Topoisomerase IB Research Articles

Differential cellular localization of DNA gyrase and topoisomerase IB in response to DNA damage in Deinococcus radiodurans.

Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.

Synthesis and Biological Activities of 11- and 12-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

Herein, a series of 11- or 12-substituted benzophenanthridinone derivatives was designed and synthesized for the discovery of dual topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Enzyme-based assays indicated that two compounds 12 and 38 showed high TOP1 inhibitory potency (+++), and four compounds 35, 37, 39 and 43 showed good TDP1 inhibition with IC50 values ranging from 10 to 18 μM. 38 could induce cellular TOP1cc formation, resulting in the highest cytotoxicity against HCT-116 cells (0.25 μM). The most potent TDP1 inhibitor 43 (10 μM) could induce cellular TDP1cc formation and enhance topotecan-induced DNA damage and showed strong synergistic cytotoxicity with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Design, synthesis, and biological evaluation of 3,3'-diindolylmethane N-linked glycoconjugate as a leishmanial topoisomerase IB inhibitor with reduced cytotoxicity.

Leishmaniasis, one of the neglected diseases, ranks second to malaria in the cause of parasitic mortality and morbidity. The present chemotherapeutic regimen faces the limitations of drug resistance and toxicity concerns, raising a great need to develop new chemotherapeutic leads that are orally administrable, potent, non-toxic, and cost-effective. Several research groups came forward to fill this therapeutic gap with new classes of active compounds against leishmaniasis, one such being 3,3'-diindolylmethane (DIM) derivatives. We tried to link this concept with another promising approach of glycoconjugation to study how glycosylated groups work differently from non-glycosylated ones. In the present study, a series of 3,3'-DIM derivatives have been synthesized and screened for their anti-leishmanial potency on Leishmania donovani promastigotes. Next, we synthesized the β-N,N' glycoside of potent compound 3d using indole-indoline conversion, Fischer-type glycosylation, 2,3-dichloro-5,6-dicyano-1,4-benzoquionone (DDQ) oxidation, and molecular iodine catalyzed coupling with a suitable aldehyde in reasonable overall yield. The biological evaluation revealed that glycosides had reduced cytotoxic effects on the J774A.1 macrophage cell line. The enzyme inhibition study confirms that the glycoside derivatives have significant inhibitory activity against the leishmanial topoisomerase IB enzyme. Molecular docking further displayed the better binding efficiency of glycoside 13 with the target enzyme, suggesting the involvement of more H-bond interactions in the case of glycosides as compared to free drugs. Therefore, this work helps in proposing the fact that the addition of sugar moieties adds some favorable characteristics to free inhibitors, making it a promising approach for future clinical diagnostic and therapeutic applications, which can prove to be a valuable arsenal in combating such neglected diseases.

Synthetic beidellite clay as nanocarrier for delivery of antitumor oxindolimine-metal complexes

Studies on the immobilization of oxindolimine‑copper(II) or zinc(II) complexes [ML] in synthetic beidellite (BDL) clay were developed to obtain a suitable inorganic carrier capable of promoting the modified-release of metallopharmaceuticals. Previous investigations have shown that the studied metal complexes are promising antitumor agents, targeting DNA, mitochondria, and some proteins. They can bind to DNA, causing oxidative damage via formation of reactive oxygen species (ROS). In mitochondria they lead to a decrease in membrane potential, acting as decoupling agents, and therefore efficiently inducing apoptosis. Additionally, they inhibit human topoisomerase IB and cyclin dependent kinases, proteins involved in the cell cycle. BDL clays in the sodium form were synthesized under hydrothermal conditions and characterized by a set of physicochemical techniques while the BDL-[ML] hybrid materials were prepared by ion exchange method. The characterization of pristine clay and the obtained hybrids were performed by Infrared, Raman, electron paramagnetic resonance and energy dispersive X-ray spectroscopies, thermogravimetric analysis, scanning electron microscopy, X-ray powder diffraction, specific surface area, zeta potential and surface ionic charge measurements. The [ML] release assays under the same cell incubation conditions were performed monitoring metals by X-ray fluorescence. The BDL-[CuL] hybrid materials were stable and able to derail tumor HeLa cells, with corresponding IC50 values in the 0.11–0.41 mg mL−1 range. By contrast, the analogous hybrid samples of zinc(II) and the pristine BDL proved to be non-toxic facing the same cells. These results indicate a promising possibility of using synthetic beidellite as a carrier of such antitumor metal complexes.

