A-ring and E-ring modifications of the cytotoxic alkaloid Luotonin A: Synthesis,computational and biological studies

A series of new Luotonin A derivatives with substituents at rings A and E was synthesized, together with some E-ring-unsubstituted derivatives. Subsequently, the compound library was examined in silico for their binding into a previously proposed site in the DNA/topoisomerase I binary complex. Whereas no convincing correlation between docking scores and biological data from in vitro assays could be found, one novel 4,9-diamino Luotonin A derivative had strong antiproliferative activity based on massive G2/M phase arrest. As this biological activity clearly differs from the reference compound Camptothecin, this strongly indicates that at least some Luotonin A derivatives may be potent antiproliferative agents, however with a different mode of action.     

Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

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.     

Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme.

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.     

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.     

Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides

DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein. We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length. DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer ( approximately 20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III. P1 nuclease and KMnO(4) footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2-3 nucleotides downstream of the cleavage site. Binding characteristics indicate that the enzyme interacts efficiently with both single-stranded and double-stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase. The oligonucleotides containing STS effectively inhibit the M. smegmatis topoisomerase I DNA relaxation activity.     

Eukaryotic topoisomerase I-DNA interaction is stabilized by helix curvature.

The influence of DNA structure on topoisomerase I-DNA interaction has been investigated using a high affinity binding site and mutant derivatives thereof. Parallel determinations of complex formation and helix structure in the absence of superhelical stress suggest that the interaction is intensified by stable helix curvature. Previous work showed that a topoisomerase I binding site consists of two functionally distinct subdomains. A region located 5' to the topoisomerase I cleavage site is essential for binding. The region 3' to the cleavage site is covered by the enzyme, but not essential. We report here that the helix conformation of the latter region is an important modulator of complex formation. Thus, complex formation is markedly stimulated, when an intrinsically bent DNA segment is installed in this region. A unique pattern of phosphate ethylation interferences in the 3'-part of the binding site indicates that sensing of curvature involves backbone contacts. Since dynamic curvature in supercoiled DNA may substitute for stable curvature, our findings suggest that topoisomerase I is able to probe DNA topology by assessment of writhe, rather than twist.     

Analysis of etoposide binding to subdomains of human DNA topoisomerase II alpha in the absence of DNA

Epipodophyllotoxins are effective anti-tumor drugs that inhibit eukaryotic DNA topoisomerase II by trapping the enzyme in a covalent complex with DNA. We show that both the recombinant N-terminal ATPase domain and the B'A' core domain of human topoisomerase IIalpha (htopoIIalpha) bind radiolabeled etoposide specifically, even in the absence of DNA. The addition of ATP impairs etoposide binding to the holoenzyme and the N-terminal domain, but not to the core domain. To see if this interference resembles that between novobiocin and ATP in the bacterial GyrB subunit, we modeled the structure of the N-terminal domain of htopoIIalpha and performed molecular docking analysis with etoposide. Mutagenesis of critical amino acids, predicted to stabilize the drug within the N-terminal domain, reveals a less efficient binding of etoposide to the mutated proteins as monitored by direct drug binding assays, although the binding of ATP is not affected.     

Topoisomerase II is a cellular target for antiproliferative cobalt salicylaldoxime complex.

Topoisomerase II is a cellular target for a number of clinically relevant antitumor drugs. To elucidate the possible cellular target for the antiproliferation activity of cobalt salicylaldoxime (CoSAL), which inhibits 50% of leukemic cell proliferation at a concentration of 60 microM, DNA binding studies and studies of the action of this complex on topoisomerase II catalytic activities were carried out. The results from DNA binding studies show that CoSAL binds DNA strongly with a stoichiometric ratio of two drug molecules for five nucleotide bases and shows a mode of interaction similar to that of DNA groove binding agents. The results from topoisomerase II inhibition studies show that the complex inhibits the relaxation activity of topoisomerase II in a dose-dependent manner and poisons its activity through cleavage complex formation. To see if the hydroxyl group present on imine nitrogen is involved in topoisomerase II poisoning, we synthesized an analogue of CoSAL in which the hydroxyl group was replaced with semicarbazone. This complex too binds DNA with an affinity similar to that of CoSAL, but with a small difference in the mode of interaction; however, it marginally inhibits leukemic cell proliferation and does not inhibit topoisomerase II activity, which suggests the involvement of a hydroxyl group. An immunoprecipitation assay was conducted which showed that the cleavage complex formed in the presence of CoSAL contained 75% of the complex, while the other complex shows only 7. 65%. Cyclic voltametric spectra of the complexes in the presence of DNA show that they do not oxidize DNA. These results suggest that CoSAL shows a bidirectional mode of interaction with enzyme and DNA and inhibits topoisomerase II activity by forming a drug-mediated cleavage complex. Our data strongly suggest that topoisomerase II may be one of the cellular targets for antiproliferation activity of CoSAL.     

