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Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Received September 16, 2005; accepted November 2, 2005
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Abstract |
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). These large DNA viruses encode for many of their own enzymes of DNA metabolism, including a small type IB topoisomerase (vTopo) that is closely related to the human type I topoisomerase (hTopo) (Shuman, 1998). The viral enzyme has been shown to be important for virus replication (Shuman et al., 1989; Da Fonseca and Moss, 2003) and is a potential target for drug treatment against smallpox and molluscum contagiosum virus, a pox virus that infects immunocompromised patients causing painful and disfiguring skin lesions (Hwang et al., 1998). The poxvirus topoisomerases are distinguished by their specificity for forming reversible phosphotyrosine linkages with the backbone of DNA at the pentapyrimidine consensus sequence (T/C)CCTT
, where the arrow denotes the site of nucleophilic attack (Shuman, 1998).
Although the active sites of the poxvirus and human enzymes are congruous, there are sufficient differences in their modes of DNA recognition that allow selective targeting of small molecules. Examples include the potent poisoning of hTopo by the anticancer drug camptothecin, which is essentially ineffective against the pox enzyme (Gupta et al., 1992; Liu et al., 2000). Likewise, vTopo is competitively inhibited by two coumarin drugs, novobiocin and coumermycin, which show little activity against hTopo (Sekiguchi et al., 1996). Other DNA binding ligands, such as the intercalator nogalomycin, can affect the activity of both enzymes, although the mechanisms are quite distinct (Sim et al., 1997, 2000; Yakovleva et al., 2004). Taking together, these findings indicate that there is significant opportunity for discovery of ligands that interact selectively with these enzymes. Despite this realization, there are no robust technologies available for efficient attainment of this goal.
We reported recently the first continuous fluorometric assay for topoisomerase that allows monitoring of the reaction under multiple-turnover conditions (Kwon et al., 2004). The assay takes advantage of sensitive molecular beacon technology and the surprising ribonuclease reaction of vTopo that allows recycling of the enzyme for multiple turnovers, as shown in Fig. 1. Here, we use this assay to blindly screen a 1990-member small-molecule library and to identify several specific inhibitors of the poxvirus topoisomerase that are orders of magnitude more potent than those known previously. Structural comparison of library hits revealed seven compounds with significant similarity to four known families of topoisomerase IB inhibitors, establishing the validity of the ribonuclease assay in the discovery of ligands that affect the DNA reactions of this enzyme. It is noteworthy that the remaining hits were structurally distinct from any known topo ligands, and four of these were found to be active in the nanomolar concentration range. A mechanistic characterization of the most potent inhibitors is described.
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Materials and Methods |
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; Kwon et al., 2002). The enzyme concentration was determined by UV absorbance using an extinction coefficient of 28,140 M–1cm–1 in a buffer containing 20 mM sodium phosphate, pH 6.0, and 6 M guanidinium hydrochloride (Kwon and Stivers, 2002). Human topoisomerase was obtained from Sigma. pUC19 plasmid DNA was purified from Escherichia coli strain DH5
(pUC19). RNA and DNA Substrates with Fluorescent Tags. The sequence of the 18 mer DNA/RNA substrate containing a single uridine ribonucleotide substitution for the 3' thymidine residue of the consensus cleavage sequence is shown here, where FAM is 6-carboxyfluorescein and DAB is the universal fluorescence quencher, dabcyl1: 18U-FAM/18-DAB, 5' CGTGTCGCCCTUATTCCG-FAM-3', and 3' GCACAGCGGGAATAAGGC-DAB-5'.
For the DNA cleavage and religation reactions, a 18/24 mer duplex was synthesized in which the scissile strand contained two modifications. First, the 5'-end was modified with a FAM label, and second, the nucleotide just 3' of the ultimate T of the pentameric consensus sequence (underlined), was labeled with the fluorescent adenine analog 2-aminopurine (P): FAM-18AP/24, 5'-FAM-CGTGTCGCCCTTPTTCCG-3', and 3'-GCACAGCGGGAATAAGGCTATCAC-5'.