Synthesis of 11-aminoalkoxy substituted benzophenanthridine derivatives as tyrosyl-DNA phosphodiesterase 1 inhibitors and their anticancer activity

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme that repairs DNA lesions caused by the trapping of DNA topoisomerase IB (TOP1)-DNA break-associated crosslinks. TDP1 inhibitors have synergistic effect with TOP1 inhibitors in cancer cells and can overcome cancer cell resistance to TOP1 inhibitors. Here, we report the synthesis of 11-aminoalkoxy substituted benzophenanthridine derivatives as selective TDP1 inhibitors and show that six compounds 14, 16, 18, 20, 25 and 27 exhibit high TDP1 inhibition potency. The most potent TDP1 inhibitor 14 (IC50 = 1.7 ± 0.24 μM) induces cellular TDP1cc formation and shows synergistic effect with topotecan in four human cancer cell lines MCF-7, A549, H460 and HepG2.

Design, Synthesis and Cytotoxicity of Thiazole-Based Stilbene Analogs as Novel DNA Topoisomerase IB Inhibitors

A series of new thiazole-based stilbene analogs were designed, synthesized and evaluated for DNA topoisomerase IB (Top1) inhibitory activity. Top1-mediated relaxation assays showed that the synthesized compounds possessed variable Top1 inhibitory activity. Among them, (E)-2-(3-methylstyryl)-4-(4-fluorophenyl)thiazole (8) acted as a potent Top1 inhibitor with high Top1 inhibition of ++++ which is comparable to that of CPT. A possible binding mode of compound 8 with Top1–DNA complex was further provided by molecular docking. An MTT assay against human breast cancer (MCF-7) and human colon cancer (HCT116) cell lines revealed that the majority of these compounds showed high cytotoxicity, with IC50 values at micromolar concentrations. Compounds 8 and (E)-2-(4-tert-butylstyryl)-4-(4-fluorophenyl)thiazole (11) exhibited the most potent cytotoxicity with IC50 values of 0.78 and 0.62 μM against MCF-7 and HCT116, respectively. Moreover, the preliminary structure–activity relationships of thiazole-based stilbene analogs was also discussed.

Three-Component Synthesis of 2-Amino-3-cyano-4H-chromenes, In Silico Analysis of Their Pharmacological Profile, and In Vitro Anticancer and Antifungal Testing.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a–o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a–h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a–o and 6a–h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a–o and 6a–h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo.

Ruthenium(II) complexes (Ru1-Ru5), with the general formula [Ru(N-S)(dppe)2]PF6, bearing two 1,2-bis(diphenylphosphino)ethane (dppe) ligands and a series of mercapto ligands (N-S), have been developed. The combination of these ligands in the complexes endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. For the A549 (lung) and MDA-MB-231 (breast) cancer cell lines, the IC50 values of the complexes were 288- to 14-fold lower when compared to cisplatin. Furthermore, the complexes were selective for the A549 and MDA-MB-231 cancer cell lines compared to the MRC-5 nontumor cell line. The multitarget character of the complexes was investigated by using calf thymus DNA (CT DNA), human serum albumin, and human topoisomerase IB (hTopIB). The complexes potently inhibited hTopIB. In particular, complex [Ru(dmp)(dppe)2]PF6 (Ru3), bearing the 4,6-diamino-2-mercaptopyrimidine (dmp) ligand, effectively inhibited hTopIB by acting on both the cleavage and religation steps of the catalytic cycle of this enzyme. Molecular docking showed that the Ru1-Ru5 complexes have binding affinity by active sites on the hTopI and hTopI-DNA, mainly via π-alkyl and alkyl hydrophobic interactions, as well as through hydrogen bonds. Complex Ru3 displayed significant antitumor activity against murine melanoma in mouse xenograph models, but this complex did not damage DNA, as revealed by Ames and micronucleus tests.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 μM; LRL-TP-94: 2.9 μM; and LRL-TP-101: 35.3 μM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 μM in extracellular promastigote assay and 34.0, 53.7, and 11.4 μM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Synthesis of Methoxy-, Methylenedioxy-, Hydroxy-, and Halo-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Inhibitors and Their Biological Activity for Drug-Resistant Cancer.

