Topoisomerase I/II inhibition by a novel naphthoquinone containing a modified anthracycline ring system

In an attempt to create more effective chemotherapeutic compounds, the naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was synthesized. Cell viability studies revealed TU100 is specific for eukaryotes and induces cell death. Based on its structural similarities to the anthracyclines and isoquinolines, the ability of TU100 to inhibit topoisomerase I and II was examined. TU100 was an effective inhibitor of both enzymes, as indicated by its ability to prevent topoisomerase-mediated relaxation of supercoiled plasmid DNA. The mechanism of action does not involve TU100 intercalation into DNA, unlike anthracyclines. Pre-incubation of topoisomerase with TU100 dramatically decreased the IC₅₀, suggesting the drug is a novel slow acting topoisomerase inhibitor that works in the absence of DNA. Taken together these results indicate the novel naphthoquinone adduct TU100 is a dual topoisomerase I/II inhibitor with a unique mechanism of action and chemotherapeutic potential.     

Quadruplex-Duplex Motifs as New Topoisomerase I Inhibitors

In this article, 13 short chains that can form G-quadruplex and quadruplex-duplex motif have been designed. Fourteen oligonucleotides, including 13 short chains as well as a reference short chain all have certain level of inhibition to topoisomerase I, whether or not they form G-quadruplex and quadruplex-duplex motif, and the G-quadruplex and quadruplex-duplex motif show better activity than single short chain. The result confirmed that after forming G-quadruplex and quadruplex-duplex motif these 14 oligonucleotides are competitive inhibition, that is, through the priority binding with the topoisomerase I and precluding from its binding with the normal substrate pBR322 and, therefore, inhibiting the next reaction.     

Glutamate Regulates the Activity of Topoisomerase I in Mouse Cerebellum

Topoisomerase I (topo I) is a nuclear enzyme which participates in most DNA transactions. It was shown to be inhibited in depolarized neurons by poly adenosine diphosphate (ADP)-ribosylation of the enzyme protein. We demonstrated previously an age and sex dependent topo I activity and enzyme protein level in the various regions of mouse brain. A specific distribution pattern of topo I was observed and the inhibitory neurons exhibited the highest enzyme activity and protein level in both the nucleus and the cytoplasm. Here, we show that neurotransmitters (glutamate and gamma-aminobutyric acid (GABA)) regulate the activity of topo I in mouse cerebellum sections. Glutamate exhibited a significant time-dependent inhibition of topo I activity but no effect of the enzyme protein level. GABA in contrary only slightly and transiently inhibited topo I activity. The inhibitory effect of glutamate was mediated by Ca⁺² and by ADP-ribosylation of topo I protein and the glutamate ionotropic receptors were involved. Glutamate also diminished the inhibitory effect of topotecan on topo I. These results point to distinct and highly specific effects of the major neurotransmitters on topo I activity in the cerebellum suggesting that topo I possesses a specific role in the brain which differs from its known biological functions.     

The phosphoCTD-interacting domain of Topoisomerase I

The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.     

Molecular docking approach on the Topoisomerase I inhibitors series included in the NCI anti-cancer agents mechanism database

Topoisomerase I (Top1) is an essential enzyme participating to all those processes associated with separation of DNA strands. It manages superhelical tensions through the transient breakage of one strand of duplex DNA, followed by the unwinding of supercoiled DNA. Camptothecins, a class of alkaloids extracted from the wood of a Chinese tree, were found to be potent inhibitors of Topoisomerase I. The National Cancer Institute (NCI) Anti-cancer Agents Mechanism Database contains several camptothecins derivatives, classified as selective Top1 inhibitors. In this work we performed molecular docking studies on 24 camptothecin-like inhibitors present in this database (using Autodock 3.0.5). In order to consider the different orientations of the active site residues, docking was performed using four different structures of a Top1-DNA complex. The results obtained allowed us to analyze some conformations adopted by the inhibitors during active site binding, confirming the role of hydrogen bond and contributed to clarify the loss of activity due to single point mutations.     

Structural Basis for Topoisomerase I Inhibition by Nucleoside Analogs

Abstract

Nucleoside analogs such as 1-β-D-arabinofuranosyl cytidine (AraC) and 2′, 2′-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top 1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2′-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top 1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3′ endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.     

Rational design and synthesis of topoisomerase I and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.     

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.     

Topoisomerase I amplification in melanoma is associated with more advanced tumours and poor prognosis

In this study, we used array-comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array-comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1-targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.     

Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I

Linear expression constructs can facilitate gene function studies. We describe a method to generate linear expression constructs for mammalian cells by one-step polymerase chain reaction (PCR) with vaccinia DNA topoisomerase I (TOPO). Cytomegalovirus (CMV) 5′ promoter, the gene of interest, and V5 bovine growth hormone (BGH) polyA 3′ terminator elements were PCR-amplified with target-specific primers containing vaccinia DNA TOPO-specific sequence and complementary sequence to each other. We amplified specific and complementary sequences. These three elements were directionally joined with vaccinia TOPO. The joined products were then directly transfected into Chinese hamster ovary cells. Compared with the transfection of supercoiled plasmids, comparable expression signals were obtained for green fluorescent protein, chloramphenicol acetyltransferase, and β-galactosidase proteins using Western blots. This is a quick and efficient method to generate linear expression construct. Unlike Invitrogen TOPO Tools, our method avoided the secondary round of PCR and more rapidly yielded correct joining products. This method can be easily used in the function test of uncharacterized open reading frames.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Computer Analysis of Conformational and Physicochemical Properties of Nucleotide Sequences Cleavable by DNA Topoisomerase I

DNA binding with enzymes is followed by specific adaptation of the DNA structure, including partial or almost complete melting, structural changes in the sugar-phosphate backbone, stretching, compressing, bending or kinking, base flipping, etc. The set of conformational changes is individual for each enzyme and is aimed at efficiently adjusting the orbitals of the reacting groups of the enzyme and the specific DNA site to 10°–15°. The efficiency of nucleotide sequence adaptation determined by the enzyme depends on several structural characteristics. Optimal adjustment is achieved only in the case of specific DNA sequences; as a result, the reaction rate is four to eight orders of magnitude higher with specific than with nonspecific sequences. DNA topoisomerase I (Topo) is a sequence-dependent enzyme. Although less efficiently, Topo cleaves sequences which differ considerably from the optimal sequence. A method based on the analysis of conformational and physicochemical properties of the DNA helix was used to examine many nucleotide sequences cleavable by Topo. The method yields detailed information on similarity or difference of DNA structural units. The cleavable sequences proved to be similar in roll, slide, twist, and rise. In addition, all sequences had sterically disadvantageous contacts between N3 and NH₂ of guanines and N3 of adenines in the minor groove, which corresponded to the presence of dinucleotides Py-Pu in the cleavage site. DNA bending towards the major groove is easier in the case of the optimal sequence. This method is promising for analyzing the efficiency of nucleic acid cleavage by various DNA- and RNA-dependent enzymes.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 488–496.Original Russian Text Copyright © 2005 by Oshchepkov, Bugreev, Kolchanov, Nevinsky.