These two probes allowed measurement of protein binding by monitoring the increase in fluorescein anisotropy of the DNA and DNA cleavage by the increase in 2-aminopurine fluorescence when the 6 mer leaving group rapidly dissociates after strand scission (Kwon and Stivers, 2002). The oligonucleotide strands were synthesized using an ABI 394 synthesizer (Applied Biosystems, Foster City, CA) using nucleoside phosphoramidites obtained from Glen Research (Sterling, VA). The oligonucleotides were purified using anion exchange high-performance liquid chromatography and desalted using disposable gel-filtration columns (G-10). The purity of oligonucleotides was confirmed using electrophoresis through a 20% polyacrylamide gel containing 7 M urea and matrix-assisted laser desorption ionization/time of flight analysis. The DNA duplexes were prepared in buffer A (20 mM Tris-HCl and 200 mM NaCl, pH 9.0) by mixing the two strands in a molar ratio of 1.15:1(the dabcyl-labeled strand was in excess).
Steady-State Fluorescence Anisotropy Measurements for Noncovalent DNA Binding. Samples (150 µl containing 0.1 µM FAM-18AP/24) were excited with vertically polarized light at 492 nm (1-nm bandpass), and both vertical and horizontal emissions were monitored at 517 nm (3-nm bandpass). Three replicate measurements were made for each addition of topoisomerase in the range 0 to 0.15 µM, and the values were then averaged. The G factor was calculated, and its value was used to calculate the anisotropy. To determine whether library compounds displaced the bound DNA from Y274F, a complex was formed using 0.1 µM FAM-18AP/24 and 2 µM Y274F, and the anisotropy was measured as increasing concentrations of compound were added to the solution (0–5 µM). As a positive control, after the addition of compound, the noncovalent DNA complex was completely disrupted by the addition of the competitive inhibitor ATP in the concentration range of 0 to 100 mM. In all cases, anisotropy measurements were carried out in triplicate, and the results were averaged.
High-Throughput Screening. For screening of the 1990-compound NCI Diversity Library, a Fluoromax-3 fluorimeter with a Micro-Max 96-well fluorescence plate reader attachment was used. Each reaction well in the screen contained enough test compound to give a 100 µM concentration after the addition of 100 µl of reaction solution containing enzyme (10 nM) and buffer A. Reactions were then initiated by the addition of 18U-FAM/18-DAB (0.5 µM final concentration in the well). For validation, positive and negative control wells were also included that consisted of enzyme and substrate without inhibitor and substrate without enzyme. The rates of fluorescence change at 517 nm with excitation at 492 nm were followed by taking six fluorescence readings in each well over a 30-min time period.
Inhibition of Steady-State Plasmid Supercoil Relaxation. Supercoil relaxation reactions were performed with 0.5 µg of supercoiled pUC19, 1 nM vTopo, or 2 U of hTopo and various concentrations of inhibitor. The reaction mixtures were incubated for 30 min (20 min for hTopo) at room temperature and were then quenched with one-fifth volume of 5x loading buffer containing 0.4% SDS, 5% glycerol, and Tris-acetate-EDTA. The relaxed DNA and supercoil DNA were resolved on 1% agarose gel at 135 V for 2 h in a Tris-acetate-EDTA running buffer. After ethidium bromide staining and fluorescence imaging using a GelDoc instrument (Bio-Rad, Hercules, CA), the bands corresponding to supercoiled and relaxed DNA in each lane were quantified using the software supplied with the instrument. Inhibition results using the supercoiled relaxation assay were repeated three times to estimate the variability in the measurements.
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). The reactions were performed using an stopped-flow fluorescence instrument in the two-syringe mode (Applied Photophysics, Leatherhead, Surrey, UK). Equal volumes of 1 µM vTopo and 50 nM substrate in the presence of varying concentrations of compound were rapidly mixed, and the increase in 2-aminopurine fluorescence was followed using excitation at 315 nm with monitoring at emission wavelengths greater than 360 nm using a cut-on filter (Kwon and Stivers, 2002). On average, five or more kinetic transients were averaged and fitted to a first-order rate equation to obtain the reported rate constants and errors.