Design, synthesis, and biological evaluation of a novel series of bisintercalating DNA-binding piperazine-linked bisanthrapyrazole compounds as anticancer agents

A series of bisintercalating DNA binding bisanthrapyrazole compounds containing piperazine linkers were designed by molecular modeling and docking techniques. Because the anthrapyrazoles are not quinones they are unable to be reductively activated like doxorubicin and other anthracyclines and thus they should not be cardiotoxic. The concentration dependent increase in DNA melting temperature was used to determine the strength of DNA binding and the bisintercalation potential of the compounds. Compounds with more than a three-carbon linker that could span four DNA base pairs achieved bisintercalation. All of the bisanthrapyrazoles inhibited human erythroleukemic K562 cell growth in the low to submicromolar concentration range. They also strongly inhibited the decatenation activity of topoisomerase IIα and the relaxation activity of topoisomerase I. However, as measured by their ability to induce double strand breaks in plasmid DNA, the bisanthrapyrazole compounds did not act as topoisomerase IIα poisons. In conclusion, a novel group of bisanthrapyrazole compounds were designed, synthesized, and biologically evaluated as potential anticancer agents.     

Modification-dependent restriction endonuclease,MspJI, flips 5-methylcytosine out of the DNA helix

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme''s two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.     

Mechanism of inhibition of DNA gyrase by ES-1273, a novel DNA gyrase inhibitor

We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor.     

Human topoisomerase I poisoning by protoberberines: potential roles for both drug-DNA and drug-enzyme interactions

Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I poisoning. We have employed a broad range of biophysical, biochemical, and computer modeling techniques to characterize and cross-correlate the DNA-binding and topoisomerase poisoning properties of four protoberberine analogues that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an observation consistent with an intercalative mode of interaction. (ii) Enthalpically favorable interactions, such as stacking interactions between the intercalated ligand and the neighboring base pairs, provide <50% of the thermodynamic driving force for the complexation of the protoberberines to duplex DNA. Computer modeling studies on protoberberine-DNA complexes suggest that only rings C and D intercalate into the host DNA helix, while rings A and B protrude out of the helix interior into the minor groove. (iii) All four protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of the D-ring influence both DNA binding and topoisomerase I poisoning properties. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoning activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines. (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a profound influence on topoisomerase I poisoning activity. Specifically, protoberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3, 4-methylenedioxy substituents all bind DNA with a similar affinity. By contrast, these analogues exhibit markedly different topoisomerase I poisoning activities, with these activities following the hierarchy: 3,4-methylenedioxy > 2,3-dimethoxy > 3, 4-dimethoxy. These differences in topoisomerase I poisoning activity may reflect the differing abilities of the analogues to interact with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consistent with a mechanistic model in which both ligand-DNA and ligand-enzyme interactions are important for the poisoning of topoisomerase I by protoberberines, with the DNA-directed interactions involving ring D and the enzyme-directed interactions involving ring A. It is reasonable to suggest that the poisoning of topoisomerase I by a broad range of other naturally occurring and synthetic ligands may entail a similar mechanism.     

Structure of a complex between E. coli DNA topoisomerase I and single-stranded DNA

In order to gain insights into the mechaism of ssDNA binding and recognition by Escherichia coli DNA topoisomerase I, the structure of the 67 kDa N-terminal fragment of topoisomerase I was solved in complex with ssDNA. The structure reveals a new conformational stage in the multistep catalytic cycle of type IA topoisomerases. In the structure, the ssDNA binding groove leading to the active site is occupied, but the active site is not fully formed. Large conformational changes are not seen; instead, a single helix parallel to the ssDNA binding groove shifts to clamp the ssDNA. The structure helps clarify the temporal sequence of conformational events, starting from an initial empty enzyme and proceeding to a ssDNA-occupied and catalytically competent active site.