Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies.

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

DNA preference of indenoisoquinolines: a computational approach.

The human topoisomerase IB (hTopoIB) enzyme is a monomeric protein that relaxes the supercoils on double-stranded DNA by forming a covalent DNA/hTopoIB complex by introducing a nick on the DNA strand. Inhibition of hTopoIB results in cell death, which makes this protein a strong target for the treatment of various cancer types, including small-cell lung cancers and ovarian cancers. Camptothecin (CPT) and indenoisoquinoline (IQN) classes of compounds inhibit the hTopoIB activity by intercalating to nicked DNA pairs; however, these inhibitors show different preferences towards DNA bases when bound to the DNA/hTopoIB complex. Here, we investigated the affinities of CPT and one IQN derivative towards different DNA base pairs. The two inhibitors showed different stacking behaviors in the intercalation site and interaction pattern with binding pocket residues, indicating that they have different inhibition mechanisms in the binding pocket that affects the base-pair selectivity. The results obtained from this study are expected to guide researchers in designing gene-specific and more potent compounds to fight cancer through hTopoIB poisoning.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Binding profile of a mixed-ligand silver(I) complex with DNA and Topoisomerase I.

A new mixed-ligand Ag(I) complex, [Ag(daf)(phen)]NO3 (daf = 4,5-diazafluoren-9-one and dian = N-(4,5-diazafluoren-9-ylidene)aniline), was synthesized. The elemental analysis, FTIR, 1HNMR, UV-Vis spectroscopy, cyclic voltammetry, and DFT (Density Functional Theory) geometry optimization method were applied in order to predict the Ag(I) complex structure which concluded to a distorted tetrahedral N4 coordination around the Ag(I) center. A detailed in silico analysis of the bioaffinity of the complex to DNA and human DNA-Topoisomerase I was conducted using molecular docking simulations and ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) techniques. In this overall scenario, the results suggest the dominance of π-π stacking interactions of the heteroaromatic ligands in the intercalating pocket of DNA and the active site of the enzyme and the rational correlation between being a good intercalator and a potent Topoisomerase I inhibitor. In vitro DNA-binding experiments by spectrophotometric, spectrofluorometric, Voltammetric, and viscometric techniques at physiological pH also confirmed the computational results. The complex inhibited MCF-7 cell growth in a dose-dependent manner while being nontoxic on HUVEC normal cells.

Evolution of TOP1 and TOP1MT Topoisomerases in Chordata.

Type IB topoisomerases relax the torsional stress associated with DNA metabolism in the nucleus and mitochondria and constitute important molecular targets of anticancer drugs. Vertebrates stand out among eukaryotes by having two Type IB topoisomerases acting specifically in the nucleus (TOP1) and mitochondria (TOP1MT). Despite their major importance, the origin and evolution of these paralogues remain unknown. Here, we examine the molecular evolutionary processes acting on both TOP1 and TOP1MT in Chordata, taking advantage of the increasing number of available genome sequences. We found that both TOP1 and TOP1MT evolved under strong purifying selection, as expected considering their essential biological functions. Critical active sites, including those associated with resistance to anticancer agents, were found particularly conserved. However, TOP1MT presented a higher rate of molecular evolution than TOP1, possibly related with its specialized activity on the mitochondrial genome and a less critical role in cells. We could place the duplication event that originated the TOP1 and TOP1MT paralogues early in the radiation of vertebrates, most likely associated with the first round of vertebrate tetraploidization (1R). Moreover, our data suggest that cyclostomes present a specialized mitochondrial Type IB topoisomerase. Interestingly, we identified two missense mutations replacing amino acids in the Linker region of TOP1MT in Neanderthals, which appears as a rare event when comparing the genome of both species. In conclusion, TOP1 and TOP1MT differ in their rates of evolution, and their evolutionary histories allowed us to better understand the evolution of chordates.

Holanamine, a Steroidal Alkaloid from the Bark of Holarrhena pubescens Wall. ex G. Don Inhibits the Growth of Leishmania donovani by Targeting DNA Topoisomerase 1B.