Equilibrium Cleavage Measurements. The equilibrium cleavage reactions were performed in the presence and absence of inhibitors 13778, 48300, 14163, and 88915 in the concentration range 0 to 20 µM (Kwon and Stivers, 2002) using a buffer containing 20 mM Tris-HCl and 200 mM NaCl at pH 8. Wild-type topoisomerase (60 nM) and 5'-FAM-labeled 32/32-mer DNA duplex (80 nM) with the scissile strand sequence of 5'-FAM-CGTGTCGCCCTTNTTCCGATAGTGACTACAGC-3' were incubated for 30 min at room temperature, and the covalent complex present at equilibrium was trapped by the rapid addition of 1 volume of 10% SDS (20 µl). The reactions in the presence of compound were performed by preincubating the compound with the enzyme or DNA for 10 min before initiating the reaction. The free and covalently bound DNA were separated by electrophoresis using a 10% polyacrylamide gel containing 0.1% SDS. The fraction covalent complex was then determined using eq. 1 by phosphorimaging: [Frac complex = counts covalent complex/(counts covalent complex + counts free DNA)] (eq. 1). The equilibrium cleavage measurements were repeated three times for error estimation.
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Results |
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One hundred thirteen "hits" in the primary screen were intensely colored compounds, which is a very large fraction of the total number of colored compounds in the library. One explanation for the promiscuous inhibition by colored compounds is an inner filter effect on the high-throughput fluorescence assay (i.e., an absorption band overlap with the fluorescein fluorophore). To estimate how many false positives were contained within the colored hits, we selected a subset of 43 on the basis of their conformance with Lipinski's Rule of Five and assayed these in a secondary plasmid relaxation assay using 20 µM compound (Lipinski, 2003) (see also below). At this reduced concentration, 6 compounds (15%) showed complete inhibition, 26 (60%) showed partial inhibition, and 11 (25%) showed no inhibition at all. Of the six compounds showing complete inhibition of plasmid supercoil relaxation at 20 µM compound concentration, only one of these (327447) showed significant activity at lower concentrations (IC50 
2 µM; Table 1). We conclude that the promiscuous inhibition by colored compounds does not solely arise from an inner filter effect but may also be primarily caused by a propensity of these extensively aromatic compounds to form nonspecific inhibitory aggregates (Seidler et al., 2003; Feng et al., 2005). In fact, a recent study estimated that 19% of randomly selected small molecules commonly found in chemical libraries are nonspecific, aggregation-based inhibitors at 30 µM concentrations (Feng et al., 2005). This percentage is somewhat higher than the 12.4% hit rate we observed with the Diversity Library at the 100 µM initial screening conditions but strongly suggests that many of these initial hits are nonspecific. In any event, the high hit rate under the primary screening conditions is not especially problematic for a library of this size because follow-up studies can quickly discriminate between weak and strong inhibitors (see below). For larger libraries, it would be advantageous to use more stringent screening conditions (i.e., concentrations of compound less than 100 µM) and to include a low concentration of detergent in the RNase assay buffer to disperse aggregates (Seidler et al., 2003; Feng et al., 2005).
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We brought forward these 21 compounds for testing in a DNA supercoil release assay (Fig. 3). These compounds were initially tested at 10 µM concentration, and representative data from 13 of these are shown in Fig. 3A. Although 19 compounds showed similar inhibitory potencies as observed in the RNase assay (Table 1), 2 did not inhibit supercoil relaxation at 10 µM concentration (13776 and 7810) (Fig. 3A). Further testing of the active compounds at serial 10-fold dilutions revealed that four compounds (13778, 48300, 14163, and 88915) showed significant inhibition of plasmid supercoil relaxation even at 100 nM concentrations (Fig. 3, B and C). These final four compounds had IC50 values around 10 nM (data not shown) and were subjected to a thorough mechanistic analysis to determine the selectivity and mode of inhibition as described below.
Selectivity of Inhibitors. We were interested whether the four most potent vTopo inhibitors showed selectivity for the pox family enzyme. Thus, inhibition of supercoil relaxation by the human enzyme was tested (Table 1). For 13778 and 88915, partial or complete inhibition of hTopo was only observed at relatively high compound concentrations in the range 5 to 20 µM (Fig. 4, A and B). For 48300 and 14163, no inhibition was observed even at compound concentrations as high as 20 µM (Fig. 4, C and D). Because vTopo shows approximately 50% inhibition using 10 nM concentrations of these compounds, these results indicated selectivity in the range of 500- to >2000-fold compared with hTopo.
