Search Results (9)

Tyrosyl-DNA phosphodiesterase 1 (TDP1) represents a compelling pharmacological target for the development of agents designed to circumvent tumor resistance to topoisomerase 1 (TOP1) inhibitors, a major class of clinically relevant antineoplastic drugs. This paper describes the design and synthesis of novel hybrid TDP1 inhibitors combining coumarin and monoterpene moieties via rigid isoxazole and 1,2,3-triazole heterocyclic linkers. The synthesis was accomplished via [3 + 2] cycloaddition of nitrile oxides to alkynes and copper-catalyzed click chemistry. Biological tests have demonstrated the crucial role of linker nature in the activity of the compounds. Isoxazole-linked conjugates showed strong inhibitory effects on TDP1, with IC₅₀ values in the submicromolar to low micromolar range (0.8–3.2 μM). Overall, these values slightly surpassed those of the triazole-linked analogues, whose IC₅₀ values ranged from 1.1 to 23.3 μM. At noncytotoxic doses, compounds 26e and 16b enhanced the sensitivity of human cervical cancer (HeLa) cells to the antitumor agent topotecan, a TOP1 inhibitor, thereby supporting the promise of this structural class as components of combination chemotherapy. Full article

Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are important DNA repair enzymes that remove various adducts from the 3′- and 5′-ends of DNA, respectively. The suppression of the activity of these enzymes is considered as a promising adjuvant therapy for oncological diseases in combination with topoisomerase inhibitors. The simultaneous inhibition of TDP1 and TDP2 may result in greater antitumor effects, as these enzymes can mimic each other’s functions. We have previously shown that usnic acid-based sulfides can act as dual inhibitors, with TDP1 activity in the low micromolar range and their TDP2 at 1 mM. The oxidation of their sulfide moieties to sulfoxides led to an order of magnitude decrease in their cytotoxicity potential, while their TDP1 and TDP2 activity was preserved. In this work, we synthesized new series of usnic acid-based sulfides and their oxidized analogues, i.e., sulfoxides and sulfones, to systematically study these irregularities. The new compounds inhibit TDP1 with IC₅₀ values (the concentration of inhibitor required to reduce enzyme activity by half) in the 0.33–25 μM range. Most sulfides and some sulfoxides and sulfones inhibit TDP2 with an IC₅₀ = 138−421 μM. In addition, the most active compounds synergized (×4) with topotecan on the HeLa cell line as well as causing dose-dependent DNA damage, as confirmed by Comet assay. Sulfides with the 6-methylbenzoimidazol-2-yl substituent (8f, IC₅₀ = 0.33/138 μM, TDP1/2) and sulfones containing a pyridine-2-yl fragment (12k, IC₅₀ = 2/228 μM, TDP1/2) are the most potent derivatives and, therefore, are promising for further development. Full article

Deoxycholic acid derivatives containing various heterocyclic functional groups at C-3 on the steroid scaffold were designed and synthesized as promising dual tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) inhibitors, which are potential targets to potentiate topoisomerase poison antitumor therapy. The methyl esters of DCA derivatives with benzothiazole or benzimidazole moieties at C-3 demonstrated promising inhibitory activity in vitro against TDP1 with IC₅₀ values in the submicromolar range. Furthermore, methyl esters 4d–e, as well as their acid counterparts 3d–e, inhibited the phosphodiesterase activity of both TDP1 and TDP2. The combinations of compounds 3d–e and 4d–e with low-toxic concentrations of antitumor drugs topotecan and etoposide showed significantly greater cytotoxicity than the compounds alone. The docking of the derivatives into the binding sites of TDP1 and TDP2 predicted plausible binding modes of the DCA derivatives. Full article

Tyrosyl-DNA phosphodiesterase 1 and 2 (Tdp1 and Tdp2) are DNA repair enzymes that repair DNA damage caused by various agents, including anticancer drugs. Thus, these enzymes resist anticancer therapy and could be the reason for resistance to such widely used drugs such as topotecan and etoposide. In the present work, we found compounds capable of inhibiting both enzymes among derivatives of (−)-usnic acid. Both (+)- and (−)-enantiomers of compounds act equally effectively against Tdp1 with IC₅₀ values in the range of 0.02–0.2 μM; only (−)-enantiomers inhibited Tdp2 with IC₅₀ values in the range of 6–9 μM. Surprisingly, the compounds protect HEK293FT wild type cells from the cytotoxic effect of etoposide (CC₅₀ 3.0–3.9 μM in the presence of compounds and 2.4 μM the presence of DMSO) but potentiate it against Tdp2 knockout cells (CC₅₀ 1.2–1.6 μM in the presence of compounds against 2.3 μM in the presence of DMSO). We assume that the sensitizing effect of the compounds in the absence of Tdp2 is associated with the effective inhibition of Tdp1, which could take over the functions of Tdp2. Full article

A series of deoxycholic acid (DCA) amides containing benzyl ether groups on the steroid core were tested against the tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) enzymes. In addition, 1,2,4- and 1,3,4-oxadiazole derivatives were synthesized to study the linker influence between a para-bromophenyl moiety and the steroid scaffold. The DCA derivatives demonstrated promising inhibitory activity against TDP1 with IC₅₀ in the submicromolar range. Furthermore, the amides and the 1,3,4-oxadiazole derivatives inhibited the TDP2 enzyme but at substantially higher concentration. Tryptamide 5 and para-bromoanilide 8 derivatives containing benzyloxy substituent at the C-3 position and non-substituted hydroxy group at C-12 on the DCA scaffold inhibited both TDP1 and TDP2 as well as enhanced the cytotoxicity of topotecan in non-toxic concentration in vitro. According to molecular modeling, ligand 5 is anchored into the catalytic pocket of TDP1 by one hydrogen bond to the backbone of Gly458 as well as by π–π stacking between the indolyl rings of the ligand and Tyr590, resulting in excellent activity. It can therefore be concluded that these derivatives contribute to the development of specific TDP1 and TDP2 inhibitors for adjuvant therapy against cancer in combination with topoisomerase poisons. Full article

Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2′,3′,5′-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC₅₀) in the lower micromolar and submicromolar range. 2′,3′,5′-Tri-O-benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC₅₀ = 0.6 μM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC₅₀ > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50–100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells. Full article

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5′ phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently. Full article

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this ‘abortive catalysis’ can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones. Full article

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5′-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5′-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5′ tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition. Full article