Inhibitors Of Human DNA Topoisomerase Research Articles

Utilizing coordination chemistry through formation of a CuII-quinalizarin complex to manipulate cell biology: An in vitro, in silico approach

Quinalizarin, an analogue of anthracycline anticancer agents, is an anticancer agent itself. A CuII complex was prepared and characterized by elemental analysis, UV-Vis & IR spectroscopy, mass spectrometry, EPR and DFT. The intention behind the preparation of the complex was to increase cellular uptake, compare its binding with DNA against that of quinalizarin, modulation of semiquinone formation, realization of human DNA topoisomerase I & human DNA topoisomerase II inhibition and observation of anticancer activity. While the first two attributes of complex formation lead to increased efficacy, decrease in semiquinone generation could results in a compromise with efficacy. Inhibition of human DNA topoisomerase makes up this envisaged compromise in free radical activity since the complex shows remarkable ability to disrupt activities of human DNA topoisomerase I and II. The complex unlike quinalizarin, does not catalyze flow of electrons from NADH to O2 to the extent known for quinalizarin. Hence, decrease in semiquinone or superoxide radical anion could make modified quinalizarin [as CuII complex] less efficient in free radical pathway. However, it would be less cardiotoxic and that would be advantageous to qualify it as a better anticancer agent. Although binding to calf thymus DNA was comparable to quinalizarin, it was weaker than anthracyclines. Low cost of quinalizarin could justify consideration as a substitute for anthracyclines but the study revealed IC50 of quinalizarin/CuII-quinalizarin was much higher than anthracyclines or their complexes. Even then, there is a possibility that CuII-quinalizarin could be an improved and less costly form of quinalizarin as anticancer agent.

N-(benzazol-2-yl)-2-substituted phenylacetamide derivatives: Design, synthesis and biological evaluation against MCF7 breast cancer cell line

This work describes the straightforward and efficient one-pot synthesis of a new library of N-(benzazol-2-yl)-2-substituted phenylacetamide derivatives (19–27). Using the MTT assay, these compounds were evaluated for their in vitro anticancer activity against the MCF7 human breast cancer cell line, and the results were compared to the standard doxorubicin. The majority of compounds exhibited an inhibitory effect against the cancer cell line, with compounds 19, 22, and 26 exhibiting exceptional cytotoxicity against MCF7 cells. Using flow cytometry, the most potent compound 19 on the induction of apoptosis in the breast cancer cell line was determined. Compound 19 induced G1-phase cell cycle arrest followed by apoptotic cell death. In silico analyses of potent compounds 19, 22, and 26 were conducted to investigate their interactions with Human DNA topoisomerase II. The energy calculations were found to be in excellent agreement with the calculated IC50 values. In addition, drug similarity parameter values for the three active compounds were determined using in silico ADME prediction studies. Considering all of these results, it appears that these N-(benzazol-2-yl)-2-substituted phenylacetamide derivatives may be effective anticancer agents. This work may possibly generate new concepts for the enhancement of inhibitors of human DNA topoisomerase II for breast cancer treatment.

ATP-competitive inhibitors of human DNA topoisomerase IIα with improved antiproliferative activity based on N-phenylpyrrolamide scaffold

ATP-competitive inhibitors of human DNA topoisomerase II show potential for becoming the successors of topoisomerase II poisons, the clinically successful anticancer drugs. Based on our recent screening hits, we designed, synthesized and biologically evaluated new, improved series of N-phenylpyrrolamide DNA topoisomerase II inhibitors. Six structural classes were prepared to systematically explore the chemical space of N-phenylpyrrolamide based inhibitors. The most potent inhibitor, 47d, had an IC50 value of 0.67 μM against DNA topoisomerase IIα. Compound 53b showed exceptional activity on cancer cell lines with IC50 values of 130 nM against HepG2 and 140 nM against MCF-7 cancer cell lines. The reported compounds have no structurally similarity to published structures, they are metabolically stable, have reasonable solubility and thus can serve as promising leads in the development of anticancer ATP-competitive inhibitors of human DNA topoisomerase IIα.

Dual Inhibitors of Human DNA Topoisomerase II and Other Cancer-Related Targets.