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). In this analysis, the change in fluorescence anisotropy upon Y274F binding to the 5'-FAM 18AP/24 duplex is followed (Fig. 5). The addition of increasing concentrations of Y274F to a solution of 5'-FAM 18AP/24 resulted in a saturable increase in the steady-state fluorescence anisotropy of the DNA (Fig. 5A), as would be expected upon the formation of a high molecular weight enzyme-DNA complex.
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Effect of Active Compounds on DNA Transesterification. A second potential mechanism of inhibition involves the reversible DNA transesterification step catalyzed by topo I. This step involves DNA cleavage (kcl) to form a 3'-phosphotyrosyl linkage between the enzyme and DNA, and DNA religation (kr) in which the expelled 5'-hydroxyl of the DNA attacks the phosphotyrosyl linkage to reform the intact DNA backbone. The forward-cleavage reaction may be studied under irreversible cleavage conditions using "suicide substrates" that have weakly base-paired DNA strands 3' to the site of cleavage that dissociate to solution after cleavage (Shuman, 1991). If the leaving strand contains a 2-aminopurine fluorescent label on the 5' end, rapid strand dissociation leads to an increase in the 2-aminopurine fluorescence that is a measure of the preceding rate-limiting cleavage reaction (Kwon and Stivers, 2002). Otherwise, reversible conditions may be used in which the covalent complex accumulates to its equilibrium level dictated by the ratio of forward and reverse rate constants kcl/kr = Kcl = [covalent complex]/[noncovalent complex](Kwon and Stivers, 2002). In this case, larger substrates are used in which the strands remain paired after DNA cleavage.
We first performed stopped-flow fluorescence analysis of the irreversible cleavage reaction of the FAM-18AP/24 suicide substrate by vTopo in the presence of 13778, 48300, 14163, and 88915. In this assay, compound concentrations as high as 100 times the IC90 value for supercoil release were used. Representative stopped-flow data are shown in Fig. 6 in the presence of 0, 1, and 10 µM concentrations of compound 13778. The cleavage rate in the absence of 13778 was 1.4 s–1, and was found to be unaffected by the presence of 13778. Identical results were obtained for 48300, 14163, and 88915 (data not shown). Therefore, these data establish that inhibition of the steady-state RNase and supercoil release reactions involves a step after formation of the covalent complex. As observed for DNA binding, the results were the same regardless of whether the compound was preincubated with the enzyme or DNA (data not shown).
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). The covalent complex is then separated from the free DNA using denaturing polyacrylamide gel electrophoresis, and the fluorescence in each species was quantified by fluorescence imaging. In the absence of inhibitor, 18% of the total DNA fluorescence migrates in a covalent complex with the enzyme (Fig. 7, A–D). Compounds 13778, 48300, and 88915 had no discernible effect on the equilibrium amount of covalent complex (Fig. 7, A, B, and D). In stark contrast, compound 14163 produced a concentration-dependent decrease in the amount of covalent complex (Fig. 7C), such that at concentrations greater than 2 µM, no covalent complex could be detected. The decrease in the amount of covalent complex observed with 14163 cannot arise from inhibition of noncovalent DNA binding (Fig. 5B). Although this effect of 14163 could, in principle, arise from an increase in the rate of religation relative to cleavage, we favor a mechanism involving hindrance of the cleavage chemistry, either by direct or indirect interactions with active site groups. Even though 14163 showed no effect on the single turnover cleavage rate, it is quite possible that the cleavage rate is affected in the equilibrium substrate because the binding site for this compound may be created after cleavage and/or requires the presence of a paired DNA strand 3' to the cleavage site. On the other hand, the binding rate of 14163 could be slow relative to the single turnover cleavage rate, making it an ineffective inhibitor under this type of assay condition. Whatever the detailed mechanism, these findings accentuate the importance of measuring inhibitory activities in multiple assay formats. The distinct inhibition mechanisms for these four active compounds are discussed further below.
