Search

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated

The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.

Hydrolytic loss of nucleobases from the deoxyribose backbone of DNA is one of the most common unavoidable types of damage in synthetic and cellular DNA. The reaction generates abasic sites in DNA and it is important to understand the properties of these lesions. The acidic nature of the α-protons of the ring-opened abasic aldehyde residue facilitates β-elimination of the 3’-phosphoryl group. This reaction is expected to generate a DNA strand break with a phosphoryl group on the 5’-terminus and a trans-α,β-unsaturated aldehyde residue on the 3’-terminus; however, a handful of studies have identified noncanonical sugar remnants on the 3’-terminus, suggesting that the products arising from strand cleavage at AP sites in DNA may be more complex than commonly thought. We characterized the strand cleavage induced by treatment of an abasic site-containing DNA oligonucleotide with heat, NaOH, piperidine, spermine, and the base excision repair glycoslyases Fpg and Endo III. The results showed that, under multiple conditions, cleavage at an abasic site in a DNA oligomer generated noncanonical sugar remnants including cis-α,β-unsaturated aldehyde, 2-deoxyribose, and 3-thio-2,3-dideoxyribose products on the 3’-terminus of the strand break.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron-sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

We demonstrate the remarkable ability of surface-enhanced Raman spectroscopy (SERS) to track the allosteric changes in restriction endonuclease KpnI (R.KpnI) caused by metal ions. R.KpnI binds and promiscuously cleaves DNA upon activation by Mg2+ ions. However, the divalent ion Ca2+ induces high fidelity cleavage, which can be overcome by higher concentrations of Mg2+ ions. In the absence of any 3D crystal structure, for the first time, we have elucidated the structural underpinnings of such a differential effect of divalent ions on the endonuclease activity. A combined SERS and molecular dynamics (MD) approach showed that Ca2+ ion activates an enzymatic switch in the active site, which is responsible for the high fidelity activity of the enzyme. Thus, SERS in combination with MD simulations provides a powerful tool for probing the link between the structure and activity of enzyme molecules that play vital roles in DNA transactions.

Titanium is one of the most abundant elements on Earth but is commonly thought to have no role in biology because of its propensity to hydrolyze. Nature stabilizes hard Lewis acidic metals from hyd...

RNA repeat expansions cause more than 30 neurological and neuromuscular diseases with no known cures. Since repeat expansions operate via diverse pathomechanisms, one potential therapeutic strategy is to rid them from disease-affected cells, using bifunctional small molecules that cleave the aberrant RNA. Such an approach has been previously implemented for the RNA repeat that causes myotonic dystrophy type 1 [DM1, r(CUG)(exp)] with Cugamycin, which is a small molecule that selectively binds r(CUG)(exp) conjugated to a bleomycin A5 cleaving module. Herein, we demonstrate that, by replacing bleomycin A5 with deglycobleomycin, an analogue in which the carbohydrate domain of bleomycin A5 is removed, the selectivity of the resulting small-molecule conjugate (DeglycoCugamycin) was enhanced, while maintaining potent and allele-selective cleavage of r(CUG)(exp) and rescue of DM1-associated defects. In particular, DeglycoCugamycin did not induce the DNA damage that is observed with high concentrations (25 μM) of Cugamycin, while selectively cleaving the disease-causing allele and improving DM1 defects at 1 μM.

The ruthenium(II) complexes [RuCl(L1)(L3)]Cl (1), [RuCl(L1)(L4)]Cl (2), [RuCl(L2)(L4)]Cl (3), [RuCl(L1)(L5)]Cl (4), and [RuCl(L2)(L5)]Cl (5) of NNN-donor dipicolylamine (dpa) bases (L4, L5) having BODIPY (boron-dipyrromethene) moieties, NN-donor phenanthroline derivatives (L1, L2), and benzyldipicolylamine (bzdpa, L3) were prepared and characterized by spectroscopic techniques and their cellular localization/uptake and photocytotoxicity studied. Complex 1, as its PF6 salt (1a), has been structurally characterized with help of a single-crystal X-ray diffraction technique. It has a RuN5Cl core with the Cl bonded trans to the amine nitrogen atom of bzdpa. The complexes showed intense absorption spectral bands near 500 nm (e ≈ 58000 M-1 cm-1) in 2 and 3 and 654 nm (e ≈ 80000 M-1 cm-1) in 4 and 5 in 1/1 DMSO/DPBS (v/v). Complex 5 having biotin and PEGylated-disteryl BODIPY gave a singlet oxygen quantum yield (ΦΔ) of ∼0.65 in DMSO. Complex 5 exhibited remarkable PDT (photodynamic therapy) activity (IC50 ≈ 0.02 μM) with a photocytotoxicity index (PI) value of >5000 in red light of 600-720 nm in A549 cancer cells. The biotin-conjugated complexes showed better photocytotoxicity in comparison to nonbiotinylated analogues in A549 cells. The complexes displayed less toxicity in HPL1D normal cells in comparison to A549 cancer cells. The emissive BODIPY complexes 3 and 5 (ΦF ≈ 0.07 in DMSO) showed significant mitochondrial localization.

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (

The Cas9 nuclease binds and cleaves DNA through its large-scale structural rearrangements. However, its unique property of not releasing the cleaved DNA has forbidden spectroscopic detection of the cleavage event. Here, we employ a novel fluorescence probe based on pyrene excimer emission to detect a minute structural change not detectable by other methods and demonstrate its applicability to spectroscopic tracking of the Cas9 nuclease activity in time. We show that the intensity of excimer emission depends sensitively on a subtle change in the structural environment of the target nucleic acid, which enables discrimination between cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. Kinetic parameters were obtained from the temporal evolution of the excimer emission, which revealed that DNA binding is hardly affected by PAM-distal mismatches, whereas the rate of cleavage by Cas9 decreases dramatically even with a 1-bp mismatch. Spectroscopic studies using the pyrene-based probe should be promising for biomolecular systems affected by subnm structural changes.

A Cu(II) complex, that is, [Cu(CNC-B3)(IM-B7)](PF6)2, 1, containing CNC-B3 (L2) = CNC–pincer–vitamin B3 and IM-B7 (L3) = 1H-imidazole-1-methanamine-vitamin B7 conjugates, was developed as a potenti...

Bridging organic and inorganic chemistry as well as molecular biology, this undergraduate experiment deals with the synthesis of ligands for metal complexes that can cleave DNA. Students gain theoretical and experimental expertise about metal–DNA interactions and the mechanisms behind DNA cleavage. This experiment lays a basis for the understanding and the rational design of artificial DNA scissors and provides insight into interdisciplinary research in the field of bioinorganic chemistry.

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays, we showed that YM155 causes extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions, indicating that hydrogen peroxide and oxygen are not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations, a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon 2-electron reductive activation.

Photosensitizers with high photocleavage ability are urgently needed to improve photodynamic therapy efficacy. Herein, a supramolecular complex was constructed through host-guest self-assembly using hexa-β-CD-appended ruthenium polypyridyl (6CD-Ru) and adamantane-modified anthracene (ADA-AN) in water. The targeted DNA-intercalation of peripheral anthracenes can remarkably enhance photocleavage ability and antitumor activity of the complex irradiated with visible light.

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

Topoisomerase II is a critical enzyme in replication, transcription, and the regulation of chromatin topology. Several anticancer agents target topoisomerases in order to disrupt cell growth. Cannabidiol is a major non-euphoriant, pharmacologically active component of cannabis. Previously, we examined the cannabidiol derivative HU-331 in order to characterize the mechanism of the compound against topoisomerase IIα. In this current work, we explore whether cannabidiol (CBD) impacts topoisomerase II activity, and we additionally examine the activity of these compounds against topoisomerase IIβ. CBD does not appear to strongly inhibit DNA relaxation and is not a poison of topoisomerase II DNA cleavage. However, oxidation of CBD allows this compound to inhibit DNA relaxation by topoisomerase IIα and β without poisoning DNA cleavage. Additionally, we found that oxidized CBD, similar to HU-331, inhibits ATP hydrolysis and can result in inactivation of topoisomerase IIα and β. We also determined that oxidized CBD and HU-331 are both able to stabilize the N-terminal clamp of topoisomerase II. Taken together, we conclude that while CBD does not have significant activity against topoisomerase II, both oxidized CBD and HU-331 are active against both isoforms of topoisomerase II. We hypothesize that oxidized CBD and HU-331 act against the enzyme through interaction with the N-terminal ATPase domain. According to the model we propose, topoisomerase II inactivation may result from a decrease in the ability of the enzyme to bind to DNA when the compound is bound to the N-terminus.

Spatial and temporal control over DNA cleavage by photoactivated enediynes can be complemented by additional factors such as the release of internal strain, chelation, pH changes, intramolecular H-bonds, and substituent effects. This review presents design and reactivity of photoactivated enediynes/enynes and analyses the chemical, biological, and photophysical challenges in their applications.

The 7-azaindenoisoquinolines are cytotoxic topoisomerase I (Top1) inhibitors. Previously reported representatives bear a 3-nitro group. The present report documents the replacement of the potentially genotoxic 3-nitro group by 3-chloro and 3-fluoro substituents, resulting in compounds with high Top1 inhibitory activities and potent cytotoxicities in human cancer cell cultures and reduced lethality in an animal model. Some of the new Top1 inhibitors also possess moderate inhibitory activities against tyrosyl-DNA phosphodiesterase 1 (TDP1) and tyrosyl-DNA phosphodiesterase 2 (TDP2), two enzymes that are involved in DNA damage repair resulting from Top1 inhibitors, and they produce significantly more DNA damage in cancer cells than in normal cells. Eighteen of the new compounds had cytotoxicity mean-graph midpoint (MGM) GI(50) values in the submicromolar (0.033–0.630 μM) range. Compounds 16b and 17b are the most potent in human cancer cell cultures with MGM GI(50) values of 0.063 and 0.033 μM, respectively. Possible binding modes to Top1 and TDP1were investigated by molecular modeling.

Oligonucleotide conjugates of small molecules are widely used in chemical biology and have found increasing interest in the context of DNA-encoded chemical libraries for drug discovery. Attachment of molecules to DNA bound to the solid support is an attractive small-molecule conjugation method that permits the use of organic solvents, rigorous reaction conditions, and simple workup. However, the conjugated structures must be resistant to the harsh DNA deprotection/cleavage conditions and the stabilities of building blocks under various deprotection conditions are mostly unexplored. In the present study, we analyzed the stability of 131 structurally diverse fragments that contain amides and amide-like elements during DNA deprotection protocols. Structural features susceptible to decomposition in DNA deprotection conditions were identified and a protocol that enabled the synthesis of DNA conjugates with labile fragments on solid support was identified.

