an extraordinary wavenumber shift for the adriamycin complex compared to that of daunomycin. The structures of both anthracyclines, however, are very closely related and differ only by a surplus hydroxyl group of adriamycin in the C14 position. The wavenumber shift observed for the DNA-adriamycin complex is unequivocally attributed to an additional linkage of the C14-OH of adriamycin to the phosphate group of DNA. Thus, serveral of the hypothetical structural models for the DNA-adriamycin complex for which a hydrogen bond between the C14 hydroxyl of the drug and DNA phosphate was postulated (S. Neidle, Cancer Treatment Rep. 61, 928 (1977); G. J. Quigley et. al., Proc. Natl. Acad Sci. USA 77, 7204 (1980)) get the first clear-cut experimental evidence.     

Interaction of adriamycin with DNA as studied by resonance Raman spectroscopy.

Raman and resonance Raman spectra of the complex DNA-adriamycin in aqueous solution have been recorded and analysed. Calf thymus DNA was used and it is found that in the complex DNA-adriamycin the chromophore of adriamycin is intercalated in the GC sequences. The substituents on the rings give hydrogen bonding interactions with the base pairs above and below the intercalation site. It is suggested from the Raman and resonance Raman spectral modifications that the phenolic groups of the chromophore are involved in the drug-DNA intercalation, in addition to pi-pi, hydroxyl and amino group interactions.     

Subsidiary hydrogen bonding of intercalated anthraquinonic anticancer drugs to DNA phosphate

Several anthraquinone derivatives are active against different kinds of human cancer. The cancerostatic activity has been mainly attributed to their ability to bind strongly to DNA by intercalation. Here, infrared spectroscopy was used to detect further, more specific DNA interactions with the prominent anticancer drugs daunomycin, adriamycin, aclacinomycin A and mitoxantrone as well as with the cytotoxic violamycin BI. The most striking result was a significant decrease in wave number of the band arising from antisymmetric stretching vibration of the PO2- groups of DNA upon complexation with adriamycin, aclacinomycin A, violamycin BI and mitoxantrone. This became evident after separation of the contributions from conformational changes of DNA to the influence on the wave number of that band. The drug-induced shift was interpreted in terms of the formation of a hydrogen bond between the intercalated drug molecules and the PO2- moiety of DNA via the following terminal hydroxyl groups: C14-OH for adriamycin, C4-OH for both aclacinomycin A and violamycin BI and, more tentatively, the external side-chain OH of mitoxantrone. Theoretical considerations, consisting of semi-empirical CNDO/2 calculations as well as normal coordinate analyses performed with molecular model fragments, provided results confirming and rationalising the experimental findings. The capacities of the anthracyclines for restriction of the conformational flexibility of DNA differ, presumably due to variations in the spatial dimensions of the sugar moieties of the drugs. The compatibility of the present results with data obtained from current geometrical models, especially those for the DNA-daunomycin and DNA-adriamycin complexes, is discussed in detail.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

Role of iron in adriamycin biochemistry

Adriamycin forms a chelate with Fe(III) that exhibits complex redox chemistry. The drug ligand is able to directly reduce the bound Fe(III) with the concomitant production of a one-electron oxidized drug radical. This Fe(II) can reduce oxygen to hydrogen peroxide and cleave the peroxide to yield the hydroxyl radical. In addition, the drug X Fe complex can catalyze the transfer of electrons from reduced glutathione to molecular oxygen to yield superoxide, hydrogen peroxide, and hydroxyl radicals. The adriamycin X Fe complex binds to DNA to form a ternary drug X Fe X DNA complex, which is also able to catalyze the thiol-dependent reduction of oxygen and the formation of hydroxyl radical from hydrogen peroxide. As a consequence of this chemistry, the adriamycin X Fe complex can cleave DNA on the addition of glutathione or hydrogen peroxide. Although less well defined, the adriamycin X Fe complex can bind to cell membranes and cause oxidative destruction of these membranes in the presence of thiols or hydrogen peroxide.     

Sequence specific DNA binding of Ets-1 transcription factor: molecular dynamics study on the Ets domain--DNA complexes

