On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I-DNA covalent binary complex

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I-DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.     

Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site

Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.     

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)–DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5′ DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism.     

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.     

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.     

Lectin-carbohydrate interactions. Studies of the nature of hydrogen bonding between D-galactose and certain D-galactose-specific lectins, and between D-mannose and concanavalin A

The binding of galactose-specific lectins from Erythrina indica (EIL), Erythrina arborescens (EAL), Ricinus communis (agglutinin; RCA-I), Abrus precatorius (agglutinin; APA), and Bandeiraea simplicifolia (lectin I; BSL-I) to fluoro-, deoxy-, and thiogalactoses were studied in order to determine the strength of hydrogen bonds between the hydroxyl groups of galactose and the binding sites of the proteins. The results have allowed insight into the nature of the donor/acceptor groups in the lectins that are involved in hydrogen bonding with the sugar. The data indicate that the C-2 hydroxyl group of galactose is involved in weak interactions as a hydrogen-bond acceptor with uncharged groups of EIL and EAL. With RCA-I, the C-2 hydroxyl group forms two weak hydrogen bonds in the capacity of a hydrogen-bond acceptor and a donor. On the other hand, there is a strong hydrogen bond between the C-2 hydroxyl group of galactose, which acts as a donor, and a charged group on BSL-I. The C-2 hydroxyl group of the sugar is also a hydrogen-bond donor to APA. The lectins are involved in strong hydrogen bonds through charged groups with the C-3 and C-4 hydroxyl groups of galactose, with the latter serving as hydrogen-bond donors. The C-6 hydroxyl group of the sugar is weakly hydrogen bonded with neutral groups of EIL, EAL, and APA. With BSL-I, however, a strong hydrogen bond is formed at this position with a charged group of the lectin. The C-6 hydroxyl groups is a hydrogen-bond acceptor for EIL and EAL, a hydrogen-bond donor for APA and BSL-I, and appears not to be involved in binding to RCA-I. The data with the thiosugars indicate the involvement of the C-1 hydroxyl group of galactose in binding to EIL, EAL, and BSL-I, but not to RCA-I and APA. We have also performed a similar analysis of the binding data of fluoro- and deoxysugars to concanavalin A [Poretz, R. D. and Goldstein, I. J. (1970) Biochemistry 9, 2890-2896]. This has allowed comparison of the donor/acceptor properties and free energies of hydrogen bonding of the hydroxyl groups of methyl alpha-D-mannopyranoside to concanavalin A with the results in the present study. On the basis of this analysis, new assignments are suggested for amino acid residues of concanavalin A [corrected] that may be involved in hydrogen bonding to the sugar.     

Crucial structural factors and mode of action of polyene amides as inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Natural antibiotic polyene amides such as myxalamides are potent inhibitors of mitochondrial complex I. Because of the significant instability of this series of compounds due to an extended pi-conjugation skeleton, a detailed characterization of their inhibitory action has not been performed. To elucidate the action mechanism as well as binding manner of polyene amides with complex I, identification of the roles of each functional group in the inhibitory action is needed. We here synthesized a series of amide analogues and carried out structure-activity studies with bovine heart mitochondrial complex I. With respect to the left-hand portion, the natural pi-conjugation skeleton common to many natural products is not required for the inhibition and can be substituted with a simpler substructure such as a conjugated diene. The geometry and shape of the left-hand portion were shown to be important for the inhibition, suggesting that this portion may bind to a narrow hydrophobic pocket in the enzyme rather than merely partitioning into the lipid membrane phase. Concerning the right-hand portion of the inhibitor, the presence of the 2-methyl, amide NH, and (S)-1'-methyl groups was crucial for the activity, suggesting that both methyl groups neighboring the amide group finely adjust the hydrogen-bonding ability of the amide group. In contrast, modifications of the 2'-OH group did not significantly influence the activity, suggesting that the role of this functional group is not to serve as a hydrogen bond donor to the enzyme but to act as a hydrophilic anchor directing the right-hand portion at or near the membrane surface. Detailed characterization of the action mechanism indicated that the polyene amides share a common binding domain with other complex I inhibitors, though their binding position (or manner) within the domain may differ considerably from that of other inhibitors.     

Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin.