Leishmaniasis is a group of neglected tropical diseases (NTDs) caused by about 20 species of obligate intracellular protozoan parasites of the genus Leishmania, which occurs in cutaneous, mucocutaneous, and visceral forms. Many researchers have sought to utilize natural products for novel and effective treatments to combat many infectious diseases, including leishmaniasis. Holarrhena pubescens Wall. ex G. Don (Apocynaceae) bark is a rich source of bioactive steroidal alkaloids. The total alkaloidal extract (IC50 6.12 ± 0.117 µg/mL), and the isolated alkaloid, holanamine, showed significant antileishmanial activity (IC50 2.66 ± 0.112 µM against AG83 and 3.80 ± 0.126 µM against BHU-575) against the Leishmania donovani parasite, better than miltefosine (IC50 19.61 ± 0.093 µM against AG83 and 23.20 ± 0.094 µM against BHU-575). Holanamine inhibited the L. donovani topoisomerase 1B (LdToP1B) in a non-competitive manner (IC50 2.81 ± 0.105 µM), indicating that it interacts with the free enzyme and enzyme-DNA complex without inhibiting human topoisomerase. Hydrogen bonding and hydrophobic interactions of holanamine with the N-terminal and hinge region of the large subunit of LTop1B is responsible for its potent antileishmanial activity, as shown by docking studies. Treatment with holanamine causes apoptotic-like cell death by generating cellular and mitochondrial reactive oxygen species, disrupting the mitochondrial membrane potential and inducing ultrastructural alterations in the promastigotes. Holanamine effectively clears intracellular amastigotes but minimally affects host macrophages with no significant cytotoxicity in HEK 293 and L929 cell lines. Thus, our studies show that holanamine can further be used to develop effective antileishmanial agents against evolving drug-resistant parasites.

A Yeast-Based Screening System for Differential Identification of Poisons and Suppressors of Human Topoisomerase I.

BACKGROUND: Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern)-induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1-DNA covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poisons developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. METHODS: In this study we set up a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. The effect of test compounds on growth is monitored with and without overexpression of the recombinant human TOP1. RESULTS: This yeast-based screening system can identify human TOP1 poisons for anticancer efficacy as well as TOP1 suppressors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. CONCLUSIONS: This yeast-based screening system can distinguish between TOP1 suppressors and TOP1 poisons. The assay can also identify compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

Discovery of novel rigid analogs of 2-naphthol with potent anticancer activity through multi-target topoisomerase I & II and tyrosine kinase receptor EGFR & VEGFR-2 inhibition mechanism.

A novel oxygen-containing heterocyclic linked 1H-benzo[f]chromene moieties (4a-g) and (6a-g) with anti-proliferative activity against cancer cell lines was designed, synthesized, and established on the basis of spectral data. All the prepared compounds were evaluated in vitro for their anti-proliferative activity against MCF-7, HCT-116, HepG-2 cell lines and normal cell lines HFL-1, WI-38. Compounds 4a, 4b, and 6e exhibited good activity against MCF-7, HCT-116, and HepG-2 cell lines, comparable to that of Vinblastine and Doxorubicin, and weak active against normal cell lines. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 4a, 4b, and 6e on the more sensitive cell line MCF-7 were studied. We found that compounds 4a, 4b, and 6e induce cell cycle arrest at G2/M phases for MCF-7 treated cells compared to untreated cells, which causes apoptosis and inhibits both the topoisomerase I and II enzymes. In addition, compounds 4a and 4b exhibited comparable inhibitory activity on tyrosine kinase receptors EGFR and VEGFR-2 kinases to that of the reference protein kinases inhibitor Sorafenib. The in silico molecular docking of the most active compounds into the active sites of EGFR kinase and Topo I & II enzymes provides us with a reasonable clarification of the interpreted biological data.

Higher detection of JC polyomavirus in colorectal cancerous tissue after pretreatment with topoisomerase I enzyme; colorectal tissue serves as a JCPyV persistence site.

BACKGROUND: The JC polyomavirus has been blamed to contribute in colorectal cancer (CRC), however, the topic is still controversial. Varying detection rate of JCPyV genome has been reported mainly due to technical reasons. Here, we provide summative data on the topic, with emphasize on technical issues. METHODS: Formalin-fixed paraffin-embedded tissue samples from 50 patients with CRC, consisting of tumoral and non-cancerous marginal tissue (totally 100 samples) were included in the study. After DNA extraction, specific JCPyV T-Ag sequences were targeted using Real-time PCR. To unwind the supercoiled JCPyV genome, pretreatment with topoisomerase I, was applied. Immunohistochemical (IHC) staining was performed using an anti-T-Ag monoclonal antibody. RESULTS: In the first attempts, no samples were found to be positive in Real-time PCR assays. However, JCPyV sequences were found in 60% of CRC tissues and 38% of non-cancerous colorectal mucosa after application of pre-treatment step with topoisomerase I enzyme (P = 0.028). T-Ag protein was found in the nuclear compartment of the stained cells in IHC assays. CONCLUSIONS: The presence of JCPyV in CRC tissues, as well as T-Ag localization in the nucleolus, where its oncogenic effect takes place, may provide supporting evidence for JCPyV involvement in CRC development. The study highlights the importance of using topoisomerase I to enhance JCPyV genome detection. Also, colorectal tissue is one of the permissive human tissue for JC resistance after preliminary infection.