Molecular dynamics (MD) simulations for Ets-1 ETS domain-DNA complexes were performed to investigate the mechanism of sequence-specific recognition of the GGAA DNA core by the ETS domain. Employing the crystal structure of the Ets-1 ETS domain-DNA complex as a starting structure we carried out MD simulations of: (i). the complex between Ets-1 ETS domain and a 14 base-pair DNA containing GGAA core sequence (ETS-GGAA); (ii). the complex between the ETS domain and a DNA having single base-pair mutation, GGAG sequence (ETS-GGAG); and (iii). the 14 base-pair DNA alone (GGAA). Comparative analyses of the MD structures of ETS-GGAA and ETS-GGAG reveal that the DNA bending angles and the ETS domain-DNA phosphate interactions are similar in these complexes. These results support that the GGAA core sequence is distinguished from the mutated GGAG sequence by a direct readout mechanism in the Ets-1 ETS domain-DNA complex. Further analyses of the direct contacts in the interface between the helix-3 region of Ets-1 and the major groove of the core DNA sequence clearly show that the highly conserved arginine residues, Arg391 and Arg394, play a critical role in binding to the GGAA core sequence. These arginine residues make bidentate contacts with the nucleobases of GG dinucleotides in GGAA core sequence. In ETS-GGAA, the hydroxyl group of Tyr395 is hydrogen bonded to N7 nitrogen of A(3) (the third adenosine in the GGAA core), while the hydroxyl group makes a contact with N4 nitrogen of C(4') (the complementary nucleotide of the fourth guanosine G(4) in the GGAG sequence) in the ETS-GGAG complex. We have found that this difference in behavior of Tyr395 results in the relatively large motion of helix-3 in the ETS-GGAG complex, causing the collapse of bidentate contacts between Arg391/Arg394 and the GG dinucleotides in the GGAG sequence.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Theoretical and spectroscopic evidence for coordination ability of 3,3'-benzylidenedi-4-hydroxycoumarin. New neodymium (III) complex and its cytotoxic effect

Theoretical and spectroscopic studies of 3,3'-benzylidenedi-4-hydroxycoumarin (bhc) have been performed. B3LYP/6-31G* calculations reproduced the experimental molecular structure of bhc and showed two O-H...O asymmetrical intramolecular hydrogen bonds with O...O distances 2.638 and 2.696 A. The calculated Fukui functions and Molecular Electrostatic Potential for bhc and its deprotonated form, bhc(2-), predicted that the most probable reactive sites for electrophilic attack and hydrogen bonds are the carbonyl oxygens, followed by the hydroxyl oxygens. The coordination ability of 3,3'-benzylidenedi-4-hydroxycoumarin has been proved in a complexation reaction with neodymium (III) ion. The new neodymium (III) complex of bhc was studied by elemental analyses, conductivity and other physical properties, mass spectra, (1)H, (13)C NMR, UV-Vis and IR spectroscopy. The data obtained are in agreement with the metal:ligand ratio of 1:1, and the formula Nd(bhc(2-))(OH)(H(2)O), where bhc(2-)=C(25)H(14)O(6)(2-). The vibrational analysis of the neodymium (III) complex, free bhc, and its monomeric building block, 4-hydroxycoumarin, showed that in the Nd(III) complex the ligand coordinates to the metal ion through both deprotonated hydroxyl groups. The participation of both carbonyl groups in coordination to the metal ion was confirmed by the significant shift of nu(C=O) to lower wavenumber. The evaluation of the cytotoxic activity of the new Nd(III) complex on SKW-3 and HL-60/Dox cells revealed, that it is a potent cytotoxic agent and should be subset further to more detailed pharmacological and toxicological study.     

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.     

Initial studies of a phytoestrogen-deoxyribonucleic acid interaction

Molecular modeling studies show that estrogens such as estradiol complement the topography of spaces between base pairs in unwound DNA and simultaneously hydrogen bond phosphate moieties on opposite strands. We demonstrate here that the phytoestrogen coumestrol has this capability, in addition to its documented properties of UV absorbance at lambda greater than 300 nm and fluorescence. The latter properties enable spectroscopic examination of interactions with DNA by methods not possible with estrogenic steroids. On exposure to calf thymus DNA, the UV spectrum of coumestrol displays a bathochromic shift and simultaneous hypochromic effect with an isosbestic point at 370 nm, suggesting a shift between coexisting free and bound states. Similar results are observed with the intercalating agents adriamycin, ethidium bromide, and acridine. The fluorescence spectrum of coumestrol is quenched on exposure to DNA as are those of adriamycin and acridine. Coumestrol differs from the intercalators in that denatured DNA does not affect its UV spectrum or alter its relative fluorescence yield. Unlike classical intercalators, coumestrol has no influence on the thermal stability of calf thymus DNA. Preliminary electrophoretic analysis of DNA plasmid conformers indicates that coumestrol is incapable of significantly altering DNA superhelical density, in contrast to ethidium bromide. These initial physicochemical data provide evidence for the DNA base-estrogen electronic and/or hydrophobic interactions suggested by modeling studies, yet tend to rule out classical intercalation as an explanation for these phenomena.     