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Discussion |
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). Ligands that fall into the latter category are called "topoisomerase poisons" because they stabilize single-strand nicks in duplex DNA that in turn give rise to toxic double-strand breaks when encountered by replication forks during DNA replication. This is the mode of action of all clinically useful drugs that target topoisomerases (Chen and Liu, 1994).
In our blind screen of the NCI library, we detected inhibitors of vTopo that are structurally related to four known classes of mammalian topo I poisons and structurally novel inhibitors (Fig. 8). One library member, camptothecin, is a clinically used topo I poison that showed partial inhibition of vTopo using the primary screening conditions. In addition, two other structural congeners of camptothecin were also detected as inhibitors (Fig. 8). In this respect, it is interesting to note that camptothecin is widely considered a highly specific poison of mammalian type I enzymes. Previous work has shown that camptothecin does not affect wild-type vTopo DNA relaxation activity at concentrations 
100 µM, but it can inhibit relaxation by a mutant enzyme (D221V) that more closely matches the coding sequence in a highly conserved region of eukaryotic topo I enzymes (Gupta et al., 1992). The sensitive ribonuclease high-throughput screen has uncovered that camptothecin is weakly active against vTopo, which may indicate that its efficacy is greater for the ribonuclease activity than for the DNA unwinding action of vTopo, or that the ribonuclease assay is simply more sensitive.
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Aside from camptothecin, three other known structural classes of topo I poisons were also detected in the primary screen at 100 µM compound concentration. These compounds are related to the protoberberine, nogalamycin, and bibenzimidazole chemical scaffolds (Fig. 8). The five-ring protoberberines and the closely related benzo[i]phenanthridine derivatives are potent hTopo poisons with activity against several human tumor cell lines (Li et al., 2000, 2003; Ruchelman et al., 2004). Their mechanism of action involves DNA intercalation by the C and D rings (Fig. 8) (Li et al., 2000) and enzyme-specific interactions with the methylenedioxybenzene A ring. The A and D ring features are recapitulated in the smaller two- and three-ring system inhibitors detected here, suggesting a shared mechanism of inhibition between the vaccinia and human enzymes. Nogalomycin has been shown to intercalate into DNA using its four-ring aglycone ring system, which positions its nogalose and amino glucose sugar rings into the minor and major grooves, respectively (Fig. 8) (Sim et al., 1997). Its mode of action with hTopo seems to involve induced DNA bending that stabilizes the covalent complex (Sim et al., 2000). Recent studies have also examined nogalomycin inhibition of vTopo and concluded that it does not stabilize the covalent complex but instead inhibits DNA cleavage with an IC50 value of 0.7 µM (Yakovleva et al., 2004). The most obvious feature of the nogalomycin congeners detected here is their lack of the nogalose sugar substituent (Fig. 8), indicating that this minor groove binding group is not essential for vTopo inhibition. Finally, bi- and terbenzimidazoles have been shown to be DNA minor groove binders acting by stabilizing the covalent complex between hTopo and DNA via a long range conformational change rather than direct intercalation at the cleavage site, as with camptothecin (Xu et al., 1998). The ribonuclease assay readily detected the mono- and bibenzimidazole library compounds shown in Fig. 3, and the bibenzimidazole compound has been reported previously as a potent hTopo inhibitor (Jin et al., 2000). The related terbenzimidazole derivatives of this library member have been shown to increase the potency as an hTopo poison, and the activity is strongly correlated with the greater binding affinity of the terbenzimidazole for the DNA minor groove. We conclude that the ribonuclease assay robustly detected all of the known topoisomerase inhibitors and poisons present in the Diversity Library and that this screen has significant potential to quickly reveal structure-activity relationships relevant for development of specific inhibitors of pox family topoisomerases.