In order to investigate the mechanism of topoisomerase I inhibition by camptothecin, we studied the induction of DNA cleavage by purified mammalian DNA topoisomerase I in a series of oligonucleotides and analyzed the DNA sequence locations of preferred cleavage sites in the SV40 genome. The oligonucleotides were derived from the sequence of the major camptothecin-induced cleavage site in SV40 DNA (Jaxel, C., Kohn, K. W., and Pommier, Y. (1988) Nucleic Acids Res. 16, 11157 to 11170) with the cleaved bond in their center. DNA length was critical since cleavage was detectable only in 30 and 20 base pair-(bp) oligonucleotides, but not in a 12-bp oligonucleotide. Cleavage was at the same position in the oligonucleotides as in SV40 DNA. Its intensity was greater in the 30- than in the 20-bp oligonucleotide, indicating that sequences more than 10 bp away from the cleavage site may influence intensity. Camptothecin-induced DNA cleavage required duplex DNA since none of the single-stranded oligonucleotides were cleaved. Analysis of base preferences around topoisomerase I cleavage sites in SV40 DNA indicated that camptothecin stabilized topoisomerase I preferentially at sites having a G immediately 3' to the cleaved bond. Experiments with 30-bp oligonucleotides showed that camptothecin produced most intense cleavage in a complementary duplex having a G immediately 3' to the cleavage site. Weaker cleavage was observed in a complementary duplex in which the 3'G was replaced with a T. The identity of the 3' base, however, did not affect topoisomerase I-induced DNA cleavage in the absence of drug. These results indicate that camptothecin traps preferentially a subset of the enzyme cleavage sites, those having a G immediately 3' to the cleaved bond. This strong preference suggests that camptothecin binds reversibly to the DNA at topoisomerase I cleavage sites, in analogy to a model previously proposed for inhibitors of topoisomerase II (Capranico, G., Kohn, K.W., and Pommier, Y. (1990) Nucleic Acids Res. 18, 6611-6619).     

Interaction between SV40 DNA and camptothecin, an antitumor alkaloid

Camptothecin specifically interacted with closed superhelical circular SV40 DNA during incubation in 1.0 M NaCl at 37 degrees C and induced an alkali-labile linkage in the E strand. No interaction occurred in the reaction mixture containing 0.1 M NaCl, or at 4 degrees C. As camptothecin did not affect linear SV40 DNA, the superhelical structure of DNA appeared to be essential. The site of the alkali-labile linkage induced in SV40 DNA I through interaction with camptothecin was near the origin of replication on the basis of the results of experiments with restriction enzymes. Neither sulfhydryl reagents nor EDTA affected the interaction between camptothecin and SV40 DNA I, so the action of camptothecin is different from those of antitumor antibiotics, bleomycin or neocarzinostatin. Analysis of the s20,0w value of SV40 DNA I after the interaction with camptothecin and the sedimentation profiles of DNA after heating in the reaction mixture indicated that the interaction between camptothecin and SV40 DNA I was different from those of intercalating or alkylating agents such as ethidium bromide and methylmethanesulfonate. Replacement of the OH group at C-20 in the E ring of camptothecin by H-, CH3-, and Cl- resulted in the reduction, in this order, of the potency for interaction with SV40 DNA I to induce an alkali-labile linkage.     

DNA binding properties of novel distamycin analogs that lack the leading amide unit at the N-terminus

First examples of distamycin (Dst) analogs which lack hydrogen bond donor or acceptor groups at the N-terminus have been synthesized. The first molecule of this series, which is a bispyrrole peptide, did not exhibit any detectable binding with double-stranded (ds) DNA. However, all other analogs did bind strongly to AT-rich sequences of ds-DNA, with the binding affinities increasing as a function of the number of repeating pyrrole carboxamide units. These results imply that a hydrogen bond donor or acceptor atom per se at the N-terminus is not a prerequisite for DNA binding in the case of pyrrole carboxamide-based Dst analogs. However, in the absence of H-bond donor or acceptor at the N-terminus, a minimum of three pyrrole carboxamide units is necessary for the onset of DNA binding. Beyond this minimum number, the binding affinity increases as a function of the number of pyrrole units, as a result of the greater availability of hydrogen bonding and van der Waals surface. Experiments with poly[d(G-C)] have shown that the presence of the N-terminus formamide group is not inevitable for GC binding of this class of molecules. The observation that the N-terminus formamide unit can be dispensed with suggests that these molecules, which are much easier to synthesize and functionalize, can be used in place of the conventional analogs of distamycin for the development of novel minor groove binders with extended sequence recognition properties.     

Thermodynamic interactions in the glutamate dehydrogenase-NADPH-oxalylglycine complex

