Induced DNA Cleavage Research Articles

A Comparison of In Vitro Studies between Cobalt(III) and Copper(II) Complexes with Thiosemicarbazone Ligands to Treat Triple Negative Breast Cancer

Metal complexes have gained significant attention as potential anti-cancer agents. The anti-cancer activity of [Co(phen)2(MeATSC)](NO3)3•1.5H2O•C2H5OH 1 (where phen = 1,10-phenanthroline and MeATSC = 9-anthraldehyde-N(4)-methylthiosemicarbazone) and [Cu(acetylethTSC)Cl]Cl•0.25C2H5OH 2 (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide) was investigated by analyzing DNA cleavage activity. The cytotoxic effect was analyzed using CCK-8 viability assay. The activities of caspase 3/7, 9, and 1, reactive oxygen species (ROS) production, cell cycle arrest, and mitochondrial function were further analyzed to study the cell death mechanisms. Complex 2 induced a significant increase in nicked DNA. The IC50 values of complex 1 were 17.59 μM and 61.26 μM in cancer and non-cancer cells, respectively. The IC50 values of complex 2 were 5.63 and 12.19 μM for cancer and non-cancer cells, respectively. Complex 1 induced an increase in ROS levels, mitochondrial dysfunction, and activated caspases 3/7, 9, and 1, which indicated the induction of intrinsic apoptotic pathway and pyroptosis. Complex 2 induced cell cycle arrest in the S phase, ROS generation, and caspase 3/7 activation. Thus, complex 1 induced cell death in the breast cancer cell line via activation of oxidative stress which induced apoptosis and pyroptosis while complex 2 induced cell cycle arrest through the induction of DNA cleavage.

Synthesis and DNA Cleavage Activity of Cationic Methylene-Blue-Backboned Polymers.

Methods of DNA cleavage have broad bioapplications in gene editing, disease treatment, and biosensor design. The traditional method for DNA cleavage is mainly through oxidation or hydrolysis mediated by small molecules or transition metal complexes. However, DNA cleavage by artificial nucleases using organic polymers has been rarely reported. Methylene blue has been extensively studied in the fields of biomedicine and biosensing due to its excellent singlet oxygen yield, redox properties, and good DNA affinity. Methylene blue mainly relies on light and oxygen for DNA cleavage, and the cutting rate is slow. Here, we synthesize cationic methylene-blue-backboned polymers (MBPs) that can bind DNA efficiently and induce DNA cleavage through free radical mechanisms in the absence of light and exogenous reagents, showing high-efficiency nuclease activity. In addition, MBPs with different structures showed selectivity for DNA cleavage, and the cleavage efficiency of the flexible structure was significantly higher than that of the rigid structure. Studies on the DNA cleavage mechanism have shown that the cleavage mechanism of MBPs is not through the common ROS-mediated oxidative cleavage pathway, but through the radical of MBP• inducing DNA cleavage. Meanwhile, MBPs can simulate topoisomerase I (Topo I)-mediated topological rearrangement of superhelical DNA. This work paved a way for the application of MBPs in the field of artificial nucleases.

Flavokavain C Suppresses Breast Cancer Cell Viability and Induces Cell Apoptosis by Triggering DNA Damage.

Breast cancer, presented by multiple breast cancer subtypes that coexist within a diagnosed tumor in clinical, has ranked as the most common malignancy in women in recent years. Evidence suggested that limited effective drugs caused the unsatisfactory therapeutic efficacy of breast cancer. Flavokavain C exhibited anticancer activity on colon cancer cells HCT116. It is yet unknown if it can be used to treat breast cancer. This study aims to believe the mechanisms by which Flavokavain C suppresses cell proliferation and the pathways that impact on this effect in breast cancer. 3-(4,5-Dimethythiazol)-2,5-diphenyltetrazolium bromide assay was chosen to evaluate cell cytotoxicity. Colony formation and cell proliferation assays using 5-ethynyl-2'-deoxyuridine staining were performed. Cell cycle progression and apoptosis were examined via flow cytometry and Western blotting, respectively. Five methods (comet assay, immunofluorescence, Western blotting, agarose gel electrophoresis and molecular docking) were used to quantify DNA damage and its cellular response. Compared to cisplatin, Flavokavain C possessed a comparable or more substantial inhibitory effect on breast cancer cell viability while having lower cytotoxicity on human mammary cells. Breast cancer cells treated with Flavokavain C had their colony formation suppressed, DNA replication blocked, the G2/M phase cell cycle arrested, and apoptosis. Furthermore, the results indicated that Flavokavain C would directly interact with DNA and induce DNA cleavage, demonstrating that DNA is an attractive substrate for Flavokavain C. These results suggested that Flavokavain C had strong anticancer activity against multiple subtypes of breast cancer cells.

