Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors

Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 μM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.     

Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIalpha expression

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.     

Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome inhibitors

DNA topoisomerase I (Top1) is the target of camptothecin, and novel Top1 inhibitors are in development as anticancer agents. Top1 inhibitors damage DNA by trapping covalent complexes between the Top1 catalytic tyrosine and the 3'-end of the broken DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) can repair Top1-DNA covalent complexes by hydrolyzing the tyrosyl-DNA bond. Inhibiting Tdp1 has the potential to enhance the anticancer activity of Top1 inhibitors (http://discover.nci.nih.gov/pommier/pommier.htm) and to act as antiproliferative agents. In the present study, we report that neomycin inhibits Tdp1 more effectively than the related aminoglycosides paromomycin and lividomycin A. Inhibition of Tdp1 by neomycin is observed both with single- and double-stranded substrates but is slightly stronger with duplex DNA, which is different from aclarubicin, which only inhibits Tdp1 with the double-stranded substrate. Inhibition by neomycin can be overcome with excess Tdp1 and is greatest at low pH. To our knowledge, aminoglycoside antibiotics and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin are the first reported pharmacological Tdp1 inhibitors.     

Explorations of peptide and oligonucleotide binding sites of tyrosyl-DNA phosphodiesterase using vanadate complexes

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate and functions as a DNA repair enzyme that cleaves stalled topoisomerase I-DNA complexes. We previously determined a procedure to crystallize a quaternary complex containing Tdp1, vanadate, a DNA oligonucleotide, and a tyrosine-containing peptide that mimics the transition state for hydrolysis of the Tdp1 substrate. Here, the ability of vanadate to accept a variety of different ligands is exploited to produce several different quaternary complexes with a variety of oligonucleotides, and peptides or a tyrosine analogue, in efforts to explore the binding properties of the Tdp1 DNA and peptide binding clefts. Eight crystal structures of Tdp1 with vanadate, oligonucleotides, and peptides or peptide analogues were determined. These structures demonstrated that Tdp1 is able to bind substituents with limited sequence variation in the polypeptide moiety and also bind oligonucleotides with sequence variation at the 3' end. Additionally, the tyrosine analogue octopamine can replace topoisomerase I derived peptides as the apical ligand to vanadate. The versatility of this system suggests that the formation of quaternary complexes around vanadate could be adapted to become a useful method for structure-based inhibitor design and has the potential to be generally applicable to other enzymes that perform chemistry on phosphate esters.     

Structural modifications of camptothecin and effects on topoisomerase I inhibition.

Camptothecin (1), a potent antitumor alkaloid, is known to inhibit topoisomerase I, an enzyme that relaxes supercoiled DNA. Modifications have been made to the B, D, and E rings of this natural product. Specifically, compounds 2-10 either have an ester moiety in place of the E ring lactone, a methyl ester attached to position 14, a saturated (or nonexistent) deaza B ring, or contain a combination of these permutations. We have conducted in vitro assays against the topoisomerase I relaxation reaction which verify the necessity for a lactone in the E ring. Furthermore, steric requirements at position 14 are shown to be crucial for activity, and planarity of the A and B rings of camptothecin is also implicated in the ability of the drug to inhibit topoisomerase I. Speculation on the nature of the drug binding pocket is presented.     

Cellular topoisomerase I inhibition and antiproliferative activity by MJ-III-65 (NSC 706744), an indenoisoquinoline topoisomerase I poison

To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interaction, and drug resistance, attempts to synthesize compounds that are CPT-like in their specificity and potency yet display a unique profile have been underway. In this pursuit, we have identified one of the idenoisoquinoline derivatives, MJ-III-65 (NSC 706744; 6-[3-(2-hydroxyethyl)amino-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline) with both similarities and differences from CPT. MJ-III-65 traps topoisomerase I (Top1) reversibly like CPT but with different DNA sequence preferences. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with MJ-III-65 at nanomolar concentrations. These MJ-III-65-induced protein-linked DNA breaks were resistant to reversal after an hour of drug removal, compared with CPT, which completely reversed. Studies in human cells in culture found MJ-III-65 to be cytotoxic. Furthermore, limited cross-resistance was observed in camptothecin-resistant cell lines. MJ-III-65 also exhibits antitumor activity in mouse tumor xenografts.     

Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.     

Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes

It has previously been shown that intracellular perfusion of isolated cardiac myocytes with cGMP reduces the amplitude of the trans-sarcolemmal calcium current (ICa) elevated by cAMP-dependent mechanisms. To test the hypothesis that cGMP acts by stimulating a cyclic nucleotide phosphodiesterase (PDE) activity, PDE activity and the effects of methylisobutylxanthine (MIX), a PDE inhibitor, on ICa were examined in cardiomyocytes dissociated from frog ventricle. PDE activity was determined by measuring hydrolysis of [33P]cAMP in subcellular fractions. Using 100 microM cAMP as substrate, approximately 50% of the PDE activity was found in the 20,000 x g particulate fraction. Basal activity in this fraction had a Vmax of 15.4 nmol [corrected] of cAMP hydrolyzed/min/mg of protein and a Km of 113 microM cAMP. The PDE activity of the particulate fraction was stimulated significantly by cGMP. Half-maximal stimulation was observed with 1.1 microM cGMP. This value is virtually identical to the value for the concentration of intracellular cGMP that produced a half-maximal reduction of cAMP-elevated ICa in electrophysiological experiments. The cGMP-stimulated PDE activity had a Vmax of 9.5 nmol/min/mg [corrected] and a Km of 12.3 microM cAMP. MIX (100 microM) selectively inhibited the cGMP-stimulated PDE activity (IC50 = 20 microM). To determine whether PDEs modulate the amplitude of ICa, the effects of MIX were examined on basal ICa and cAMP-elevated ICa. MIX produced small increases in the basal ICa and increased ICa in the presence of 1 microM intracellular cAMP. MIX at 100 microM potentiated the effects of submaximal doses of isoproterenol and shifted the dose-response curve for cAMP to the left but did not affect the dose-response curve for 8-bromo-cAMP. MIX reversed the effect of cGMP on the cAMP-elevated ICa. We conclude that cyclic nucleotide PDEs play an important role in modulating the cardiac calcium current. The hypothesis that cGMP inhibits the cAMP-elevated ICa by activating a PDE is supported by the finding that MIX inhibited both the cGMP-stimulated PDE activity and the effect of cGMP on ICa at similar concentrations.     

Role of curcumin-dependent modulation of tumor microenvironment of a murine T cell lymphoma in altered regulation of tumor cell survival

Using a murine model of a T cell lymphoma, in the present study, we report that tumor growth retarding action of curcumin involves modulation of some crucial parameters of tumor microenvironment regulating tumor progression. Curcumin-administration to tumor-bearing host caused an altered pH regulation in tumor cells associated with alteration in expression of cell survival and apoptosis regulatory proteins and genes. Nevertheless, an alteration was also observed in biophysical parameters of tumor microenvironment responsible for modulation of tumor growth pertaining to hypoxia, tumor acidosis, and glucose metabolism. The study thus sheds new light with respect to the antineoplastic action of curcumin against a tumor-bearing host with progressively growing tumor of hematological origin. This will help in optimizing application of the drug and anticancer research and therapy.     

Targeting topoisomerase I: molecular mechanisms and cellular determinants of response to topoisomerase I inhibitors

Background: Topoisomerase I is required for DNA relaxation during critical cellular functions. The identification of camptothecins as specific enzyme inhibitors and their clinical efficacy have stimulated extensive efforts to exploit topoisomerase I as a tumor target and explain the putative mechanisms of antitumor-specific action. Objective: This review provides an overview of the recent achievements in the development of topoisomerase I inhibitors and in the explanation of the biological pathways involved in tumor response. Results/conclusion: In spite of the difficulty to identify novel topoisomerase I inhibitors with improved pharmacological properties, a growing body of evidence supports the possibility of optimizing the therapeutic profile of available agents. The explanation of defense mechanisms and the molecular determinants of tumor cell response is expected to provide a basis for the design of combination approaches for optimization of topoisomerase I inhibitors-based therapy.     

Combined topoisomerase I inhibition for the treatment of metastatic colon cancer

The objective of this study was to define the maximally tolerated dose (MTD) and response rate of a combination of two topoisomerase I inhibitors, topotecan and irinotecan, in patients with metastatic colon cancer. Eleven patients, the majority with previously progressive disease on 5-fluorouracil-based regimens, were enrolled onto a phase I/II dose escalation trial utilizing continuous infusion topotecan for 2 weeks and weekly irinotecan x 3 with cycles repeated every 28 days. Dosages of topotecan utilized included 0.2 and 0.25mg/m2/day. Irinotecan was administered at a dose of 62 mg/m2 by i.v. bolus. Patients were followed for toxicity and response. The MTD of the combination of agents was found to be 0.25mg/m2/day for topotecan and 62 mg/m2 for irinotecan. The most common serious toxicities were diarrhea and nausea/vomiting. Only one patient experienced grade III neutropenia. There were no complete or partial responses. However, four patients had prolonged disease stabilization (SD) of up to 324 days and this group remained on protocol therapy for an average of 227 days (p=0.0005 versus patients not achieving SD). We concluded that the MTD for this combination of topoisomerase I inhibitors, given on this particular schedule, has been defined. This combination cannot be recommended as a first- or second-line therapy for patients with metastatic colon cancer based on the responses observed. However, approximately one-third of patients achieved prolonged disease stabilization. Topotecan with irinotecan may be useful as a palliative regimen for a subgroup of colon cancer patients.     

90-kDa heat shock protein inhibition abrogates the topoisomerase I poison-induced G2/M checkpoint in p53-null tumor cells by depleting Chk1 and Wee1

The G(2)/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G(2)/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G(2)/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G(2)/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.     

Circadian regulation of mouse topoisomerase I gene expression by glucocorticoid hormones

Because glucocorticoid hormones modulate various biological processes, the endogenous rhythm of their secretion is thought to be an important factor affecting the efficacy and/or toxicity of many drugs. Topoisomerase I (Topo I) is a nuclear target of the anticancer drug camptothecin (CPT). In this study, we demonstrate that Topo I expression in tumor-bearing mice and the efficacy of CPT on the tumor are affected by the 24-h variation in circulating glucocorticoid levels. A single administration of corticosterone (CORT) to the tumor-bearing mice resulted in a significant increase in Topo I mRNA levels not only in the tumor masses but also in other healthy tissues such as liver and skeletal muscle. The CORT-induced increase in Topo I mRNA was suppressed by pretreating the mice with RU486, a glucocorticoid receptor antagonist. Significant 24-h oscillations in the Topo I mRNA levels were observed in the tumor and healthy liver without exogenous CORT, and were eliminated by adrenalectomy of the mice. This result suggests that endogenous glucocorticoid hormones are involved in the circadian regulation of Topo I gene expression. Furthermore, the anti-tumor efficacy of the Topo I inhibitor CPT-11 on the tumor-bearing mice was enhanced by administering the drug at the time when the Topo I activity was increased. Our present results demonstrate that glucocorticoid is involved in the 24-h oscillation mechanism of Topo I gene expression and suggest that monitoring the circadian rhythm in Topo I activity is useful for choosing the most appropriate time of day to administer of Topo I inhibitors.     

Diarylsemicarbazones: synthesis, antineoplastic activity and topoisomerase I inhibition assay.

A series of diarylsemicarbazones was synthesized and tested against human neoplastic cell lines. The more active members have a l-naphthyl ring at the carbamidic nitrogen, and chloro, dimethylamino or nitro group substituents at the benzylidene moiety. None of these showed affinity to DNA. One of the more active compounds was tested as a topoisomerase I inhibitor and showed a potent effect. SAR studies demonstrated linear correlation between lypophilicity and activity on the most sensitive lines and a definite conformational shape for antineoplastic action.     

Dichloromethane fraction from Gardenia jasminoides: DNA topoisomerase 1 inhibition and oral cancer cell death induction

Context: A growing body of evidence shows that compounds of plant origin have the ability to prevent cancer. The fruit of gardenia, Gardenia jasminoides Ellis (Rubiaceae), has long been used as a food additive and herbal medicine, and its pharmacological actions, such as protective activity against oxidative damage, cytotoxic effect, and anti-inflammatory and anti-tumor activity, have already been reported.

Objective: The purpose of the present study was to investigate the presence of DNA topoisomerase 1 inhibitor in various solvent fractions of Gardenia extract and examine the induction of oral cancer cell death upon treatment with Gardenia extract.

Materials and methods: The methanol extract of Gardenia was partitioned with n-hexane, dichloromethane, ethyl acetate, n-butanol, and water.

Results: In the DNA topoisomerase 1 assay, n-hexane and dichloromethane fractions inhibited topoisomerase 1 and led to a decrease in the cell viability of KB cells. The dichloromethane fraction (0.1?mg/mL) also showed 77% inhibition of cell viability in KB cells compared with HaCaT cells. Treatment with dichloromethane fraction led to apoptotic cell death as evidenced by flow cytometric analysis and morphological changes. In addition, treatment with Gardenia extract dichloromethane fraction led to the partial increase of caspase-3, caspase-8 and caspase-9 activities and the cleavage of poly (ADP-ribose) polymerase.

Conclusion: Taken together, these results suggest that the dichloromethane fraction from Gardenia extract induces apoptotic cell death by DNA topoisomerase 1 inhibition in KB cells. These findings suggest the possibility that Gardenia extract could be developed as an anticancer modality.     