CRISPR-Cas9 is a cutting-edge genome-editing technology, which employs the endonuclease Cas9 to cleave DNA sequences of interest. However, the catalytic mechanism of DNA cleavage and the critical role of the Mg2+ ions have remained elusive. Here, quantum–classical QM(Car-Parrinello)/MM simulations are used to disclose the two-Mg2+ aided mechanism of phosphodiester bond cleavage in the RuvC domain. We reveal that the catalysis proceeds through an associative pathway activated by H983 and fundamentally assisted by the joint dynamics of the two Mg2+ ions, which cooperatively act to properly orient the reactants and lead the chemical step to completion. Cross-validation of this mechanism is achieved by evaluating alternative reaction pathways and in light of experimental data, delivering fundamental insights on how CRISPR-Cas9 cleaves nucleic acids. This knowledge is critical for improving the Cas9 catalytic efficiency and its metal-dependent function, helping also the development of novel Cas9-based genome-editing tools.

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N40 or N100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O2-• or HOO•) and hydrogen peroxide (H2O2) and leads to the same set of products (3′-phosphoglycolate, 5′-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4′ radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H2O2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H2O2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn2+ and Mg2+, although including Mn2+ increases the activity, and Mn2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

Precolibactins and colibactins represent a family of natural products that are encoded by the clb gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb+ E. coli induce megalocytosis and DNA double-strand breaks in eukaryotic cells, but paradoxically, this gene cluster is found in the probiotic Nissle 1917. Evidence suggests precolibactins are converted to genotoxic colibactins by colibactin peptidase (ClbP)-mediated cleavage of an N-acyl- D-Asn side chain, and all isolation efforts have employed ΔclbP strains to facilitate accumulation of precolibactins. It was hypothesized that colibactins form unsaturated imines that alkylate DNA by cyclopropane ring opening (2 → 3). However, as no colibactins have been isolated, this hypothesis has not been tested experimentally. Additionally, precolibactins A–C (7–9) contain a pyridone that cannot generate the unsaturated imines that form the basis of this hypothesis. To resolve this, we prepared 13 synthetic colibactin derivatives and evaluated their DNA binding and alkylation activity. We show that unsaturated imines, but not the corresponding pyridone derivatives, potently alkylate DNA. The imine, unsaturated lactam, and cyclopropane are essential for efficient DNA alkylation. A cationic residue enhances activity. These studies suggest that precolibactins containing a pyridone are not responsible for the genotoxicity of the clb cluster. Instead, we propose that these are off-pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The results presented herein provide a foundation to begin to connect metabolite structure with the disparate phenotypes associated with clb+ E. coli.

Type II DNA topoisomerases resolve topological knots and tangles in DNA that result from routine cellular processes and are effective targets for anticancer therapeutics. To this end, thiosemicarbazones have been identified as having the ability to kill cancer cells from several cell lines. Literature evidence suggests that at least some thiosemicarbazones have an impact on topoisomerase II activity. However, the mechanism is not as clearly defined. Therefore, we set out to analyze the activity of four α-(N)-heterocyclic thiosemicarbazone compounds against topoisomerase IIα. The ligands, acetylpyridine-ethylthiosemicarbazone (APY-ETSC) and acetylpyrazine-methylthiosemicarbazone (APZ-MTSC), and their copper(II) [Cu(II)] complexes [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were examined for the ability to impact the catalytic cycle of human topoisomerase IIα. Both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were more effective at inhibiting DNA relaxation compared with the ligands alone. Further, both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] increased double-stranded DNA cleavage levels without inhibiting topoisomerase IIα-mediated DNA ligation. The Cu(II) complexes inactivate enzyme activity over time suggesting a critical interaction with the enzyme. Additionally, we found that the Cu(II)-thiosemicarbazone complexes do not significantly impact DNA cleavage by the catalytic core of the enzyme. This evidence is supported by the fact that both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl], and to a lesser extent the ligands, inhibit topoisomerase IIα-mediated ATP hydrolysis. Based upon kinetic analysis, the Cu(II) complexes appear to be noncompetitive inhibitors of the ATPase domain of topoisomerase IIα. Taken together, our results provide evidence that Cu(II) complexes of α-(N)-heterocyclic thiosemicarbazones catalytically inhibit the enzyme through the ATPase domain but also promote double-stranded DNA cleavage by the enzyme.

A series of four new mononuclear copper(II) polypyridyl complexes (1-4) have been designed, developed, and thoroughly characterized by several physicochemical techniques. The CT-DNA binding properties of 1-4 have been investigated by absorption, emission spectroscopy, and viscosity measurements. All the complexes especially 1 and 4 exhibit cytotoxicity toward several cancer cell lines, suggesting their anticancer properties as observed by several in vitro assays. Additionally, the complexes show inhibition of endothelial cell (HUVECs) proliferation, indicating their antiangiogenic nature. In vivo chick embryo angiogenesis assay again confirms the antiangiogenic properties of 1 and 4. The formation of excessive intracellular ROS (H2O2 and O2(•-)) and upregulation of BAX induced by copper(II) complexes may be the plausible mechanisms behind their anticancer activities. The present study may offer a basis for the development of new transition metal complexes through suitable choice of ligands for cancer therapeutics by controlling tumor angiogenesis.

