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For decades prebiotic chemists have attempted to achieve replication of RNA under prebiotic conditions with only limited success. One of the long-recognized impediments to achieving true replication of a duplex (copying of both strands) is the so-called strand inhibition problem. Specifically, while the two strands of an RNA (or DNA) duplex can be separated by heating, upon cooling the strands of a duplex will reanneal before mononucleotide or oligonucleotide substrates can bind to the individual strands. Here we demonstrate that a class of plausible prebiotic solvents, when coupled with thermal cycling and varying levels of hydration, circumvents the strand inhibition problem, and allows multiple rounds of information transfer from both strands of a duplex (replication). Replication was achieved by simultaneous ligation of oligomers that bind to their templates with the aid of the solvents. The solvents used consisted of concentrated solutions of urea and acetamide in water (UAcW), components that were likely abundant on the early Earth. The UAcW solvent system favors the annealing of shorter strands over the re-annealing of long strands, thereby circumventing strand inhibition. We observed an improvement of DNA and RNA replication yields by a factor of 100× over aqueous buffer. Information transfer in the UAcW solvent system is robust, being achieved for a range of solvent component ratios, various drying conditions, and in the absence or presence of added salts., (© 2021 Wiley-VCH GmbH.)

Androgen receptor (AR), a ligand-activated transcription factor, is a master regulator in the development and progress of prostate cancer (PCa). A major challenge for the clinically used AR antagonists is the rapid emergence of resistance induced by the mutations at AR ligand binding domain (LBD), and therefore the discovery of novel anti-AR therapeutics that can combat mutation-induced resistance is quite demanding. Therein, blocking the interaction between AR and DNA represents an innovative strategy. However, the hits confirmed targeting on it so far are all structurally based on a sole chemical scaffold. In this study, an integrated docking-based virtual screening (VS) strategy based on the crystal structure of the DNA binding domain (DBD) of AR was conducted to search for novel AR antagonists with new scaffolds and 2-(2-butyl-1,3-dioxoisoindoline-5-carboxamido)-4,5-dimethoxybenzoicacid (Cpd39) was identified as a potential hit, which was competent to block the binding of AR DBD to DNA and showed decent potency against AR transcriptional activity. Furthermore, Cpd39 was safe and capable of effectively inhibiting the proliferation of PCa cell lines (i.e., LNCaP, PC3, DU145, and 22RV1) and reducing the expression of the genes regulated by not only the full-length AR but also the splice variant AR-V7. The novel AR DBD-ARE blocker Cpd39 could serve as a starting point for the development of new therapeutics for castration-resistant PCa., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

The NRF2 transcription factor is a key regulator in cellular oxidative stress response, and acts as a tumor suppressor. Aberrant activation of NRF2 has been implicated in promoting chemo-resistance, tumor growth, and metastasis by activating its downstream target genes. Hence, inhibition of NRF2 promises to be an attractive therapeutic strategy to suppress cell proliferation and enhance cell apoptosis in cancer. Direct targeting of NRF2 with small-molecules to discover protein-DNA interaction inhibitors is challenging as it is a largely intrinsically disordered protein. To discover molecules that bind to NRF2 at the DNA binding interface, we performed an NMR-based fragment screen against its DNA-binding domain. We discovered several weakly binding fragment hits that bind to a region overlapping with the DNA binding site. Using SAR by catalogue we developed an initial structure-activity relationship for the most interesting initial hit series. By combining NMR chemical shift perturbations and data-driven docking, binding poses which agreed with NMR information and the observed SAR were elucidated. The herein discovered NRF2 hits and proposed binding modes form the basis for future structure-based optimization campaigns on this important but to date 'undrugged' cancer driver., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Emerging evidence has revealed that oxidative damages of DNA correlate with the pathogenesis of some diseases, and numerous investigations have also suggested that supplementation of antioxidants is beneficial for keeping health by rectifying in vivo redox status. Here, we construct antioxidative dipeptides with the Ugi four-component reaction (comprising p -aminobenzyl alcohol, benzaldehyde, or vanillin, a series of antioxidative carboxylic acids and isocyanides as reagents) and then attempt to attach the dipeptides to [60]fullerene by the Bingel reaction. However, this endeavor does not lead to the amelioration of the radical-scavenging property because abilities of fullerenyl dipeptides to trap 2,2'-diphenyl-1-picrylhydrazyl and galvinoxyl radicals are still dependent upon the phenolic hydroxyl group in the dipeptide scaffold rather than upon the fullerenyl group. Alternatively, when the obtained fullerenyl dipeptides are evaluated in a peroxyl radical-induced oxidation of DNA, it is found that introducing a fullerene moiety into dipeptide enables antioxidative effect to be enhanced 20-30% because the fullerene moiety facilitates the corresponding dipeptide to intercalate with DNA strands, and thus, to increase the antioxidative efficacy. Our results suggest that connecting an antioxidative skeleton with the hydrophobic fullerene moiety might lead to a series of novel antioxidant hybrids applied for the inhibition of DNA oxidation.