Human DNA topoisomerase II is an important target in anticancer therapy. Despite the clinical success of drugs that target topoisomerase II, the development of resistant cancer cells can limit their clinical efficacy. To maximize the therapeutic potential of anticancer drugs, combination therapies and multitarget drugs have been suggested in many studies, where the use of multitarget drugs is advantageous from a pharmacokinetic point of view. There are various different options for the preparation of dual-target or multiple-target inhibitors, as topoisomerase II is both structurally (e.g., topoisomerase I, Hsp90, and kinases) and functionally (e.g., histone deacetylases and proteasome) connected to many validated anticancer targets. In this Perspective, we discuss the scientific background behind targeting topoisomerase II together with a number of other targets important in cancer therapy, review the present status, and discuss further options in the field.

Identification of potent catalytic inhibitors of human DNA topoisomerase II by structure-based virtual screening

Human DNA topoisomerase II (Top2) is a promising target for cancer treatment. To overcome the limitations of known Top2 inhibitors, novel Top2 catalytic inhibitors with new scaffolds were identified by structure-based virtual screening. In particular, compound 8 showed good in vitro antiproliferative activity with a broad spectrum. Top2-mediated cleavage assay and molecular modeling rationalized the mode of action. The new Top2 inhibitors are considered as good starting points for further hit-to-lead optimization in anticancer drug discovery.

Polypharmacology: The Rise of Multitarget Drugs over Combination Therapies

Polypharmacology: The Rise of Multitarget Drugs over Combination Therapies

Recent Advances in the Development of Catalytic Inhibitors of Human DNA Topoisomerase IIα As Novel Anticancer Agents

DNA topoisomerases comprise an important family of enzymes that catalyse the induction of topological changes (e.g. relaxation/ supercoiling, catenation/decatenation and knotting/unknotting) in the DNA molecule. These enzymes perform their functions by creating transient either single-stranded or double-stranded breaks in the DNA molecule. Due to their ability to modulate the topology of the DNA molecule, DNA topoisomerases play vital roles in replication, transcription, chromosome separation and segregation, and thus represent an important collection of design targets for novel anticancer drugs. The aim of this review is to provide an overview of the development of catalytic inhibitors of the human topoisomerase IIα enzyme--an important member of the DNA topoisomerase family--as potential novel anticancer agents. The group of catalytic topoII inhibitors is classified into four types according to their molecular mechanism of action: inhibitors that bind to the ATP binding site, inhibitors that prevent the ATP hydrolysis step and trap the enzyme in a closed clamp, inhibitors that block the DNA cleavage and inhibitors that prevent the enzyme binding to the DNA. One of the important considerations highlighted throughout this review is the structure-based perspective of inhibitor design, giving the reader a medicinal chemist's perspective on this vibrant and active field of drug design research.

Abstract C39: Catalytic inhibition of human DNA topoisomerase II α by salicylate and related nonsteroidal anti-inflammatory drugs

Abstract Salicylate-based drugs, including aspirin (acetylsalicylic acid), have long been used in the treatment of mild to moderate cancer pain and have been shown in population-based studies to have cancer chemopreventive properties. Several mechanisms have been hypothesized to underlie these effects, including the inhibition of cyclooxygenases and the inhibition of the transcription factor NF-\#954;B. We have recently demonstrated, however, that salicylate, the primary metabolite of aspirin, is a novel catalytic inhibitor of human DNA topoisomerase IIα (topo II), a nuclear enzyme essential for cell proliferation and division and the target of several widely used anti-cancer chemotherapeutics. As a consequence of this inhibition, and independent of its capacity to inhibit cyclooxygenases and NF-\#954;B, we have demonstrated that a brief pretreatment of human breast cancer cells with salicylate attenuates the cytotoxicity of the topo II poisons, doxorubicin and etoposide. We have observed that salicylate prevents doxorubicin-induced DNA double-strand break generation, which is attributable to salicylate-mediated inhibition of doxorubicin-stabilized topo II-DNA cleavable complex formation in vivo. Together, these data suggest that co-administration of salicylates could negatively impact the efficacy of cancer treatment regimens incorporating topo II-targeting therapeutics. We have now extended our investigation of salicylate, using a biochemical approach to determine the mechanism whereby salicylate inhibits the catalytic activity of topo II. As the inhibition of topo II can occur at one of several stages in its catalytic cycle, we have undertaken multiple independent approaches, including an examination of DNA binding, DNA intercalation, measurement of ATPase activity and evaluation of salicylate's capacity to stabilize topo II in a closed clamp formation without causing DNA double-strand breaks. In addition to delineating the mechanism of salicylate-mediated inhibition of topo II, we have investigated whether common salicylate- and non-salicylate-based non-steroidal anti-inflammatory drugs (NSAIDs) possess similar topo II inhibitory properties, initially by examining the effects of short-term exposure on doxorubicin-induced DNA damage signaling followed by a direct examination of their effects on topo II catalytic activity. Our experiments demonstrate that inhibition of topo II is readily observed with multiple salicylate-based therapies at clinically achieved concentrations. These investigations identify a novel cellular target of salicylate and will inform future studies that may reveal evidence warranting the discouragement of NSAID co-administration in patients undergoing treatment for any of the broad-reaching malignancies using topo II poisons in their therapeutics regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C39.