The high-throughput screen has also uncovered 21 structurally novel inhibitors of vTopo that had IC90 values for the ribonuclease reaction of less than 10 µM. Only 2 of these 21 compounds failed to show comparable inhibition in the supercoil relaxation assay (Fig. 3A). The remaining 19 compounds showed potencies of inhibition against DNA relaxation that were comparable with or even significantly enhanced compared with their potencies in the ribonuclease screen. It is not surprising that there are differences between the inhibitory effects of the same compound in the two assays. If the compound interacts with the covalent complex and not the free enzyme, as we have generally found (see below), the presence of a 2' hydroxyl with the ribonuclease substrate could have a differential effect on the interaction of the inhibitor compared with the 2' hydrogen in DNA. Nevertheless, the results here show that any differences in inhibitor interactions between these two substrates do not prevent the detection of both novel and established topo I ligands that act by widely different mechanisms.
The structural features of the 21 most potent vTopo inhibitors are varied (Table 1). Several compounds contain pentavalent antimony or arsenic, and two of these (48300 and 13778) are specific inhibitors of vTopo, with IC50 values in the nanomolar range against DNA relaxation. It is noteworthy that compound 13778 has been identified recently as a human immunodeficiency virus-1 entry inhibitor that disrupts the binding of the viral gp120 with the CD4 receptor (Yang et al., 2005) and is also an inhibitor of B-ZIP transcription factor DNA binding (Rishi et al., 2005). Three others compounds with IC50 values in the 0.5 to 10 µM range (373989, 12155, and 7810) have two or three fused aromatic rings suggestive of an intercalative mode of action. Finally, compound 14163, which shows nanomolar potency in DNA relaxation assays, is an entirely novel topoisomerase inhibitor with an unusual substituted furan ring structure (Table 1). Given the structural diversity of these compounds, it is unlikely that they use similar binding modes, suggesting that the vTopo-DNA target displays a promiscuous array of potentially inhibitory binding sites.
Inhibitory Mechanism of Highly Active vTopo-Specific Inhibitors. We used three efficient assays to ascertain the mode of inhibition for supercoil relaxation brought around by the four potent vTopo specific compounds 13778, 48300, 88915, and 14163. DNA anisotropy measurements established that none of these compounds bind to vTopo competitively with DNA (Fig. 5). Likewise, single-turnover suicide DNA cleavage measurements revealed no effect of these compounds on any step leading to the formation of the covalent enzyme-DNA adduct (Fig. 6). Finally, compounds 13778, 48300, and 88915 had no discernible effect on the equilibrium level of covalent complex (Fig. 7), but increasing concentrations of 14163 completely prevented the formation of the covalent complex (Fig. 7C). Thus, all four inhibitors bind noncompetitively or uncompetitively to the noncovalent or covalent complex. Although an intercalative mechanism for inhibition has not been directly studied here, the structures of 88915, 13778, 14163, and 48300 are not suggestive of such a mechanism, and these compounds are equally active in the ribonuclease and supercoil release assays. Compound 14163 is unique in that its binding to the enzyme-DNA complex does not significantly interfere with DNA binding but totally disrupts covalent chemistry.
It is intriguing that no compounds have yet been identified that act as vTopo poisons (i.e., act by stabilizing the covalent enzyme-DNA complex). To date, vTopo inhibitors have been characterized as 1) competitive inhibitors of DNA binding (novobiocin, coumermycin, and ATP) (Sekiguchi et al., 1996)3, 2) noncompetitive inhibitors by binding to the noncovalent complex (nogalamycin) (Yakovleva et al., 2004), or 3) uncompetitive inhibitors by binding to the noncovalent or covalent complex, as found for the inhibitors identified here. It is possible that there are unique structural features of the covalent complex between vTopo and DNA that prevent extensive intercalation of a compound into the strand break, as observed in crystal structures of camptothecin bound to hTopo and cleaved DNA (Staker et al., 2002; Chrencik et al., 2004). Instead, the uncompetitive inhibitors discovered here and previously (Kwon et al., 2004) act by inducing an alternative form of the covalent complex that is unproductive for DNA relaxation or steady-state turnover of the ribonuclease substrate. The frequency of this mode of inhibition suggests that it may turn out to be the rule, and not the exception, with vTopo.