Extracts from the rhizome of the turmeric plant are widely consumed as anti-inflammatory dietary supplements. Turmeric extract contains the three curcuminoids, curcumin (≈80% relative abundance), demethoxycurcumin (DMC; ≈15%), and bisdemethoxycurcumin (BDMC; ≈5%). A distinct feature of pure curcumin is its instability at physiological pH, resulting in rapid autoxidation to a bicyclopentadione within 10-15 min. Here, we describe oxidative transformation of turmeric extract, DMC, and BDMC and the identification of their oxidation products using LC-MS and NMR analyses. DMC autoxidized over the course of 24 h to the expected bicyclopentadione diastereomers. BDMC was resistant to autoxidation, and oxidative transformation required catalysis by horseradish peroxidase and H2O2 or potassium ferricyanide. The product of BDMC oxidation was a stable spiroepoxide that was equivalent to a reaction intermediate in the autoxidation of curcumin. The ability of DMC and BDMC to poison recombinant human topoisomerase IIα was significantly increased in the presence of potassium ferricyanide, indicating that oxidative transformation was required to achieve full DNA cleavage activity. DMC and BDMC are less prone to autoxidation than curcumin and contribute to the enhanced stability of turmeric extract at physiological pH. Their oxidative metabolites may contribute to the biological effects of turmeric extract.

The structure–activity relationships and hit-to-lead optimization of dual Top1–TDP1 inhibitors in the indenoisoquinoline drug class were investigated. A series of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines were synthesized and evaluated. Several compounds displayed potent dual Top1–TDP1 inhibition. The 9-hydroxy series exhibited potencies and cytotoxicities vs Top1 that surpassed those of camptothecin (CPT), the natural alkaloid that is being used as a standard in the Top1-mediated DNA cleavage assay. One member of this series was a more potent Top1 inhibitor at a concentration of 5 nM and produced a more stable ternary drug–DNA–Top1 cleavage complex than CPT.

Conspectus In 1996, a snapshot of the field of synthesis was provided by many of its thought leaders in a Chemical Reviews thematic issue on “Frontiers in Organic Synthesis”. This Accounts of Chemical Research thematic issue on “Synthesis, Design, and Molecular Function” is intended to provide further perspective now from well into the 21st century. Much has happened in the past few decades. The targets, methods, strategies, reagents, procedures, goals, funding, practices, and practitioners of synthesis have changed, some in dramatic ways as documented in impressive contributions to this issue. However, a constant for most synthesis studies continues to be the goal of achieving function with synthetic economy. Whether in the form of new catalysts, reagents, therapeutic leads, diagnostics, drug delivery systems, imaging agents, sensors, materials, energy generation and storage systems, bioremediation strategies, or molecules that challenge old theories or test new ones, the function of a target has been and continues to be a major and compelling justification for its synthesis. While the targets of synthesis have historically been heavily represented by natural products, increasingly design, often inspired by natural structures, is providing a new source of target structures exhibiting new or natural functions and new or natural synthetic challenges. Complementing isolation and screening approaches to new target identification, design enables one to create targets de novo with an emphasis on sought-after function and synthetic innovation with step-economy. Design provides choice. It allows one to determine how close a synthesis will come to the ideal synthesis and how close a structure will come to the ideal function. In this Account, we address studies in our laboratory on function-oriented synthesis (FOS), a strategy to achieve function by design and with synthetic economy. By starting with function rather than structure, FOS places an initial emphasis on target design, thereby harnessing the power of chemists and computers to create new structures with desired functions that could be prepared in a simple, safe, economical, and green, if not ideal, fashion. Reported herein are examples of FOS associated with (a) molecular recognition, leading to the first designed phorbol-inspired protein kinase C regulatory ligands, the first designed bryostatin analogs, the newest bryologs, and a new family of designed kinase inhibitors, (b) target modification, leading to highly simplified but functionally competent photonucleases—molecules that cleave DNA upon photoactivation, (c) drug delivery, leading to cell penetrating molecular transporters, molecules that ferry other attached or complexed molecules across biological barriers, and (d) new reactivity-regenerating reagents in the form of functional equivalents of butatrienes, reagents that allow for back-to-back three-component cycloaddition reactions, thus achieving structural complexity and value with step-economy. While retrosynthetic analysis seeks to identify the best way to make a target, retrofunction analysis seeks to identify the best targets to make. In essence, form (structure) follows function.

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

Bleomycins A5 and B2 were used to study the structural features in hairpin DNAs conducive to strong BLM–DNA interaction. Two members of a 10-hairpin DNA library previously found to bind most tightly to these BLMs were subsequently noted to share the sequence 5′-ACGC (complementary strand sequence 5′-GCGT). Each underwent double-strand cleavage at five sites within, or near, an eight base pair region of the DNA duplex which had been randomized to create the original library. A new hairpin DNA library was selected based on affinity for immobilized Fe(III)·BLM A5. Two of the 30 newly identified DNAs also contained the sequence 5′-ACGC/5′-GCGT. These DNAs bound to the Fe(II)·BLMs more tightly than any DNA characterized previously. Surface plasmon resonance confirmed tight Fe(III)·BLM B2 binding and gave an excellent fit for a 1:1 binding model, implying the absence of significant secondary binding sites. Fe(II)·BLM A5 was used to assess sites of double-strand DNA cleavage. Both hairpin DNAs underwent double-strand cleavage at five sites within or near the original randomized eight base region. For DNA 12, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA 13 underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA 14, which bound Fe·BLM poorly, was converted to a strong binder (DNA 15) by insertion of the sequence 5′-ACGC/5′-GCGT. These findings reinforce the idea that tighter DNA binding by Fe·BLM leads to increased double-strand cleavage by a novel mechanism and identify a specific DNA motif conducive to strong BLM binding and cleavage.