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.

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

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.

New Dibenzoquinoxalines Inhibit Triple-Negative Breast Cancer Growth by Dual Targeting of Topoisomerase 1 and the c-MYC G-Quadruplex.

As c-MYC is one of the central players in triple-negative breast cancer (TNBC) oncogenesis, inhibiting c-MYC expression would be an effective anticancer strategy. Transcription-induced negative supercoiling is crucial in the regulation of c-MYC transcription, which facilitates the formation of a G4 structure in NHE III1 that can silence the transcription. However, topoisomerase 1 (Topo1) can dissipate this negative supercoiling, leading to continuous activation of c-MYC transcription. Thus, dual ligands targeting both Topo1 and c-MYC G4 appear to be significant in cancer therapy. In this study, a series of new dibenzoquinoxaline derivatives were designed, synthesized, and evaluated for both Topo1 and c-MYC inhibition. Among them, 5 was identified as the most promising dual ligand, which could effectively inhibit Topo1 activity and strongly stabilize c-MYC G4, thereby inhibiting cancer cell growth. Accordingly, this work suggests that this dual-targeting strategy may be effective in cancer therapy.

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.

In-silico Studies and Wet-Lab Validation of Camptothecin Derivatives for Anti-Cancer Activity Against Liver (HepG2) and Lung (A549) Cancer Cell Lines.

BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.

Copper(I)-Catalyzed Nitrile-Addition/N-Arylation Ring-Closure Cascade: Synthesis of 5,11-Dihydro-6H-indolo[3,2-c]quinolin-6-ones as Potent Topoisomerase-I Inhibitors.

In this paper, we present a copper(I)-catalyzed nitrile-addition/N-arylation ring-closure cascade for the synthesis of 5,11-dihydro-6H-indolo[3,2-c]quinolin-6-ones from 2-(2-bromophenyl)-N-(2-cyanophenyl)acetamides. Using CuBr and t-BuONa in dimethylformamide (DMF) as the optimal reaction conditions, the cascade reaction gave the target products, in high yields, with a good substrate scope. Application of the cascade reaction was demonstrated on the concise total syntheses of alkaloid isocryptolepine. Further optimization of the products from the cascade reaction led to 3-chloro-5,12-bis[2-(dimethylamino)ethyl]-5,12-dihydro-6H-[1,3]dioxolo[4',5':5,6]indolo[3,2-c]quinolin-6-one (2k), which exhibited the characteristic DNA topoisomerase-I inhibitory mechanism of action with potent in vitro anticancer activity. Compound 2k actively inhibited ARC-111- and SN-38-resistant HCT-116 cells and showed in vivo activity in mice bearing human HCT-116 and SJCRH30 xenografts. The interaction of 2k with the Top-DNA cleavable complex was revealed by docking simulations to guide the future optimization of 5,11-dihydro-6H-indolo[3,2-c]quinolin-6-ones as topoisomerase-I inhibitors.

A NMR study of binding the metabolite of SN38 derivatives to a model nicked DNA decamer mimicking target of Topo I inhibitors.

In this account we present NMR based results of the interaction of 7-ethyl-9-hydroxymethyl-10-hydroxycamptothecin (1), a derivative of SN38, with a model nicked DNA decamer mimicking the wild type DNA target of Topoisomerase I inhibitors from the camptothecin family. The title compound 1 can be considered a main metabolite of phase I in the metabolic pathway of camptothecin derivatives bearing the alkylamino substituent. Therefore, its pharmacodynamic properties are of interest. It was established by DOSY (Diffusion Ordered Spectroscopy) that compound 1 forms a fairly stable molecular complex with a model nicked DNA decamer with affinity constant Ka 3.02 mM-1. The analysis of NOESY experiments revealed intermolecular cross peaks and mutual induced shifts on both interacting components allowing the conclusion that guest molecule 1 is stacking the nitrogen bases inside the nick. MD (Molecular Dynamics) analysis of four possible inclusions of 1 inside the nick allows establishing the detailed geometry of a complex. Two conformations are suggested as the ones best representing the results of molecular modeling reconciled with experimental NOESY results. The aromatic core of both structures is stacking the nitrogen bases in a nick facing the unbroken strand with ring A. The protons in ring E interact with ribose protons of edge bases of a nick. In conclusion, it can be asserted that SN38 derivative 1 can effectively bind the molecular target of Topo I enzyme and play a role as a Topo I inhibitor.