Antiviral Applications of vTopo Inhibitors. Although an early study indicated that the poxvirus topoisomerase was essential (Shuman et al., 1989), a recent study has established that the deletion mutant is viable but has low infectivity because of reduced early transcription (Da Fonseca and Moss, 2003). Thus, vTopo acts very early in viral infection, and drugs that merely inhibit the enzyme may not totally prevent infectivity. It has been suggested that a vTopo poison that stabilizes the covalent intermediate might block infectivity more efficiently than the null mutant because of the accumulation of toxic nicks in the DNA (Da Fonseca and Moss, 2003). Although the compounds characterized here are not classic topoisomerase poisons that increase the level of covalent species, three of these do serve to trap the enzyme in a nonproductive covalent complex on DNA that could be just as effective in producing therapeutic effects.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: vTopo, vaccinia DNA topoisomerase IB; hTopo, human topoisomerase IB; FAM, 6-carboxyfluorescein; topo, topoisomerase; DAB, dabcyl group; NCI, National Cancer Institute; HTP, high throughput; pUC19, pUC19 plasmid DNA purified from Escherichia coli strain DH5.
1 In previous work, we had screened approximately 500 compounds from this library and thoroughly characterized one hit (NSC 112983) (Kwon et al., 2004). This compound bound exclusively to the enzyme-DNA covalent complex with a modest KD value of 1.6 µM and did not inhibit DNA cleavage. The inhibitory mechanism involved recycling of the enzyme for catalytic turnover. 
2 In HTP screening, it is important to explicitly define the term hit. Here, a hit is simply a compound that gives rise to a decrease in the rate of fluorescence increase in the RNase assay under the conditions of the screen. No presumptions are made as to the mechanism of inhibition. Such mechanisms may include specific inhibition, nonspecific inhibition by compound aggregation or protein denaturation, or even inner filter effects on the fluorescent assay. Many of the uninteresting mechanisms are excluded in subsequent secondary screens and specificity studies.
3 Under certain conditions, ATP has been reported to accelerate the rate of supercoil release by vTopo (Sekiguchi and Shuman, 1994). We were surprised to find that this nucleotide is an inhibitor of the RNase reaction and noncovalent DNA binding by Y274F vTopo. These apparently contradictory findings may indicate that the rate of supercoil relaxation is limited by product release, which is in turn facilitated by ATP. This nucleotide could bind to an allosteric site on the enzyme and give rise to these effects.
Address correspondence to: Dr. James T. Stivers, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185. E-mail: jstivers{at}jhmi.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Chrencik JE, Staker BL, Burgin AB, Pourquier P, Pommier Y, Stewart L, and Redinbo MR (2004) Mechanisms of camptothecin resistance by human topoisomerase I mutations. J Mol Biol 339: 773–784.[CrossRef][Medline]
Da Fonseca F and Moss B (2003) Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription. Proc Natl Acad Sci USA 100: 11291–11296.
Feng BY, Shelat A, Doman TN, Guy RK, and Shoichet BK (2005) High-throughout assays for promiscuous inhibitors. Nat Chem Biol 1: 146–148.[CrossRef][Medline]
Fields BN and Kinpe DM (1990) Virology. Raven Press, New York.
Gupta M, Zhu CX, and Tse-Dinh YC (1992) An engineered mutant of vaccinia virus DNA topoisomerase I is sensitive to the anti-cancer drug camptothecin. J Biol Chem 267: 24177–24180.
Hwang Y, Rhodes D, and Bushman F (2000) Rapid microtiter assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation. Nucleic Acids Res 28: 4884–4892.
Hwang Y, Wang B, and Bushman FD (1998) Molluscum contagiosum virus topoisomerase: purification, activities and response to inhibitors. J Virol 72: 3401–3406.
Jin S, Kim JS, Sim SP, Liu A, Pilch DS, Liu LF, and LaVoie EJ (2000) Heterocyclic bibenzimidazole derivatives as topoisomerase I inhibitors. Bioorg Med Chem Lett 10: 719–723.[CrossRef][Medline]
Kwon K, Jiang YL, Song F, and Stivers JT (2002) 19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate. J Biol Chem 277: 353–358.
Kwon K, Nagarajan R, and Stivers JT (2004) Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay. Biochemistry 43: 14994–15004.[CrossRef][Medline]
Kwon K and Stivers JT (2002) Fluorescence spectroscopy studies of vaccinia type IB DNA topoisomerase. Closing of the enzyme clamp is faster than DNA cleavage. J Biol Chem 277: 345–352.