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or ++++), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 μM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.

Abundant Transmission of Corona Virus Nsp2 RNA Topoisomerse I120F Mutants with Concurrence D614G Spike Protein Mutation in Australia

We previously predicted Nsp2 Corona virus protein as RNA topoisomerase through amino acid homology among Vibrio haemolytica DNA topoisomerase IA/IV as well as DNA primase, DNA gyrase and bi-subunit Trypanosoma brucei DNA topoisomerase IB. Many DNA topoisomerase I/III have RNA topoisomerase activity and such ubiquitous enzymes are conserved and involved in the regulation of replication and transcription. We have checked here mutational profile of Nsp2 RNA topoisomerase analyzing >10000 orf 1a 4405 amino acid length Corona virus polyprotein. Mutant proteins were selected by BLAST search having 99.84% sequence similarity and 181- 818 aa portion NsP2 protein (protein id. QIU82057) was analyzed using CLUSTAL Omega software. We found 26 different mutations where most changes were selected at Isoleucine and Alanine into Valine or Leucine into Phenylanaline pinpointing conserved nature of the Corona virus RNA topoisomerase. Major nonsense very abundant mutations were found at I120F (Isoleucine to Phenylalanine). Other important mutations were R27C, I198V, T85I, L410F, I559V and P583S. The I120F mutation was abundant in Australian isolates and its spread was seen in the Bangladesh and other countries like USA. We suggest that abundant I120F mutation of Nsp2 Topoisomerase may increase transmission of Corona virus by stabilizing RNA structure for efficient virus packaging. Interestingly, such mutations were found in association of D614G mutation of Spike protein, known to >70% increase infectivity. On the contrary, all P583S Nsp2 mutants analyzed had no concurrence D614G spike protein mutation. Many silent mutations (5-7) were detected by genome wide analysis but no N501Y Spike protein mutation. This is first report that predicts a link of greater Corona virus transmission with Nsp2 protein I120F mutation and may be important to discover new anti-viral drugs.

Ruthenium(II) Phosphine/Mercapto Complexes: Their in Vitro Cytotoxicity Evaluation and Actions as Inhibitors of Topoisomerase and Proteasome Acting as Possible Triggers of Cell Death Induction.

In this paper, a series of new ruthenium complexes of the general formula [Ru(NS)(dpphpy)(dppb)]PF6 (Ru1-Ru3), where dpphpy = diphenyl-2-pyridylphosphine, NS ligands = 2-thiazoline-2-thiol (tzdt, Ru1), 2-mercaptopyrimidine (pySm, Ru2), and 4,6-diamino-2-mercaptopyrimidine (damp, Ru3), and dppb = 1,4-bis(diphenylphosphino)butane, were synthesized and characterized by elemental analysis, spectroscopic techniques (IR, UV/visible, and 1D and 2D NMR), and X-ray diffraction. In the characterization, the correlation between the phosphorus atoms and their respective aromatic hydrogen atoms of the compounds in the assignment stands outs, by 1H-31P HMBC experiments. The compounds show anticancer activities against A549 (lung) and MDA-MB-231 (breast) cancer cell lines, higher than the clinical drug cisplatin. All of the complexes are more cytotoxic against the cancer cell lines than against the MRC-5 (lung) and MCF-10A (breast) nontumorigenic human cell lines. For A549 tumor cells, cell cycle analysis upon treatment with Ru2 showed that it inhibits the mitotic phase because arrest was observed in the Sub-G1 phase. Additionally, the compound induces cell death by an apoptotic pathway in a dose-dependent manner, according to annexin V-PE assay. The multitargeted character of the compounds was investigated, and the biomolecules were DNA, topoisomerase IB, and proteasome, as well as the fundamental biomolecule in the pharmacokinetics of drugs, human serum albumin. The experimental results indicate that the complexes do not target DNA in the cells. At low concentrations, the compounds showed the ability to partially inhibit the catalytic activity of topoisomerase IB in the process of relaxation of the DNA plasmid. Among the complexes assayed in cultured cells, complex Ru3 was able to diminish the proteasomal chymotrypsin-like activity to a greater extent.