Design, synthesis and biological evaluation of water soluble and non-aggregated silicon phthalocyanines, naphthalocyanines against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines as potential anticancer agents.

Cancer has become an important public problem in worldwide since cancer incidence and mortality are growing rapidly. In this study, water soluble and non-aggregated silicon (IV) phthalocyanines and naphthalocyanines containing (3,5-bis{3-[3-(diethylamino)phenoxy]propoxy}phenyl)methoxy groups have been synthesized and characterized to investigate their anticancer potential. Their DNA binding/nuclease, topoisomerases inhibition were investigated using UV-Vis absorption, thermal denaturation and agarose gel electrophoresis. The in vitro cytotoxic properties of the compounds on human lung (A549), breast (BT-20), liver (SNU-398), prostate (DU-145), melanoma (SK-Mel 128) carcinoma and human fibroblast (HFC) normal cell lines were evaluated by using MTT assay. In order to determine the mechanism of cancer cell growth suppression, cell cycle analysis was carried out using flow cytometer on A549 cell line. The Kb values of SiPc1a and SiNc2a were 6.85 ± (0.35) × 106 and 1.72 ± (0.16) × 104 M-1 and Tm values of ct-DNA were calculated as 82.02 °C and 78.07 °C, respectively in the presence of both compounds. The ΔTm values of SiPc1a and SiNc2a were calculated as 6.45 and 2.50 °C, respectively. The nuclease effects of SiPc1a and SiNc2a with supercoiled plasmid pBR322 DNA demonstrated that both compounds did not trigger any DNA nuclease effects at the lowest concentrations without irradiation whereas both compounds in the presence of activating agent (H2O2) showed significant plasmid DNA nuclease actions under irradiation (22.5 J/cm2). SiPc1a and SiNc2a inhibited to topoisomerase I on increasing concentrations whilst they had lower inhibition action toward topoisomerase II that of topoisomerase I. The in vitro cytotoxicity studies displayed that SiPc1a had the highest cytotoxic effects among the tested compounds against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines with CC50 values ranged from 0.49 to 2.99 µM. Furthermore, SiPc1a inhibited cell proliferation by cell cycle arrest in G0/G1 phase. All of these results suggested that SiPc1a is a promising candidate as an anticancer agent.

Novel urea linked ciprofloxacin-chalcone hybrids having antiproliferative topoisomerases I/II inhibitory activities and caspases-mediated apoptosis.

A novel series of urea-linked ciprofloxacin (CP)-chalcone hybrids 3a-j were synthesized and screened by NCI-60 cancer cell lines as potential cytotoxic agents. Interestingly, compounds 3c and 3j showed remarkable antiproliferative activities against both colon HCT-116 and leukemia SR cancer cells compared to camptothecin, topotecan and staurosporine with IC50 = 2.53, 2.01, 17.36, 12.23 and 3.1 µM for HCT-116 cells, respectively and IC50 = 0.73, 0.64, 3.32, 13.72 and 1.17 µM for leukemia SR cells, respectively. Also, compounds 3c and 3j exhibited inhibitory activities against Topoisomerase (Topo) I with % inhibition = 51.19% and 56.72%, respectively, compared to camptothecin (% inhibition = 60.05%) and Topo IIß with % inhibition = 60.81% and 60.06%, respectively, compared to topotecan (% inhibition = 71.09%). Furthermore, compound 3j arrested the cell cycle of leukemia SR cells at G2/M phase. It induced apoptosis both intrinsically and extrinsically via activation of proteolytic caspases cascade (caspases-3, -8, and -9), release of cytochrome C from mitochondria, upregulation of proapoptotic Bax and down-regulation of Bcl-2 protein level. Thus, the new ciprofloxacin derivative 3j could be considered as a potential lead for further optimization of antitumor agent against leukemia and colorectal carcinoma.