Novel Azapodophyllotoxin Induces DNA Cleavage via Groove Binding: An Advancement in Exploration of Anticancer Activity of a Known Inhibitor of Tubulin Polymerization and Topoisomerase

AbstractThe present study embodies exploration of new potential targets for bioactive azapodophyllotoxins (AZP) that have been mainly considered as inhibitor of tubulin polymerization and topoisomerases. The interaction of a novel AZP, HTDQ, with potential target DNA (calf thymus DNA) has been investigated alongwith its mechanism of action by using combined biophysical spectroscopic techniques and in silico approach. The steady state absorption and fluorescence studies highlights binding interaction of HTDQ with the nucleic acid and the value of Stern Volmer quenching constant (8.07×103 L mol−1) hints towards minor groove binding interaction. The KI quenching studies, helix melting and viscosity measurements further supports our speculation and is substantiated by fluorescence displacement assay using standard minor groove binder, Hoechst 33258. The conformational alterations due to HTDQ‐ctDNA binding is evident from circular dichroism studies and have been further validated from the in silico molecular docking and molecular dynamics simulation studies. Additionally, with a view to get thorough understanding of the mechanism, the DNA cleavage studies have been performed using gel electrophoresis that proved instrumental in determining the ability of HTDQ to damage the structural homogeneity of the nucleic acid.

The Delivery of ABE mRNA to the Adult Murine Liver by Lipid Nanoparticles (LNPs).

A genetic disorder is a disease caused by an abnormal DNA sequence, and almost half of the known pathogenic monogenetic mutations are caused by G-to-A mutation (Landrum et al., Nucleic Acids Res 44:D862-868, 2016). Adenine base editors (ABE), developed from the CRISPR, hold the great promise to mediate the A-to-G transition in genomic DNA while not inducing DNA cleavage (Gaudelli et al., Nature 551:464-471, 2017). Additionally, lipid nanoparticles (LNPs), as a non-viral delivery, are able to deliver the ABE mRNAs and gRNA to the target tissues (Newby and Liu, Mol Ther 29:3107-3124, 2021). This chapter mainly introduces the production and LNP delivery of ABE mRNA and gRNA.

Protective Effect of Indomethacin-loaded Polymeric Nanoparticles Against Oxidative Stress-Induced Cytotoxicity in Human Breast Adenocarcinoma Cell Model

Introduction: Anti-inflammatory drugs are being utilized to treat cancer because of its inflammatory microenvironment. Objective: The objective of this study is to investigate the antioxidant potential of indomethacin and its genotoxicity, since free or loaded in polymeric nanocapsules using MCF-7 (human breast cancer) cells as an in vitro model. Method: Development of indomethacin-loaded polyepsiloncaprolactone (PCL) nanocapsules by interfacial deposition method. It is characterized by pH determination by potentiometer, mean diameter and polydispersity index by dynamic light scattering; zeta potential by electrophoretic mobility; encapsulation efficacy by high performance liquid chromatography method; corona effect formation; 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) method by spectrofluorimetric assay; nitric oxide (NO) determination by spectrophotometric and genotoxicity assay by plasmid DNA cleavage method. Results: The results showed a mild acidic pH (4.78 ± 0.10), sizes around 200 nm and PDI<0.2 with a zeta potential around -20 mV and encapsulation efficiency of 99% (1 mg mL-1), showing a dose-dependent corona formation profile in 24h incubation. Conclusion: DCFH-DA assay showed no production of reactive oxygen species (ROS) while NO determination showed that Ind-OH-NC from 26.7 to 100 μM increased reactive nitrogen species (RNS), demonstrating antioxidant potential against MCF-7 cells. No sample at the concentrations evaluated induced DNA cleavage, being considered a safe treatment.