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans

Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was demonstrated earlier in vitro. Here, we report that D. radiodurans cells lacking drTopo IB (ΔtopoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters like mutL and recQ was reduced in the presence of NMM in mutant cells. In mutant, the percentage of anucleate cells was more while the copy number of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.

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.

Comparative metabolite profiling of Callistemon macropunctatus and Callistemon subulatus volatiles from different geographical origins

Since ancient times, essential oils from medicinal plants have supplied a huge repository of secondary metabolites. Genus Callistemon, Myrtaceae, was described to have nematicidal, antithrombotic, antistaphylococcal, antioxidant, and insecticidal activities. In this study we investigate the chemical composition of essential oils from the leaves and the flowers of C. macropunctatus (Dum.Cours.) Court and Callistemon subulatus (Cheel) from two geographical locations: Australia and Egypt. GCMS analysis identified a total of sixty-one volatile components and showed that the major constituents were 1,8-cineole, α-pinene, α-terpineol, limonene and α-phellandrene in the leaves and α-pinene, 1,8-cineole, α-phellandrene, limonene and α-terpineol in the flowers of Callistemon species. To discriminate the oil complexity within factors as location, plant parts and species, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. Furthermore, the cytotoxic effect of the oil samples was assayed against the human liver cancer cell line HepG2 to reveal a potent anticancer potential with half-maximal inhibitory concentration (IC50) values ranging from 1.76 μg/mL to 13.41 μg/mL. To gain insight into the cellular mechanism of the cytotoxic activity, Callistemon volatile oils’ activities on topoisomerases I-β and II-β were explored, which demonstrated significant enzyme inhibition with IC50 values as low as 819.6 ng/mL. Molecular in-silico docking study of the major oil components within the active binding site of the human topoisomerases I-β and II-β was conducted. α-terpineol showed promising alkyl interactions in the binding pockets of topoisomerase IB and IIB, with a docking Gibb’s free energy values of -27.0913 and -26.6478 kcal/mol, respectively. Whereas it bounded to Arg503 and four nucleotide regions deoxyadenosine DA12, deoxythymidine DT9, deoxyguanosine DG13, deoxycytidine DC8 in the topoisomerase II-β enzyme pocket, it interacted with Topo I-β enzyme through hydrophobic alkyl and Pi-alkyl binding interactions with DA12, DG13 and Arg503. Furthermore, the oxygen in the hydroxyl group showed two additional non-covalent bonds: a conventional hydrogen bond with Asp533 and water hydrogen bond with a water molecule in the vicinity of the binding surface. This research highlights the future possibility of having Callistemon subulatus as a potential natural drug for treatment of liver cancer.

Ruthenium(II)-mercapto Complexes with Anticancer Activity Interact with Topoisomerase IB

Herein we present four new ruthenium(II) complexes: [Ru(mtz)2(dppb)] (1), [Ru(mmi)2(dppb)] (2), [Ru(dmp)2(dppb)] (3), and [Ru(mpca)2(dppb)] (4), where mtz = 2-mercaptothiazoline; mmi = 2-mercapto-1-methyl-imidazole; dmp = 4,6-diamino-2-mercaptopyrimidine; mpca = 6-mercaptopyridine-3-carboxylic acid; dppb = 1,4-bis(diphenylphosphino)butane. In vitro cell culture experiments revealed cytotoxic activity for complexes 2, 3 and 4 against MCF-7 (breast, non-invasive), MDA-MB-231 (breast, invasive), A549 (lung), DU-145 (prostate) and HepG2 (liver) tumor cells, in some cases lower than the half maximal inhibitory concentration (IC50) for the reference drug (cisplatin). The deoxyribonucleic acid (DNA) interactions studied by viscosity measurements, gel electrophoresis and square-wave voltammetry indicated that the DNA binding affinity primarily occurs through non-covalent interactions. Only complex 2 was able to fully inhibit the DNA supercoiled relaxation mediated by human topoisomerase IB (Top IB). The analysis indicates that complex 2 inhibits the cleavage and the reconnection steps of the catalytic cycle, being both a poison and a catalytic inhibitor.

Antiparasitic effect of synthetic aromathecins on Leishmania infantum

BackgroundCanine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB – one of the enzymes controlling DNA topology – has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins.ResultsAromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter.ConclusionsAromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.

Antileishmanial activity of terpenylquinones on Leishmania infantum and their effects on Leishmania topoisomerase IB

Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggability.