Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.     

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.     

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

Secondary metabolites from Isodon ternifolius (D. Don) Kudo and their anticancer activity as DNA topoisomerase IB and Tyrosyl-DNA phosphodiesterase 1 inhibitors

Based on DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibition of the ethanol extract of the roots of Isodon ternifolius (D. Don) Kudo (Labiatae), its secondary metabolites has been studied. Two new compounds, an ent-abietane diterpenoid isodopene A (1) and a 2,3-seco-triterpene isodopene B (13), along with 25 known compounds were isolated. Their structures were elucidated by spectroscopic analysis and theoretical calculations. The enzyme-based assays indicated that 1 and 13 showed strong (+++) and moderate (++) TOP1 inhibition, respectively. Two chalcone derivatives 11 and 12 were firstly found as dual TDP1 and TOP1 natural inhibitors, and showed synergistic effect with the clinical TOP1 inhibitors topotecan in MCF-7 cells. Compounds 8, 16, and 22 acted as TOP1 catalytic inhibitors with equipotent TOP1 inhibition to camptothecin (++++). Compounds 7 and 8 exhibited significant cytotoxicity against MCF-7, A549, and HCT116 cells with GI₅₀ values in the range of 2.2–4.8 μM. This work would provide valuable information that secondary metabolites from I. ternifolius could be developed as anticancer agents.     

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5′ adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3′- and 5′-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5′-THF and the 3′-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5′-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.     

Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure–activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC₅₀?<?50?µM), with 17z showing excellent cell permeability and no cytotoxicity.     

Synthesis and biological evaluation of new fluorinated and chlorinated indenoisoquinoline topoisomerase I poisons

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds’ activities were found to rival or surpass those of similarly substituted 3-nitroindenoisoquinolines, and several unusually potent analogs were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Synthesis and biological evaluation of novel indole-pyrazoline hybrid derivatives as potential topoisomerase 1 inhibitors

A series of novel indole-pyrazoline hybrid derivatives were designed, synthesized, and evaluated for topoisomerase 1 (Top1) inhibitory activity. Top1-mediated relaxation assays showed that our synthesized compounds had variable Top1 inhibitory activity. Among these compounds, 3-(5-(naphthalen-1-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-1-(phenylsulfonyl)-1H-indole (6n) was found to be a strong Top1 inhibitor with better inhibitory activity than CPT and hit compounds. Our further experiments rationalized the mode of action for this new type of inhibitors, which showed no significant binding to supercoiled DNA.     

Biochemical Characterization of Human Tyrosyl-DNA Phosphodiesterase 2 (TDP2/TTRAP): A Mg2+/Mn2+-DEPENDENT PHOSPHODIESTERASE SPECIFIC FOR THE REPAIR OF TOPOISOMERASE CLEAVAGE COMPLEXES

TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)m(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.     

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.     

Tipin Functions in the Protection against Topoisomerase I Inhibitor

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.     

Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3'-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3'-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.     

Effects of phosphodiesterase 3,4,5 inhibitors on hepatocyte cAMP levels,glycogenolysis, gluconeogenesis and suceptibility to a mitochondrial toxin

Various phosphodiesterase (PDE) 3,4 and 5 inhibitors have been compared with glucagon for their effectiveness at increasing hepatocyte cAMP, glycogenolysis and gluconeogenesis. Preincubation of isolated hepatocytes with PDE 3 and 4 inhibitors (50 M) for 2 h induced significant increases in cellular cAMP level. The order of effectiveness was: glucagon (78%), V11294A (42%), rolipram (40%), milrinone (36%), CDP-840 (33%), R₀ 20-1724 (31%), papaverine (27%), isobutylmethylxanthine (28%), isoliquiritigenin (25%), theophylline (22%), and amrinone (22%). The PDE 5 inhibitors dipyridamol and sildenafil had only a slight effect on cAMP levels. Glucose formation was increased as a result of increased glycogenolysis in the following order of effectiveness: glucagon (89%), V11294A (63%), rolipram (61%), milrinone (50%), CDP-840 (46%), R₀ 20-1724 (45%), sildenafil (34%), dipyridamol (31%), papaverine (30%), isobutylmethylxanthine (29%), theophylline (20%), amrinone (20%), and isoliquiritigenin (20%). Rolipram and milrinone, selective PDE 4 and PDE 3 inhibitors respectively, stimulated the gluconeogenesis of alanine, lactate + pyruvate, or fructose in hepatocytes isolated from fasted rats. On the other hand, selective cGMP specific phospodiesterase inhibitors, sildenafil and dipyridamol inhibited alanine-induced gluconeogenesis. All PDE inhibitors increased hepatocyte susceptibility to cyanide toxicity (3–4 fold) which was prevented by fructose whereas PDE 5 inhibitors did not significantly increase hepatocyte susceptibility.     

New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties

A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen. The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important target for anti-cancer therapy, was studied for the first time. It was found that the 7-hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1 with the most active derivative (+)-25c with IC₅₀ value of 0.675 μM. This compound has low cytotoxicity (CC₅₀ >100 μM) when tested against human cancer cells which is crucial for presupposed application in combination with clinically established anticancer drugs. The ability of the new compounds to enhance the cytotoxicity of camptothecin, an established topoisomerase 1 poison, was demonstrated.     

Inhibition of neoplastic cell growth by quiescent cells is mediated by serum concentration and cAMP phosphodiesterase inhibitors

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20-1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10^?4 M Ro 20-1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30-fold in nontransformed versus only 3-fold in transformed cells. The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20-1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G₁ phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.