Copper-mediated DNA damage caused by purpurin, a natural anthraquinone

BackgroundPurpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment, has historically been used as a textile dye. However, purpurin induced urinary bladder tumors in rats, and displayed a mutagenic activity in assay using bacteria and mammalian cells. Many carcinogenic dyes are known to induce bladder cancers via DNA adduct formation, but carcinogenic mechanisms of purpurin remain unknown. In this study, to clarify the mechanism underlying carcinogenicity of purpurin, copper-mediated DNA damage induced by purpurin was examined using 32P-labeled DNA fragments of human genes relevant to cancer. Furthermore, we also measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA.ResultsPurpurin plus Cu(II) cleaved 32P-labeled DNA fragments only under piperidine treatment, indicating that purpurin caused base modification, but not breakage of the DNA backbone. In the absence of Cu(II), purpurin did not induce DNA cleavage even with piperidine treatment. Purpurin plus Cu(II) caused piperidine-labile sites predominantly at G and some T residues. Bathocuproine, a Cu(I) chelator, completely prevented the occurrence of piperidine-labile sites, indicating a critical role of Cu(I) in piperidine-labile sites induced by purpurin plus Cu(II). On the other hand, methional, a scavenger of a variety of reactive oxygen species (ROS) and catalase showed limited inhibitory effects on the induction of piperidine-labile sites, suggesting that ROS could not be major mediators of the purpurin-induced DNA damage. Considering reported DNA adduct formation by quinone metabolites of several carcinogenic agents, quinone form of purpurin, which is possibly generated via purpurin autoxidation accompanied by Cu(I)/Cu(II) redox cycle, might lead to DNA adducts and piperidine-labile sites. In addition, we measured contents of 8-oxodG. Purpurin moderately but significantly increased 8-oxodG in calf thymus DNA in the presence of Cu(II). The 8-oxodG formation was inhibited by catalase, methional and bathocuproine, suggesting that Cu(I)-hydroperoxide, which was generated via Cu(I) and H2O2, caused oxidative DNA base damage.ConclusionsWe demonstrated that purpurin induces DNA base damage possibly mediated by Cu(I)/Cu(II) redox cycle both with and without ROS generation, which are likely to play an important role in its carcinogenicity.

Synthesis and biological evaluations of novel pyrazinoic acid derivatives as anticancer agents

Pyrazinoic acid or pyrazine-2-carboxylic acid (PA), due to its nitrogenous heteroaromatic ring, can be explored as an anticancer agent. Here, a series of twenty novels PA derivatives have been synthesized and characterized using IR, NMR, and mass spectrums. Their cytotoxic activity was evaluated against three different cancer cell lines, including lung (A549), breast (MCF-7), and colon (HT-29). P16, the most potent compound, showed moderate cytotoxicity with IC50 of 6.11, 10.64, and 14.92 μM, against the A549, MCF-7, and HT-29 cell lines, respectively. Furthermore, the effect of this compound against MRC5 as a non-tumoral lung cell line, exhibited a selectivity index of 9.02. The apoptotic induction activity of P16 was also performed on the A549 cell line. The results showed that as the concentration of the compound increases (from 3 to 6 μM), the percentage of induction of apoptosis increases from 8.54% to 72.4%. Electrophoretic gel mobility shift assays showed that P16 was able to reactive oxygen species (ROS) induce DNA cleavage in the presents of H2O2 (1.0 mM) in high doses. Molecular docking was also applied to anticipate the binding locations and the binding of the synthesized compound with Bcl-2 apoptosis regulator and DNA as their proposed targets.

Zinc organocomplexes containing non-steroid anti-inflammatories and plane aromatic diimines: New potential drugs