Potency of inhibition of human DNA topoisomerase I by flavones assessed through physicochemical parameters

DNA topoisomerases, enzymes involved in DNA replication and transcription, are known as targets for anticancer drugs. Among the various types of topoisomerase inhibitors, flavones (F) have been identified as promising compounds. In this study, it is shown that the potency of flavones acting as topoisomerase I inhibitors can be ranked according to their redox properties and their 3D structure. Linear correlations were observed between the topoisomerase I inhibition activity exerted by five flavones (chrysin, apigenin, kaempferol, fisetin, quercetin) and experimental and theoretical redox parameters of F. Moreover, theoretical calculations of the dihedral angle O1-2-1′-2′ in the flavone molecules indicate the importance of their structural and steric features in their potency as topoisomerase I inhibitors. It is suggested that the flavones might interact with the DNA–topoisomerase I complex after their oxidation into quinones via autoxidation, enzymatic oxidation, or reactions with reactive oxygen species. Our investigation opens a new strategy quantitatively based on redox and 3D structural parameters in the search for the most active flavones as anticancer drug candidates inhibiting topoisomerase I.

Catalytic inhibition of human DNA topoisomerase by phenolic compounds in Ardisia compressa extracts and their effect on human colon cancer cells

Plant polyphenols, as those present in teas, have been associated with several health benefits. In this study, the main objectives were to identify and characterize the phenolic compounds in Ardisia compressa tea (AC) responsible for topoisomerase inhibition using a bioassay directed approach and modern analytical techniques, and to determine the cytotoxicity against human colon carcinoma cells. Inhibition of topoisomerase was determined by yeast and human topoisomerase biochemical assays. Identification and characterization of AC phenolic compounds were carried out using combined HPLC, MS and NMR techniques. Cytotoxicity studies were conducted using two human colorectal adenocarcinoma cell lines, HT-29 and Caco-2. LC–MS analysis of AC confirmed the presence of gallic acid, epicatechin gallate, several proanthocyanidin dimers, kaempferol, naringenin and ardisin derivatives. Topoisomerase II catalytic inhibitory activity of AC was due mainly to phenolic compounds extracted in the butanolic fraction (IC 50: 1.33 μg/ml). Purification of this fraction resulted in the isolation of several compounds: peak 10 (IC 50: 8.32 μg/ml), peaks 12/14 (IC 75: 2.85 μg/ml) and peak 15 (IC 50: 7.16 μg/ml). Characterization of peak 15, the most active fraction, led to the isolation of a naringenin isomer (C 15H 12O 5), which had a significantly higher catalytic anti-topoisomerase II activity (IC 50: 7.16 μg/ml) than commercial naringenin (IC 50: 88.1 μg/ml). AC was cytotoxic to HT-29 (IC 50: 57.9 ± 11.6 μg/ml) and Caco-2 cells (IC 50: 81.0 ± 27.5 μg/ml). These findings provide basic information and suggest the potential use of active flavonoids in Ardisia compressa tea as chemopreventive agents.