Li D, Zhao B, Sim SP, Li TK, Liu A, Liu LF, and LaVoie EJ (2003) 8,9-methylenedioxybenzo[i]phenanthridines: topoisomerase I-targeting activity and cytotoxicity. Bioorg Med Chem 11: 3795–3805.[CrossRef][Medline]
Lipinski CA (2003) Chris Lipinski discusses life and chemistry after the Rule of Five. Drug Discov Today 8: 12–16.[CrossRef][Medline]
Li TK, Bathory E, LaVoie EJ, Srinivasan AR, Olson WK, Sauers RR, Liu LF, and Pilch DS (2000) Human topoisomerase I poisoning by protoberberines: potential roles for both drug-DNA and drug-enzyme interactions. Biochemistry 39: 7107–7116.[CrossRef][Medline]
Liu LF, Desai SD, Li TK, Mao Y, Sun M, and Sim SP (2000) Mechanism of action of camptothecin. Ann NY Acad Sci 922: 1–10.
Rishi V, Potter T, Laudeman J, Reinhart R, Silvers T, Selby M, Stevenson T, Krosky P, Stephen AG, Acharya A, et al. (2005) A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors. Anal Biochem 340: 259–271.[CrossRef][Medline]
Ruchelman AL, Zhu S, Zhou N, Liu A, Liu LF, and LaVoie EJ (2004) Dimethoxybenzo[i]phenanthridine-12-carboxylic acid derivatives and 6H-dibenzo-[c,h][2,6]naphthyridin-5-ones with potent topoisomerase I-targeting activity and cytotoxicity. Bioorg Med Chem Lett 14: 5585–5589.[CrossRef][Medline]
Seidler J, McGovern SL, Doman TN, and Shoichet BK (2003) Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem 46: 4477–4486.[CrossRef][Medline]
Sekiguchi J and Shuman S (1994) Stimulation of vaccinia topoisomerase I by nucleoside triphosphates. J Biol Chem 269: 29760–29764.
Sekiguchi J, Stivers JT, Mildvan AS, and Shuman S (1996) Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. J Biol Chem 271: 2313–2322.
Shuman S (1991) Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro. J Biol Chem 266: 20576–20577.
Shuman S (1998) Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme. Biochim Biophys Acta 1400: 321–337.[Medline]
Shuman S, Golder M, and Moss B (1989) Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: evidence that the gene is essential for virus growth. Virology 170: 302–306.[CrossRef][Medline]
Sim SP, Gatto B, Yu C, Liu AA, Li TK, Pilch DS, LaVoie EJ, and Liu LF (1997) Differential poisoning of topoisomerases by menogaril and nogalamycin dictated by the minor groove-binding nogalose sugar. Biochemistry 36: 13285–13291.[CrossRef][Medline]
Sim SP, Pilch DS, and Liu LF (2000) Site-specific topoisomerase I-mediated DNA cleavage induced by nogalamycin: a potential role of ligand-induced DNA bending at a distal site. Biochemistry 39: 9928–9934.[CrossRef][Medline]
Staker BL, Hjerrild K, Feese MD, Behnke CA, Burgin AB Jr, and Stewart L (2002) The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci USA 99: 15387–15392.
Stivers JT, Jagadeesh GJ, Nawrot B, Stec WJ, and Shuman S (2000) Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase. Biochemistry 39: 5561–5572.[CrossRef][Medline]
Xu Z, Li TK, Kim JS, LaVoie EJ, Breslauer KJ, Liu LF, and Pilch DS (1998) DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles. Biochemistry 37: 3558–3566.[CrossRef][Medline]
Yakovleva L, Handy CJ, Sayer JM, Pirrung M, Jerina DM, and Shuman S (2004) Benzo[c]phenanthrene adducts and nogalamycin Inhibit DNA transesterification by vaccinia topoisomerase. J Biol Chem 279: 23335–23342.
Yang QE, Stephen AG, Adelsberger JW, Roberts PE, Zhu WM, Currens MJ, Feng YX, Crise BJ, Gorelick RJ, Rein AR, et al. (2005) Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4. J Virol 79: 6122–6133.
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