Strategies have been developing to obtain new medicines. Bioisosterism represents an approach used by medicinal chemists for the rational modification of lead compounds into safer and more clinically effective agents. Evidently, the synthesis of organocomplexes has been an important strategy in the planning of new drugs, and there are examples of great improvements in therapeutic efficacy. Thus, this work is aimed at the synthesis of new zinc complexes with nonsteroidal anti-inflammatory drugs (NSAIDs), the chemical characterization and the previous toxicity by cytotoxicity, and an evaluation of the ability of these compounds to interact with DNA. As a result, four new zinc II ternary complexes containing the NSAIDs diclofenac (Diclof) and ibuprofen (Ibup) and a zinc neutral linker were obtained by the two-step solvent metal–ligand complexation method (1. Zn-Ibup-Bipy, 2. Zn-Ibup-Phen, 3. Zn-Diclof-Bipy, 4. Zn-Diclof-Phen). Molecular structures were determined by NMR, FTIR, and HR-MS which demonstrated that the complexes are binuclear systems of the general formula [Zn(RCOO−)2N-binder]. In silico studies were performed for toxicity and physicochemical properties and biological target prediction. Cytotoxicity was determined by the MTT assay. The results indicated a relative absence of toxicity for these organometallic zinc derivatives. It was also observed that the compounds Zn-Diclof-Bipy and Zn-Diclof-Phen showed high potential without theoretical toxic effects, and they should be submitted to studies for evaluation of biological activity. Plasmidi DNA breakdown capacities were evaluated by producing single and double breaks (DNA FII and FIII) from plasmid incubation with complex solutions in the concentration range of 0 to 400 μmol·L−1 in experiments with the presence and absence of light. Both experiments did not show significant differences (P ≤ 0.05) in induced DNA cleavage activity between the maximum study concentrations (400 μmol·L−1) and the negative controls for both complexes. The types of complex 1 and 2 interactions with the secondary DNA structure were determined by titrating a CT-DNA solution with complex solutions and monitoring by circular dichroism spectrometry. Complex 1 was not cytotoxic at the concentrations tested against the Vero cell line. The results showed that both complexes interact with the grooves of the secondary structure of CT-DNA by electrostatic attraction but without evidence of alteration in the primary structure. Thus, four new compounds were synthesized and characterized, and their previous toxicities were determined. These compounds are promising new drugs, and the next step is to evaluate their activity.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Synthesis, characterization, electrochemistry, biological and molecular docking studies of the novel Co(II), Ni(II) and Cu(II) complexes derived from methanethiol bridged (2-((1H-benzo[d]imidazol-2-yl)methylthio)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone

The bio-active Co(II), Ni(II) and Cu(II) complexes derived from new (2-((1H-benzo[d]imidazol-2-yl)methylthio)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone were synthesized in good yield. The structure of these metal complexes have been characterized using IR, Uv–visible, mass spectra,1H NMR, TGA and powder XRD techniques. The spectral data of the Co(II) and Ni(II) complex indicated the tri-dentate bonding mode with the ligand and that of the Cu(II) complex is in bi-dentate bonding mode. The electrocatalytic activity of the Co(II) complex was studied by cyclic voltammeter using modified GCE towards various concentration of dopamine (DA, 3,4-dihydroxyphenethylamine). The obtained Ip value linearly increased with increasing scan rates, indicating the oxidation process of dopamine (DA) which occurred by modified Co(II) complex/GCE through diffusion controlled process. The antimicrobial, antioxidant and cytotoxicity studies of the ligand and its metal complexes were performed, the obtained results showed that the complexes are more potent bio-active as compared with uncoordinated ligand. The DNA binding property of the metal complexes were performed on double strand calf thymus DNA using electronic absorption and fluorescence emission methods, in addition, photo induced DNA cleavage activity also studied on pBR322 DNA. The molecular docking of the Co(II) complex on 6-bp DNA (PDB code: 1Z3F) and C-KIT Tyrosive kinases (TRKs) (PDB code: 1t46), further confirms the inhibition ability of the metal complex. The experimental results and the drug likeness property of the metal complexes suggest the possible applications of metal complex which, could be developed as a potent drug in the near future.

Synthesis, chemical characterization and DNA interaction study of new diclofenac and ibuprofen zinc (II)-nicotinamide ternary complexes as cyclooxygenase inhibitor prototypes