Catalytic Inhibition of Human DNA Topoisomerase II by Interactions of Grape Cell Culture Polyphenols

Previously, we isolated mixed polyphenolic fractions on a toyopearl matrix (TP-2 to TP-6) from grape cell cultures that were highly potent catalytic inhibitors in a human DNA topoisomerase II assay for cancer chemoprevention. The objectives of this study were to evaluate the potency of, and potential interactions between, individual fractions and some of the purified bioactive polyphenols that comprise these fractions on human DNA topoisomerase II catalytic activity. Treatments that combined anthocyanin-rich fractions (TP-2; 0.5 or 2.0 microg of dried material/mL), fractions containing catechins, procyanidin dimers, and flavanones (TP-4; 0.25 microg of dried material/mL), and/or fractions enriched with procyanidin oligomers and polymers (TP-6; 0.15 or 0.5 microg of dried material/mL) showed additive effects toward catalytic inhibition of the enzyme. Epicatechin gallate (IC50 = 0.029 microM), myricetin (0.39 microM), procyanidin B2 (PB2, 4.5 microM), and resveratrol (65.7 microM), constituents of the most bioactive mixed fraction from grape cell culture (TP-4), each individually provided potent catalytic inhibition of topoisomerase II. In addition, potentiating interactions between the PB2 and the other polyphenolic constituents mentioned above and between myricetin and resveratrol were clearly demonstrated. A synergistic interaction between myricetin and resveratrol was also confirmed with isobolographic analysis at a molar ratio of 1:70.

Comparing expression of different forms of human DNA topoisomerase I in Pichia pastoris

In order to rapidly screen new inhibitors of human DNA topoisomerase I in vitro, the enzyme has been successfully overexpressed in the full-length form (hTopoI) and N-terminal 184 amino acids truncated form (hTopoIΔ581) in Pichia pastoris by the secretory and intracellular styles. The results showed that the recombinant enzyme containing nuclear localization signals obviously decreased its expression level. If the enzyme was fused to α-factor secretory signal or the AKL triplet amino acids (a C-terminal peroxisomal-targeting signal), its output could be highly improved. The recombinant strain SMD-hTopoIΔ(out) was best suited to express and secrete the highly active hTopoIΔ581 with part of them glycosylated (the enzyme activity of the supernatant reached 3.4×108Ul−1). SMD-hTopoI(out) could produce and secrete the full length form of hTopoI without glycosylation (the enzyme activity of the supernatant reached 4.3×107Ul−1). The yield of the recombinant hTopoI reached 11mgl−1, achieving 9.7% of the total protein in the culture supernatant, while that of the recombinant hTopoIΔ581 reached 58mgl−1, achieving 47.3% of the total protein in the supernatant. Both forms of the recombinant enzyme exhibited the same properties as the natural hTopoI.

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 Nδ2H 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.

DNA structural alterations induced by bis-netropsins modulate human DNA topoisomerase I cleavage activity and poisoning by camptothecin

Bis-netropsins (bis-Nts) are efficient catalytic inhibitors of human DNA topoisomerase I (top I). These DNA minor groove binders are considered to serve as suppressors of top I-linked DNA breaks, which is generally believed to be related to their affinity to DNA. In this study, it was found that bis-Nts exhibit sequence-specificity of suppression of the strong top I-specific DNA cleavage sites and that this sequence-specificity is determined by differential ligand-induced structural alterations of DNA. Raman scattering analysis of bis-Nts interactions with double-stranded oligonucleotides, each containing the site of specific affinity to one of bis-Nts and a distinctly located top I degenerate consensus, demonstrated that bis-Nts induce not only structural changes in duplex DNA at their loading position, but also conformational changes in a distant top I-specific DNA cleavage site. The ability to alter the DNA structure correlates with the anti-top I inhibitory activities of the ligands. In addition, DNA structural alterations induced by bis-Nts were shown to be responsible for modulation of the camptothecin (CPT)-mediated DNA cleavage by top I. This effect is expressed in the bis-Nts-induced enhancement of some of the CPT-dependent DNA cleavage sites as well as in the CPT-induced enhancement of some of the top I-specific DNA cleavage sites suppressed by bis-Nts in the absence of CPT.

Inhibition of human DNA topoisomerase I by new DNA minor groove ligands: derivatives of oligo-1,3-thiazolecarboxamides.