Role of non-selective adenosine receptor blockade and phosphodiesterase inhibition in cisplatin-induced nephrogonadal toxicity in rats

1. It is well documented that cisplatin (CDDP) treatment increases the expression of adenosine A(1) receptors in both kidney and testes. However, the effect of adenosine at these receptors is controversial. Adenosine A(1) receptors have been documented to be involved in either cytoprotection or aggravation of nephrotoxicity. The aim of the present study was to examine the effect of the non-selective adenosine receptor inhibitor theophylline and the phosphodiesterase inhibitor pentoxifylline on CDDP-induced renal and testicular toxicity. 2. Male Wister rats were divided into six groups. Two control groups received plain drinking water and a third control group received theophylline 0.8 mg/mL in the drinking water for 2 weeks. One group of animals drinking plain water was injected intraperitoneally (i.p.) with pentoxifylline 50 mg/kg per day for 2 weeks. The remaining groups were treated in the same manner and received single dose of CDDP 7 mg/kg, i.p., 1 week after starting theophylline and pentoxifylline treatment and all animals were killed 1 week after CDDP treatment. 3. Rats treated with CDDP developed nephrotoxicity, as demonstrated by increased kidney and testes weight as a percentage of total bodyweight, blood urea nitrogen and serum creatinine levels and decreased serum calcium and albumin levels. In addition, CDDP treatment resulted in an increase in the production of malondialdehyde (MDA) and decreases in total nitrate/nitrite levels, as well as depletion of reduced glutathione (GSH) content and glutathione peroxidase (GPX) activity in both the kidney and testes. Administration of theophylline in the drinking water to CDDP-treated rats resulted in exacerbation of the indices of nephrotoxicity, depletion of GSH content and GPX activity levels, with increased MDA production and platinum accumulation in both the kidney and testes. However, pentoxifylline administration reduced CDDP-induced biochemical changes and reduced platinum accumulation in both organs. Histopathological examination of the kidney revealed that CDDP treatment produced multifocal tubular atrophy, atypical reparative changes of the tubular epithelium and marked tubular necrosis. Animals treated with the theophylline/CDDP combination showed extensive widespread damage with intratubular calcification. However, pentoxifylline treatment ameliorated the overt changes induced by CDDP treatment. 4. Theophylline exacerbates the deleterious effects of CDDP on rat kidney and testes. However, pentoxifylline alleviates CDDP-induced renal and testicular toxicity.     

Dual expression and differential regulation of phosphodiesterase 3A and phosphodiesterase 3B in human vascular smooth muscle: implications for phosphodiesterase 3 inhibition in human cardiovascular tissues

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes whose physiological role is the attenuation of the signaling mediated by the ubiquitous second messengers cAMP and cGMP. Given the myriad of physiological processes regulated by cAMP and cGMP, PDEs have long been studied as potential therapeutic targets. Although phosphodiesterase 3 (PDE3) activity is abundant in human cardiovascular tissues, and acute PDE3 inhibition, with agents such as milrinone, was beneficial in heart failure patients, prolonged treatments were associated with time-dependent reductions in hemodynamic effects and increased mortality. The molecular basis of this time-dependent reduction in efficacy has not been elucidated. In this context, we used a combination of approaches to determine PDE3 expression in human cardiovascular tissues and to elucidate the effects of prolonged elevations of cellular cAMP, as would occur with PDE3 inhibition, on this activity. Although our data confirms the expression of PDE3A in human blood vessel smooth muscle cells (HASMCs), we identify a previously unrecognized role for PDE3B in cAMP hydrolysis in human cardiovascular tissues. Specifically, although both PDE3A and PDE3B were expressed in HASMCs, their subcellular expression pattern and regulated expression by cAMP were distinct, with only expression of PDE3B being subject to cAMP-regulated expression. Thus, a paradigm emerges that allows for dual expression, with distinctive regulation, of both PDE3A and PDE3B proteins in cardiovascular tissues that may have profound significance for the rational design of molecules regulating this PDE activity.     

Investigation of the lactam side chain length necessary for optimal indenoisoquinoline topoisomerase I inhibition and cytotoxicity in human cancer cell cultures

Indenoisoquinolines with lactam substituents such as ethylamino, propylamino, and butylamino have previously demonstrated potent biological activity, but an optimal length has never been established. In the present study, a series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12 carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity. Using these lengths, analogues were prepared with the amino group and portions of the linker replaced by a pyridine ring. A three-carbon spacer within the pyridine series still demonstrated potent topoisomerase I inhibition.