In the search for new drugs, strategies such as bioisosterism have been evidenced, in which the modification of molecules is already known to be active. Thus, metal complexes of known drugs have been highlighted, with examples of significant improvements in therapeutic efficacy. In this way, this work aimed at the synthesis of new zinc complexes with nonsteroidal anti-inflammatory drugs (NSAIDs), as well as the chemical characterization and the previous toxicity by cytotoxicity with Artemia salina, and evaluating the ability of these compounds to interact with DNA. As result, two new zinc II ternary complexes containing the NSAIDs diclofenac (Diclof) and ibuprofen (Ibup) and nicotinamide neutral linker (Nic) were obtained by the two-step solvent metal-ligand complexation method. Molecular structures were determined by NMR, FTIR, HR-MS, UV–Vis and X-ray diffraction, which demonstrated that both complexes are binuclear systems of general formula [Zn2(R-COO−)4(Nic)2]. Plasmidial DNA breakdown capacities were evaluated by producing single and double breaks (DNA FII and FIII) from plasmid incubation with complex solutions in the concentration range 0 to 400 μmol·L−1 in experiments with the presence and absence of light. Both experiments did not show significant differences (P ≤ 0.05) in induced DNA cleavage activity between the maximum study concentrations (400 μmol·L−1) and the negative controls for both complexes. The types of complex 1 and 2 interactions with the secondary DNA structure were determined by titrating a CT-DNA solution with complex solutions and monitored by circular dichroism spectrometry. The results showed that both complexes interact with the grooves of the secondary structure of CT-DNA by electrostatic attraction, but without evidence of alteration in the primary structure. Acute toxicity tests against Artemia salina showed that both complexes did not produce lethality >10% of the population up to a maximum concentration of 1200 μg·mL−1 within an exposure interval of 24 h. Thus, two new compounds were synthesized, characterized and had their previous toxicities determined. These compounds are promising new drugs, with the next step being evaluations of their activity.

Smoothed Potential MD Simulations for Dissociation Kinetics of Etoposide To Unravel Isoform Specificity in Targeting Human Topoisomerase II.

Human type II topoisomerases (TopoII) are essential for controlling DNA topology within the cell. For this reason, there are a number of TopoII-targeted anticancer drugs that act by inducing DNA cleavage mediated by both TopoII isoforms (TopoIIα and TopoIIβ) in cells. However, recent studies suggest that specific poisoning of TopoIIα may be a safer strategy for treating cancer. This is because poisoning of TopoIIβ appears to be linked to the generation of secondary leukemia in patients. We recently reported that enzyme-mediated DNA cleavage complexes (in which TopoII is covalently linked to the cleaved DNA during catalysis) formed in the presence of the anticancer drug etoposide persisted approximately 3-fold longer with TopoIIα than TopoIIβ. Notably, enhanced drug-target residence time may reduce the adverse effects of specific TopoIIα poisons. However, it is still not clear how to design drugs that are specific for the α isoform. In this study, we report the results of classical molecular dynamics (MD) simulations to comparatively analyze the molecular interactions formed within the TopoII/DNA/etoposide complex with both isoforms. We also used smoothed potential MD to estimate etoposide dissociation kinetics from the two isoform complexes. These extensive classical and enhanced sampling simulations revealed stabilizing interactions of etoposide with two serine residues (Ser763 and Ser800) in TopoIIα. These interactions are missing in TopoIIβ, where both amino acids are alanine residues. This may explain the greater persistence of etoposide-stabilized cleavage complexes formed with Topo TopoIIα. These findings could be useful for the rational design of specific TopoIIα poisons.

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed atexploringan alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproductformation and may pave a new avenue to E. coli engineering.

DNA binding, prominent photonuclease activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers

The chemistry of Co(II) complexes showing efficient light induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial PDT is summarized in this article. Complexes of formulation [Co(mqt)(B)2]ClO4 1–3 where mqt is 4-methylquinoline-2-thiol and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz 3) have been prepared and characterized. The DNA-binding behaviors of these three complexes were explored by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes 1, 2 and 3 were determined to be 1.6 × 103 M−1, 1.1 × 104 M−1 and 6.4 × 104 M−1 respectively. The experimental results suggest that these complexes interact with DNA through groove binding mode. The complexes show significant photocleavage of supercoiled (SC) DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against E. coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.

Oxyresveratrol-induced DNA cleavage triggers apoptotic response in Candida albicans.