Non-enzymatic ATP-mediated binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA

ATP mediates covalent binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA. This non-enzymatic reaction has been studied with 6-[14C]hydroxymethylbenzo[alpha]pyrene (]14C]BP-6-CH2OH) and 7-[14C]-hydroxymethylbenz[alpha]anthracene ([14C]BA-7-CH2OH) at 37 degrees C in Tris buffer (pH 7.0). While ADP mediates the reaction 25-50% as well as ATP, six other possible phosphate donors including AMP were inactive as cofactors. A complex response to ATP occurred in which low binding of BP-6-CH2OH or BA-7-CH2OH was observed at concentrations of ATP below 2.5 mM, but a greater than linear response to higher concentrations of ATP was observed until ATP was saturating. Binding of the substrates to RNA was much lower than to DNA. Fluorescence spectra of BP-6-CH2OH bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[alpha]pyrene bound to DNA and free 6-methylbenzo]alpha]pyrene, indicating that ATP-mediated binding of BP-6-CH2OH to DNA occurs at the 6-methyl group. The fate of ATP and ADP in the binding reaction of BP-6-CH2OH was examined by thin layer chromatography. Loss of one phosphate group occurs during the reaction. With ATP the rate of loss is about 100-fold greater than the rate of binding of BP-6-CH2OH to DNA. This implies that the binding reaction proceeds through formation of a presumed reactive and unstable phosphate ester intermediate which then inefficiently binds to DNA.     

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.     

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

Changes of activity of daunorubicin,adriamycin and stereoisomers following the introduction or removal of hydroxyl groups in the amino sugar moiety

The results of a study of the effects of hydroxyl groups at positions, 2, 4 and 6 of the amino sugar on the activity of daunorubicin, adriamycin, and stereoisomers are presented. While the 4′-deoxy derivatives showed a slightly increased biological activity as compared with the parent compounds, the derivatives containing an additional hydroxyl group were less active. It is suggested that the changes in the polarity and in the DNA binding ability of these derivatives are the main factors accounting for the difference in the in vivo activity. The possible relations among the pK_a values, the DNA binding properties, and the cellular uptake of the compounds are discussed with particular reference to their therapeutic effectiveness.     

Oxidative destruction of DNA by the adriamycin-iron complex

The 2:1 adriamycin-Fe(III) complex is able to bind to DNA and to catalyze its oxidative destruction. The binding of the drug-metal complex to DNA is indicated by characteristic spectral changes which are different from those seen with adriamycin intercalation and by the propensity of the drug-metal complex to precipitate DNA. Furthermore, intercalated adriamycin appears not to be available for iron binding. The resulting ternary complex is quite stable: it is not disrupted by incubation in the presence of EDTA and can be isolated by using Sephadex G-50 column chromatography. Disruption of the ternary complex requires vigorous conditions (extraction with phenol at 60 degrees C). The adriamycin-iron complex in free solution has the capacity to catalyze the reduction of oxygen by thiols. The DNA-bound drug-metal complex preserves this capacity over a wide range of complex/DNA ratios. As a consequence of this thiol-dependent oxygen reduction, DNA is cleaved. This thiol-dependent DNA cleavage has been shown to require hydrogen peroxide as an intermediate product. These results have led us to propose that the thiol-dependent DNA cleavage reaction has two stages involving (1) reduction of oxygen leading to hydrogen peroxide and then (2) peroxide-dependent DNA cleavage. An unusual property of this reaction is that the cleavage is not random but gives rise to a defined 2300 base pair fragment.     

Action models for the antitumor drug camptothecin: formation of alkali-labile complex with DNA and inhibition of human DNA topoisomerase I

The antitumor activity of camptothecin (CPT) and its derivatives, including water-soluble topotecan (TPT), is determined by their ability to inhibit human DNA topoisomerase I (top 1). On the other hand, TPT has been recently shown to bind to DNA. The proposed models are based on a two-step mechanism of TPT (CPT) dimer interaction with two spatially close DNA duplexes. At the first step, the CPT lactone form binds to DNA (Streltsov et al., Mol. Biol. vol. 36, no. 5 (2002)) through hydrogen bonding of its C16a carbonyl with the guanine 2-amino group. At the second step, CPT is converted to the carboxylate form. In the absence of top 1, the C17 hydroxyl of CPT is involved in ester exchange (nicking of the DNA sugar-phosphate backbone followed by covalent joining of free phosphate to C17) whereas its C20 carboxyl forms two hydrogen bonds with the same guanine nucleotide at the opposite end of the broken DNA backbone. As a result, CPT binds to both ends of the broken DNA. The resulting CPT-DNA complex is alkali-labile. In the presence of top 1, after CPT conversion to the carboxylate form and DNA nicking, the C17 hydroxyl makes a branching hydrogen bond with N1 and N3 of guanine while the C20 carboxyl makes two hydrogen bonds with the NH of Tyr723 and N(delta2)H(2) of Asp722. Owing to this, rotation of one end of the broken sugar-phosphate backbone about the other becomes impossible; hence the CPT inhibitory effect on top 1. The proposed models are consistent with the current body of experimental data.