A series of novel thiazole-containing oligopeptides (oligo-1,3-thiazolecarboxamides) interesting specifically with the minor groove of DNA was shown to inhibit human DNA topoisomerase I (topo I). Inhibitory effects of thiazole-containing oligopeptides (TCO) increase with the number of thiazole units in such compounds. Inhibitory properties of TCO containing 3 or 4 thiazole units were shown to be 3-10 times better than those of the well-known natural antibiotic, distamycin A containing pyrrole rings. The structure of various additional groups attached to the N-terminus and C-terminus of TCO had no significant effect on TCO interaction with the complex of DNA and topo I. TCO were shown to be capable of binding with double-stranded DNA (dsDNA), and the majority of TCO analyzed were more effective in binding with dsDNA than distamycin A. Possible reasons for the different effects of distamycin A and TCO on the reaction of relaxation catalyzed by topo I are discussed.

Molecular Determinants of Site-specific Inhibition of Human DNA Topoisomerase I by Fagaronine and Ethoxidine: RELATION TO DNA BINDING

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

Human DNA‐topoisomerase I activity is affected by bis‐netropsin's binding to DNA minor groove

AbstractBis‐netropsins (bis‐Nts) are known to be efficient inhibitors of human DNA topoisomerase (topo) I with a higher antitumor activity than netropsin. New sequence‐specific derivatives of bis‐Nts were used for modulation of topo I‐mediated DNA cleavage with and without camptothecin (CPT). Relation between the bis‐Nts binding sites and topo I cleavage sites has been analyzed with the plasmid DNA constructs generated by insertion of synthetic oligonucleotides containing various topo I‐cleavage and bis‐Nt‐binding sites. These constructs offer an opportunity to study minor groove binders effects on the topo I reaction on DNA. Three major effects: (i) bis‐Nt ‐ mediated disappearance of some of the topo I cleavage sites; (ii) enhancement of some other sites, and, (iii) generation of new cleavage sites, have been fovnd and analyzed. These effects demonstrate that bis‐Nts drastically change the topo I‐mediated DNA cleavage.

Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene.

Chronic exposure of humans to benzene (BZ) causes acute myeloid leukemia (AML). Both BZ and therapy-related secondary AML are characterized by chromosomal translocations that may occur by inappropriate recombinational events. DNA topoisomerase II (topo II) is an essential sulfhydryl (SH)-dependent endonuclease required for replication, recombination, chromosome segregation, and chromosome structure. Topo II cleaves DNA at purine(R)/pyrimidine(Y) repeat sequences that have been shown to be highly recombinogenic in vivo. Certain antineoplastic drugs stabilize topo II-DNA cleavage complexes at RY repeat sequences, which leads to translocations of the type observed in leukemia. Hydroquinone (HQ) is metabolized to p-benzoquinone (BQ) in a peroxidase-mediated reaction in myeloid progenitor cells. BQ interacts wit SH groups of SH-dependent enzymes. Consequently, the aims of this research were to determine whether HQ and BQ are topo II inhibitors. The ability of the compounds to inhibit the activity of topo III was tested using an assay system that depends on the conversion, by homogeneous human topo II, of catenated kinetoplast DNA into open and/or nicked open circular DNA that can be separated from the catenated DNA by electrophoresis in a 1% agarose-ethidium bromide gel. We provide preliminary data that indicate that both HQ and BQ cause a time and concentration (microM)-dependent inhibition of topo II activity. These compounds, which potentially can form adducts with DNA, have no effect on the migration of the supercoiled and open circular forms in the electrophoretic gradient, and BQ-adducted KDNA can be decatenated by topo II. Using a pRYG plasmid DNA with a single RY repeat as a cleavage site, it was determined that BQ does not stimulate the production of linear DNA indicative of an inhibition of topo II religation of strand breaks by stabilization of the covalent topo III-DNA cleavage complex. Rather, BQ most probably inhibits the SH-dependent topo II by binding to an essential SH group. The inhibition of topo II by BQ has implications for the formation of deleterious translocations that may be involved in BZ-induced initiation of leukemogenesis.

Inhibition of Human DNA Topoisomerase II by Hydroquinone and p-Benzoquinone, Reactive Metabolites of Benzene

Inhibition of Human DNA Topoisomerase II by Hydroquinone and p-Benzoquinone, Reactive Metabolites of Benzene

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated duringin vivoexpression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.