The enzyme-reduced coenzyme-alpha-ketoglutarate ternary complex is a critical intermediate in the glutamate dehydrogenase-catalyzed reaction. Oxalylglycine, a structural analog of alpha-ketoglutarate which contains an amide carbonyl group in place of a reducible ketone group, is one of the few compounds known to complete with alpha-ketoglutarate itself. In order to examine the role of the ketone group of alpha-ketoglutarate in the ternary complex, we have carried out a calorimetric study of the corresponding oxalylglycine ternary complex, determining the complete delta H, delta G, delta S, and delta Cp profiles and the corresponding interaction parameters for that complex and have compared the various parameters with the corresponding ones previously reported for the alpha-ketoglutarate ternary complex. While the overall delta G values of the two ternary complexes differ only slightly, the enzyme-NADPH-oxalylglycine ternary complex appears to achieve much of its stability from a very tight enzyme-oxalylglycine binary complex with little or no contribution from favorable interactions in the ternary complex, while the alpha-ketoglutarate ternary complex appears to achieve the same stability by a large interaction starting from a very weak enzyme-alpha-ketoglutarate binary complex. Consideration of the enthalpic profiles, however, show that this delta G-derived picture is deceptive. The excess binding energy which stabilizes the oxalylglycine binary is in fact due to hydrogen bonding of the amide group of oxalylglycine to the enzyme; in forming the ternary complex, this hydrogen bonding is lost in favor of forming an oxalylglycine-NADPH interaction, which is very similar to the alpha-ketoglutarate-NADPH interaction which stabilizes the alpha-ketoglutarate ternary complex. We conclude that the alpha-ketoglutarate-NADPH interaction must depend on either hydrogen bonding to or steric hindrance by the ketone group and that the existence of this energetically large interaction cannot be ascribed to imine formation between the keto group and enzyme. These findings also indicate the locus on the reaction coordinate where the reduced coenzyme plays a critical role, a role other than its obvious function as a hydride donor.     

The crystal structure of the alpha-cellobiose.2 NaI.2 H(2)O complex in the context of related structures and conformational analysis

The crystal structure of beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (alpha-cellobiose) in a complex with water and NaI was determined with Mo K(alpha) radiation at 150 K to R=0.027. The space group is P2(1) and unit cell dimensions are a=9.0188, b=12.2536, c=10.9016 A, beta=97.162 degrees. There are no direct hydrogen bonds among cellobiose molecules, and the usual intramolecular hydrogen bond between O-3 and O-5' is replaced by a bridge involving Na+, O-3, O-5', and O-6'. Both Na+ have sixfold coordination. One I(-) accepts six donor hydroxyl groups and three C-H***I(-) hydrogen bonds. The other accepts three hydroxyls, one Na+, and five C-H***I(-) hydrogen bonds. Linkage torsion angles phi(O-5) and psi(C-5) are -73.6 and -105.3 degrees, respectively (phi(H)=47.1 degrees and psi(H)=14.6 degrees ), probably induced by the Na+ bridge. This conformation is in a separate cluster in phi,psi space from most similar linkages. Both C-6-O-H and C-6'-O-H are gg, while the C-6'-O-H groups from molecules not in the cluster have gt conformations. Hybrid molecular mechanics/quantum mechanics calculations show <1.2 kcal/mol strain for any of the small-molecule structures. Extrapolation of the NaI cellobiose geometry to a cellulose molecule gives a left-handed helix with 2.9 residues per turn. The energy map and small-molecule crystal structures imply that cellulose helices having 2.5 and 3.0 residues per turn are left-handed.     

Design and optimization of camptothecin conjugates of triple helix-forming oligonucleotides for sequence-specific DNA cleavage by topoisomerase I.

To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex.     

Camptothecin: current perspectives

This review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor antibiotic. Two camptothecin analogues are presently approved for use in the clinic as antitumor agents and several others are in clinical trials. Camptothecin possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA. This review summarizes the current status of studies of the mechanism of action of camptothecin, including topoisomerase I inhibition and additional cellular responses. Modern synthetic approaches to camptothecin and several of the semi-synthetic methods are also discussed. Finally, a systematic evaluation of novel and important analogues of camptothecin and their contribution to the current structure-activity profile are considered.     

Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine.

The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl methionine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCA/TGG. Sequence-specific, covalent crosslinking of the enzyme to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is demonstrated. This reaction is abolished if serine replaces the cysteine at residue #177 of the enzyme. These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophilic attack by the methyl donor SAM. The enzyme is capable of self-methylation in a DNA-independent reaction requiring SAM and the presence of cysteine at position #177.     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.     

Conjugated eicosapentaenoic acid inhibits human topoisomerase IB with a mechanism different from camptothecin

Conjugated eicosapentaenoic acid (cEPA) has been found to have antitumor effects which has been ascribed to their ability to inhibit DNA topoisomerases and DNA polymerases. We here show that cEPA inhibits the catalytic activity of human topoisomerase I, but unlike camptothecin it does not stabilize the cleavable complex, indicating a different mechanism of action. cEPA inhibits topoisomerase by impeding the catalytic cleavage of the DNA substrate as demonstrated using specific oligonucleotide substrates, and prevents the stabilization of the cleavable complex by camptothecin. Preincubation of the inhibitor with the enzyme is required to obtain complete inhibition. Molecular docking simulations indicate that the preferred cEPA binding site is proximal to the active site with the carboxylic group strongly interacting with the positively charged K443 and K587. Taken together the results indicate that cEPA inhibitor does not prevent DNA binding but inhibits DNA cleavage, binding in a region close to the topoisomerase active site.