Described here are mechanistic details of the chemical reactivities of two modified/saturated pyrimidine residues that represent naturally occurring forms of DNA damage: 5-thyminyl-5,6-dihydrothymine, commonly referred to as the “spore photoproduct” (SP), and 5,6-dihydro-2′-deoxyuridine (dHdU), formed via ionizing radiation damage to cytosine under anoxic conditions and also serving as a general model of saturated pyrimidine residues. It is shown that due to the loss of the pyrimidine C5–C6 double bond and consequent loss of ring aromaticity, the C4 position of both these saturated pyrimidines is prone to the formation of a hemiaminal intermediate via water addition. Water addition is facilitated by basic conditions; however, it also occurs at physiological pH at a slower rate. The hemiaminal species so-formed subsequently converts to a ring-opened hydrolysis product through cleavage of the pyrimidine N3–C4 bond. Further decomposition of this ring-opened product above physiological pH leads to DNA strand break formation. Taken together, these results suggest that once the aromaticity of a pyrimidine residue is lost, the C4 position becomes a “hot spot” for the formation of a tetrahedral intermediate, the decay of which triggers a cascade of elimination reactions that can under certain conditions convert a simple nucleobase modification into a DNA strand break.

Theoretical studies on the DNA-photocleavage mechanism and efficiency of some Ru(II) polypyridyl complexes as novel reagents have been carried out using the density functional theory (DFT) method. Stable DNA-docking models of Ru(II) polypyridyl complexes were obtained using the docking and DFT methods. The excited-state reduction potentials, electron-transfer (ET) activation energies, and intramolecular reorganization energies were theoretically calculated, and the corresponding frontier molecular orbitals of complexes were also presented. Based on these properties of excited states, the essential component of two different DNA-photocleavage mechanisms, i.e., the photoinduced oxidation-reduction mechanism and the singlet oxygen photosensitization mechanism, has been revealed, and the DNA-photocleavage efficiencies were reasonably explained, and hereby a complex with excellent DNA-photocleavage ability was also designed. This work offers valuable theoretical insight into the property of excited-states and the DNA-photocleavage mechanism of Ru(II) polypyridyl complexes as novel reagents.

Clinically used bleomycin A5 has been employed in a study of double-strand cleavage of a library of 10 hairpin DNAs originally selected on the basis of their strong binding to bleomycin. Each of the DNAs underwent double-strand cleavage at more than one site, and all of the cleavage sites were within, or in close proximity to, an eight-base-pair region of the duplex that had been randomized to create the original library. A total of 31 double-strand cleavage sites were identified on the 10 DNAs, and 14 of these sites were found to represent coupled cleavage sites, that is, events in which one of the two strands was always cleaved first, followed by the associated site on the opposite strand. Most of these coupled sites underwent cleavage by a mechanism described previously by the Povirk laboratory and afforded cleavage patterns entirely analogous to those reported. However, at least one coupled cleavage event was noted that did not conform to the pattern of those described previously. More surprisingly, 17 double-strand cleavages were found not to result from coupled double-strand cleavage, and we posit that these cleavages resulted from a new mechanism not previously described. Enhanced double-strand cleavages at these sites appear to be a consequence of the dynamic nature of the interaction of Fe·BLM A5 with the strongly bound hairpin DNAs.

Iron complexes of N,N-bis(2-Pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity of these and related metal complexes were carried out in 0, cell-free systems using plasmid DNA as substrate. The present study evaluated nuclear DNA cleavage activity and cell cytotoxicity of BLM and its synthetic mimics based on the ligand N4Py. The N4Py-based reagents induced nuclear DNA cleavage in living cells as efficiently as BLM and Fe(II)-BLM. Treatment of 2 cancer cell lines and 1 noncancerous cell line indicated improved cytotoxicity of N4Py when compared to BLM. Moreover, some level of selectivity was observed for N4Py on cancerous versus noncancerous cells. It was demonstrated that N4Py-based reagents and BLM induce cell death via different mechanistic pathways. BLM was shown to induce cell cycle arrest, ultimately resulting in mitotic catastrophe. In contrast, N4Py-based reagents were shown to induce apoptosis effectively. To the best of our knowledge, the present study is the first demonstration of efficient nuclear DNA cleavage activity of a synthetic BLM mimic within cells. The results presented here show that it is possible to design synthetic bioinorganic model complexes that are at least as active as the parent natural product and thereby are potentially interesting alternatives for BLM to induce antitumor activity.

Metal ion-catalyzed oxidation of hydrazine and its derivatives leads to the formation of the hydrazyl radical and subsequently to oxy-radicals in the presence of molecular oxygen. Here, we have examined the role of Cu(2+)-catalyzed oxidation of hydralazine in the induction of DNA damage. Neither 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) nor dimethyl sulfoxide (DMSO) was effective in inhibiting hydralazine-Cu(2+)-induced DNA damage. Singlet oxygen did not appear to participate in this DNA cleavage. The one-electron oxidation of hydralazine also leads to the formation of DNA radicals as confirmed by immuno-spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. Electron spin resonance (ESR) and spin-trapping studies further confirmed the formation of DNA radicals; predominantly, 2'-deoxyadenosine radical adducts were detected, while some radicals were also detected with other nucleosides. Our results suggest that free hydroxyl radicals may not be the main damaging species causing DNA cleavage and that possibly Cu-peroxide complexes, formed from Cu(+)-H2O2, are responsible for this hydralazine-Cu(2+)-induced DNA cleavage.