Oxyresveratrol is a naturally occurring phytoalexin produced by plants in response to infection. Biological activities of oxyresveratrol have been studied such as antioxidant, anticancer and anti-inflammation. However, further antimicrobial activity and its mechanism need to be investigated. This study exhibited growth inhibition against pathogenic fungi and investigated its mode of action. Oxyresveratrol inflicted cleavage on DNA, leading to G2/M phase arrest. DNA damage by oxyresveratrol was not the result of oxidative stress but it was triggered by direct binding to DNA. Oxyresveratrol-treated cells showed an apoptotic pathway characterized by phosphatidylserine exposure, apoptotic volume decrease and metacaspase activation. Mitochondria-associated apoptotic features also appeared. Oxyresveratrol-induced Ca2+ overload led to mitochondrial membrane depolarization and release of cytochrome c from mitochondria to cytosol. In conclusion, oxyresveratrol with DNA-binding affinity induces DNA cleavage, and eventually leads to mitochondria-mediated apoptosis in Candida albicans.

A Multifunctional Biomimicking Oxidovanadium(V) Complex: Synthesis, DFT Calculations, Bromo‐peroxidation and DNA Nuclease Activities

AbstractAn oxidovanadium(V) complex [VO(SGly)(bpy)]ClO4 (H2SGly=N‐(2‐hydroxybenzyl)‐L‐glycine; bpy=2,2′‐bipyridine) was synthesized and characterized by spectroscopic studies (IR, UV‐vis, ESI‐MS). The theoretical structure of the complex was optimized by density functional theory (DFT) calculations. The multifunctionality of this oxidovanadium complex was demonstrated by the application of it as a catalyst in peroxidative bromination of single‐pot conversion of phenol red to bromophenol blue, whereas its nuclease activity was established by DNA cleavage. The interaction of this oxidovanadium complex with DNA was studied by various physicochemical tools like UV‐vis, fluorescence, and viscosity measurement studies. The intrinsic binding constant of the complex with DNA was determined spectrophotometrically and calculated to be (2.3 ± 0.02) x 104 M−1. The spectroscopic studies and the viscosity measurements indicate that the complex binds to the groove of CT‐DNA, but whether to the major or minor groove that was not established. The extent of vanadium complex induced DNA cleavage was studied by gel electrophoresis techniques. The complex was found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC DNA) form I to nicked coiled (NC DNA) relaxed form II.

Abstract 4838: R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells

Abstract G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

Abstract 4855: Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression

Abstract To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives have known limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (LMP400, LMP776 and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2 and PALB2 are needed to repair DSB induced by TOP1 inhibitors (Maede, Y et al. 2014). It has been shown that BRCA1 is a determinant of response to camptothecins in addition to Olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives (Zoppoli, G et al. 2012). To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival after treatment with the LMPs. We found that SLFN11-positives cells are 10-times hypersensitive to LMPs compare to WT cells. Also, BRCA1-, BRCA2- and PALB2-deficient cells are 3- to 5- times hypersensitive to the LMPs. Adding Olaparib led to a greater cell death, especially in HR-deficient (HRD) cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with Olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers. Citation Format: Yves Pommier, Laetitia Marzi, Zoe Waever Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Mark Cushman. Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4855.

Thiocarbonyl-bound metallonitrosyl complexes with visible-light induced DNA cleavage and promising vasodilation activity

Nitric oxide has been involved in many key biological processes such as vasodilation, platelet aggregation, apoptosis, memory function, and this has drawn attention to the development of exogenous NO donors. Metallonitrosyl complexes are an important class of these compounds. Here, two new ruthenium nitrosyl complexes containing a thiocarbonyl ligand, with the formula cis-[Ru(phen)2(L)(NO)](PF6)3 (phen = phenantroline, L = thiourea or thiobenzamide), were synthesized and characterized by electronic spectroscopy, FTIR, NMR, mass spectrometry and voltammetric techniques. Theoretical calculations using Density Functional Theory (DFT) and Time-dependent Density Functional Theory (TD-DFT) were also used and further supported the characterizations of these complexes. An efficient release of nitric oxide by blue light was validated using a NO/HNO probe: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, known as cPTIO. Interestingly, the complex containing thiourea cleaved DNA even in the dark, while both complexes showed great DNA photocleavage activity in blue light. This process might work mainly through NO and hydroxyl radical production. Additionally, these complexes showed promising vasodilator activity, whose mechanism of action was investigated using N-Nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and compared to sodium nitroprusside. Both compounds were indeed NO-mediated heme-dependent activators of soluble guanylate cyclase. Additionally, they did not show any significant cytotoxicity against cancer cell lines U87 and GBM02. Altogether, these results supported both complexes having potential pharmacological applications that deserve further studies.