Cas4 proteins, a core protein family associated with the microbial system of adaptive immunity CRISPR, are predicted to function in the adaptation step of the CRISPR mechanism. Here we show that the Cas4 protein SSO0001 from the archaeon Sulfolobus solfataricus has metal-dependent endonuclease and 5' to 3' exonuclease activities against single-stranded DNA, as well as ATP-independent DNA unwinding activity toward double-stranded DNA. The crystal structure of SSO0001 revealed a decameric toroid formed by five dimers with each protomer containing one [4Fe-4S] cluster and one Mn2+ ion bound in the active site located inside the internal tunnel. The conserved RecB motif and four Cys residues are important for DNA binding and cleavage activities, whereas DNA unwinding depends on several residues located near the [4Fe-4S]-cluster. Our results suggest that Cas4 proteins might contribute to the addition of novel CRISPR spacers through the formation of 3'-DNA overhangs and to the degradation of foreign DNA.

Double strand breaks (DSBs) are the most deleterious form of DNA damage. Natural products that produce them are potent cytotoxic agents. Designing molecules that produce DSBs via a single chemical event is challenging. We determined that formation of a C4′-nucleotide radical in duplex DNA under aerobic conditions gives rise to a DSB. The original radical yields a strand break containing a peroxyl radical, which initiates opposite strand cleavage via C4′-hydrogen atom abstraction. This mechanism provides the impetus to design DNA damaging agents that produce DSBs by abstracting a single hydrogen atom from the biopolymer.

(−)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea (Camellia sinensis) leaves and many of its cellular effects are consistent with its actions as a topoisomerase II poison. In contrast to genistein and several other related bioflavonoids that act as interfacial poisons, EGCG was the first bioflavonoid shown to act as a covalent topoisomerase II poison. Although studies routinely examine the effects of dietary phytochemicals on enzyme and cellular systems, they often fail to consider that many compounds are altered during cooking or cellular metabolism. To this point, the majority of EGCG (and related catechins) in green tea leaves is epimerized during the brewing process. Epimerization reverses the stereochemistry of the bond that bridges the B- and C-rings, and converts EGCG to (−)-gallocatechin gallate (GCG). Consequently, a significant proportion of EGCG that is ingested during the consumption of green tea is actually GCG. Therefore, the effects of GCG and related epimerized green tea catechins on human topoisomerase IIα and IIβ were characterized. GCG increased levels of DNA cleavage mediated by both enzyme isoforms with an activity that was similar to that of EGCG. GCG acted primarily by inhibiting the ability of topoisomerase IIα and IIβ to ligate cleaved DNA. Several lines of evidence indicate that GCG functions as a covalent topoisomerase II poison that adducts the enzyme. Finally, epimerization did not affect the reactivity of the chemical substituents (the three hydroxyl groups on the Bring) that were required for enzyme poisoning. Thus, the activity of covalent topoisomerase II poisons appears to be less sensitive to stereochemical changes than interfacial poisons.

In the present study, molecular dynamic simulations have been performed to investigate the DNA binding affinities and cleavage activities of a new class of mononuclear copper (p-Cu(BPA) and m-Cu(BPA)) and dinuclear copper-platinum (p-Cu(BPA)-Pt and m-Cu(BPA)-Pt) metallonucleases. The simulated results reveal that the two mononuclear nucleases are noncovalent minor groove DNA binders and the two dinuclear ones tend to be bound to DNA in the major groove by a covalent bond between the platinum center and N7 of the guanine base, which is in agreement with the experimental results. The simulated results show that the binding affinities of the four studied nucleases with DNA are in the order of p-Cu(BPA)m-Cu(BPA)p-Cu(BPA)-Ptm-Cu(BPA)-Pt; the binding affinities are dominated by intermolecular binding modes of nucleases with DNA and the intermolecular hydrogen bonds. The distance probability distributions indicate that the hydrogen atoms of DNA sugar could be abstracted by the four nucleases. Specifically, the dinuclear nucleases abstract hydrogen atoms from the deoxyribose sugar linking to G(18) base while mononuclear nuclease abstracts hydrogen atoms from the deoxyribose sugars linking to C(15) and C(16) bases, suggesting that the dinuclear nucleases improve the sequence-selective cleavage of DNA compared with the mononuclear one. Moreover, the differences in calculated DNA conformational dynamics and groove parameters demonstrate that the extent of DNA conformational distortions induced by dinuclear nucleases is greater than that induced by mononuclear nucleases. This investigation provides detailed information showing that dinuclear nucleases have superior DNA binding affinities and nuclease activities as compared with their mononuclear counterparts.

The polyphenol curcumin is the principal flavor and color component of the spice turmeric. Beyond its culinary uses, curcumin is believed to positively impact human health and displays antioxidant, anti-inflammatory, antibacterial, and chemopreventive properties. It also is in clinical trials as an anticancer agent. In aqueous solution at physiological pH, curcumin undergoes spontaneous autoxidation that is enhanced by oxidizing agents. The reaction proceeds through a series of quinone methide and other reactive intermediates to form a final dioxygenated bicyclopentadione product. Several naturally occurring polyphenols that can form quinones have been shown to act as topoisomerase II poisons (i.e., they increase levels of topoisomerase II-mediated DNA cleavage). Because several of these compounds have chemopreventive properties, we determined the effects of curcumin, its oxidative metabolites, and structurally related degradation products (vanillin, ferulic acid, and feruloylmethane) on the DNA cleavage activities of human topoisomerase IIα and IIβ. Intermediates in the curcumin oxidation pathway increased the level of DNA scission mediated by both enzymes ~4-5-fold. In contrast, curcumin and the bicyclopentadione, as well as vanillin, ferulic acid, and feruloylmethane, had no effect on DNA cleavage. As found for other quinone-based compounds, curcumin oxidation intermediates acted as redox-dependent (as opposed to interfacial) topoisomerase II poisons. Finally, under conditions that promote oxidation, the dietary spice turmeric enhanced topoisomerase II-mediated DNA cleavage. Thus, even within the more complex spice formulation, oxidized curcumin intermediates appear to function as topoisomerase II poisons.

The synthesis, characterization, binding to calf thymus DNA, and plasmid DNA photocleavage studies of two ruthenium(II) pteridinylphenanthroline complexes are reported where the new pteridinylphenantholine ligands in these complexes are additions to a larger family designed to resemble DNA bases. [Ru(bpy)(2)(L-keto)](PF(6))(2)1 is synthesized from ligand substitution of Ru(bpy)(2)Cl(2) by 4-keto-pteridino[6,7-f]phenanthroline (L-keto). Increasing the reaction temperature during synthesis of 1 causes a ring scission of the L-keto ligand within the pyrimidine ring yielding a second Ru complex, [Ru(bpy)(2)(L-aap)](PF(6))(2)2 where L-aap is 2-amino-3-amidopyrazino[5,6-f]phenanthroline. The ring cleavage reaction is accompanied by the loss of one carbon in the pyrimidine ring. Complexes 1 and 2 are characterized by (1)H NMR, UV/visible absorption and FT-IR spectroscopies and by cyclic voltammetry, and these results are presented in comparison to the previously reported related complexes [Ru(bpy)(2)(L-allox)](PF(6))(2), [Ru(bpy)(2)(L-amino)](PF(6))(2), and [Ru(bpy)(2)(dppz)](PF(6))(2). In addition, 2 has been structurally characterized by X-ray diffraction. Both 1 and 2 are good intercalators of calf thymus DNA as determined by viscometry and binding constants obtained from absorption titrations. Only the ring-cleaved complex 2 exhibits a high degree of pBR322 plasmid photocleavage in contrast to the other pteridinyl-phenanthroline complexes, which exhibit no plasmid DNA photocleavage. Complex 1, however, decomposes in buffer forming the photocleaver 2, demonstrating that sample age and reactivity can affect observed photocleavage. Complex 2 appears to photocleave DNA through a singlet oxygen mechanism.

The title compound, characterized by means of an X-ray structure analysis, represents an easy example of a noncatena "1 + 2 + 1" tetranuclear copper(II) μ(3)-triazolate compound. [Cu(4)(atc)(2)(dien)(4)(ClO(4))(2)](ClO(4))(2)·2H(2)O (1), where H(2)atc = 5-amino-l,2,4-triazole-3-carboxylic acid and dien = diethylenetriamine = 1,4,7-triazaheptane, contains two copper atoms linked by a double diazinic bridge, each of which is further connected to a third and fourth copper atom (Cu') through the triply bridging triazolato ring and the bidentate carboxylato group of the atc(2-) ligands. The copper-copper distances within the tetranuclear unit are Cu-Cu = 4.059 Å, Cu-Cu' = 5.686 and 6.370 Å, and Cu'-Cu' = 11.373 Å. The compound self-assembles into a tridimensional hydrogen-bonded network to generate a MOF. 1 exhibits antiferromagnetic behavior with g = 2.10(1), J = -34.1(2) cm(-1) and j = -5.50(3) cm(-1), where J is the coupling constant of the central Cu-Cu pair and j the coupling constant of the two Cu-Cu' (Cu(central)-Cu(pheripheral)) pairs, as defined by H = -JS(2)S(2a) - j (S(1)S(2) + S(2a)S(1a)). Complex 1 has been tested as nuclease mimic. It shows good binding propensity to calf thymus DNA, with a binding constant value of 6.20 × 10(6) M(-1) (K(app)) and ΔT(m) = 18.3 °C. Moreover, the compound displays efficient oxidative cleavage of pUC18 DNA, even at low concentration, in the presence of a mild reducing agent (ascorbate), with a rate constant for the conversion of supercoiled to nicked DNA (k(obs)) of ~0.126 min(-1). The good reactivity of 1 toward DNA is explained from the electrostatic interactions of the cationic species produced in solution.

The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90-95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6'-hydroxy-ZBM, BLM Z, and 6'-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6'-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6'-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug.

Oxidized abasic sites such as 2-deoxyribonolactone (L) are produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor natural products. 2-Deoxyribonolactone is labile under alkaline conditions, but its half-life in free DNA at pH 7.5 is approximately 1 week. Independent generation of L at defined positions within nucleosomes reveals that the histone proteins catalyze strand scission and increase the rate between 11- and ∼43-fold. Mechanistic studies indicate that DNA-protein cross-links are not intermediates en route to strand scission and that C2 deprotonation is the rate-determining step. The use of mutant histone H4 proteins demonstrates that the lysine-rich tail that is often post-translationally modified in cells contributes to the cleavage of L but is not the sole source of the enhanced cleavage rates. Consideration of DNA repair in cells suggests that L formation in nucleosomal DNA as part of bistranded lesions by antitumor antibiotics results in de facto double strand breaks, the most deleterious form of DNA damage.

The complexes [Cu(salH)(2)(H(2)O)] (1), [Cu(dipsH)(2)(H(2)O)] (2), {Cu(3-MeOsal)(H(2)O)(0.75)}(n) (3), [Cu(dipsH)(2)(BZDH)(2)] (4), [Cu(dipsH)(2)(2-MeOHBZDH)(2)]·EtOH (5), [Cu(sal)(phen)] (6), [Cu(dips)(phen)]·H(2)O (7), and [Cu(3-MeOsal)(phen)]·H(2)O (8) (salH(2) = salicylic acid; dipsH(2) = 3,5-diisopropylsalicylic acid; 3-MeOsalH(2) = 3-methoxysalicylic acid; BZDH = benzimidazole; 2-MeOHBZDH = 2 methanolbenzimidazole and phen =1,10-phenanthroline) were prepared and characterized. Structures of 4, 5, and 8 were determined by X-ray crystallography. Compounds 1-8 are potent superoxide dismutase mimetics, and they are inactive as inhibitors of COX-2 activity. Compounds 1, 4, and 5 exhibit moderate inhibition of COX-1. Complexes 6-8 display rapid micromolar cytotoxicity against cisplatin sensitive (breast (MCF-7), prostate (DU145), and colon (HT29)) and cisplatin resistant (ovarian (SK-OV-3)) cell lines compared to 1-5, and they exhibit potent in vitro DNA binding and cleavage capabilities.

We describe a family of hybrid compounds for the most efficient light-activated double-strand (ds) DNA cleavage known to date. This family represents the second generation of "switchable" molecular systems for pH-gated ds DNA-cleavage which combine a potent DNA-photocleaver and a pH-regulated part derived from a dipeptide. Design of the pH-switchable part utilizes amino groups of different basicity. Whereas the basic amino groups are protonated throughout the biologically relevant pH range, the pH-gating amines undergo protonation at the pH threshold which separates cancer and normal cells. Control over the reactivity and selectivity is achieved via transformation of the initial protonation state (a monocation or a dication) into a trication at the acidic pH. This change leads to an extraordinary increase in the efficiency of ds DNA cleavage leading to the ds:ss ratios comparable with the most efficient nonenzymatic ds DNA cleavers. Statistical analysis reveals that these high ds:ss ratios result from the combination of several factors: (a) true double-stranded cleavage, and (b) conversion of single-stranded (ss)-scission into ds cleavage. Considerable part of ds cleavage is also produced via the combination of ss cleavage events.

A new ligand, 2-oxo-1,2-dihydroquinoline-3-carbaldehyde semicarbazone (OQsc-H) (1);, its N(4)-phenyl derivative (OQsc-Ph) (2); and their corresponding copper(II) complexes [CuCl(2)(OQsc-H)]·H(2)O·CH(3)OH (3), [CuCl(2)(OQsc-Ph)(H(2)O)]·CH(3)OH (4), and [CuNO(3)(OQsc-Ph)(H(2)O)]NO(3)·H(2)O·C(2)H(5)OH (5) have been synthesized and characterized by structural, analytical, and spectral methods, in order to investigate the influence of N(4)-phenyl substitution on structure and pharmacological properties. In all of the complexes, the ligands coordinated to the Cu(II) ion in a neutral fashion via ONO donor atoms. The single-crystal X-ray structures of neutral complex (3) and cationic complex (5) exhibit a slightly distorted square-pyramidal structure, while neutral complex (4) revealed an octahedral structure. The interaction of the compounds with calf thymus DNA (CT-DNA) has been explored by absorption and emission titration methods, which revealed that compounds 1-5 could interact with CT-DNA through intercalation. A gel electrophoresis pictogram demonstrated the ability of the complexes (3-5) to cleave the pBR322 plasmid DNA through a hydrolytic process. The interactions of the compounds with bovine serum albumin (BSA) were also investigated using UV-visible, fluorescence, and synchronous fluorescence spectroscopic methods. The results indicated that all of the compounds could quench the intrinsic fluorescence of BSA in a static quenching process. Investigations of antioxidative properties showed that all of the compounds have strong radical scavenging potencies against hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl radicals, nitric oxide, and superoxide anion radicals. Further, the cytotoxic effect of the compounds examined on cancerous cell lines such as human cervical cancer cells (HeLa), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (Hep G2), human skin cancer cells (A431), and noncancerous NIH 3T3 mouse embryonic fibroblasts cell lines showed that all three complexes exhibited substantial cytotoxic activity. Further, all of the pharmacological investigations support the fact that there exists a strong influence of N(4)-phenyl substitution in semicarbazone.

A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological co-reactants were quantified by gel electrophoresis, yielding second-order rate constants for DNA(supercoiled) to DNA(nicked) conversion up to 2.5 × 10(6) M(-1) min(-1), and for DNA(nicked) to DNA(linear) up to 7 × 10(5) M(-1) min(-1). Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probes TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 3 × 10(-4) to 0.13 min(-1) were determined, although many complexes demonstrated no measurable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal-associated reactive oxygen species (ROS) in the case of Cu(2+)(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe(2+)(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox co-reactants ascorbate and peroxide (-66 to +380 mV).