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks-deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

(1) Background: Chiral nanoparticular systems have recently emerged as a compelling platform for investigating stereospecific behavior at the nanoscopic level. We describe chiroselective supramolecular interactions that occur between DNA oligonucleotides and chiral polyurea nanocapsules. (2) Methods: We employ interfacial polyaddition reactions between toluene 2,4-diisocyanate and lysine enantiomers that occur in volatile oil-in-water nanoemulsions to synthesize hollow, solvent-free capsules with average sizes of approximately 300 nm and neutral surface potential. (3) Results: The resultant nanocapsules exhibit chiroptical activity and interact differentially with single stranded DNA oligonucleotides despite the lack of surface charge and, thus, the absence of significant electrostatic interactions. Preferential binding of DNA on D-polyurea nanocapsules compared to their L-counterparts is demonstrated by a fourfold increase in capsule size, a 50% higher rise in the absolute value of negative zeta potential (ζ-potential), and a three times lower free DNA concentration after equilibration with the excess of DNA. (4) Conclusions: We infer that the chirality of the novel polymeric nanocapsules affects their supramolecular interactions with DNA, possibly through modification of the surface morphology. These interactions can be exploited when developing carriers for gene therapy and theranostics. The resultant constructs are expected to be highly biocompatible due to their neutral potential and biodegradability of polyurea shells.

There are few methods available for the rapid discovery of multitarget drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG·CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1. A positive cross-selection was performed using a small library of 30 monomeric alkyne- and azide-containing ligands capable of producing >5000 possible di- and trimeric click products. The monomers were incubated with d(CTG) 16 or r(CUG) 16 under physiological conditions, and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2] cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multitarget lead therapeutic agents.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

The aims of this study were to compare diagnostic value of anti-ribosomal P protein antibody (anti-P), anti-Smith antibody (anti-Sm), anti-double-stranded DNA antibody (anti-dsDNA), anti-nucleosome antibody (ANuA), and anti-histone antibody (AHA) for systemic lupus erythematosus (SLE) as well as explore the correlation between anti-P and SLE.A retrospective study was performed with 487 SLE patients, 235 non-SLE rheumatic diseases, and 124 healthy subjects from January 2015 to December 2018. Clinical manifestations, laboratory results and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000 scores were analyzed between anti-P/+/ and anti-P/-/ patients. SPSS19.0 statistical software was used for data analysis.The sensitivities of anti-P, anti-Sm, anti-dsDNA, ANuA, and AHA in SLE were 31.6%, 20.7%, 45.0%, 27.9%, and 14.6%, and the specificities were 99.2%, 99.4%, 98.9%, 98.3%, and 96.7%, respectively. Only 27.9% of SLE had a single positive anti-P while the other 4 antibodies were all negative. There were significant differences in the age of onset, skin erythema, urinary protein, creatinine and serum IgG, IgM, C3, C4 between anti-P/+/ and anti-P/-/ patients (P < .05). When anti-Sjogren syndrome A antibody, anti-P were positive and anti-dsDNA was negative, the incidence of skin erythema was the highest (35.1%). Compared with anti-P/-/ patients, anti-P/+/ patients had higher SLEDAI scores (P < .001).Anti-P, anti-Sm, anti-dsDNA, ANuA, and AHA have high specificity but poor sensitivity in the diagnosis of SLE; combined detection can greatly improve the detection rate. Anti-P is more valuable in the diagnosis of SLE when other specific autoantibodies are negative. SLE patients with positive anti-P have an earlier onset age and are more prone to skin erythema, lupus nephritis as well as higher disease activity.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.

The water-compatible optically pure metallohelices made by self-assembly of simple nonpeptidic organic components around Fe(II) ions are now recognized as a distinct subclass of helicates that exhibit similar architecture to some natural cationic antimicrobial peptides. Notably, a new series of metallohelices was recently shown to exhibit biological activity, displaying high, structure-dependent activity against bacteria. It is also important that, thanks to their properties, such metallohelices can exhibit specific interactions with biomacromolecules. Here, following our prior report on the metallohelices that have high, structure-dependent activity against bacteria, we investigated the interactions of the series of iron(II) metallohelices with DNA, which is a potential pharmacological target of this class of coordination compounds. The results obtained with the aid of biophysical and molecular biology methods show that the investigated metallohelices accumulate in eukaryotic cells and that a significant fraction of the metallohelices accumulates in the cell nucleus, allowing them to interact also with nuclear DNA. Additionally, we have demonstrated that some metallohelices have a high affinity to DNA and are able to condense/aggregate DNA molecules more efficiently than conventional DNA-condensing agents, such as polyamines. Moreover, this capability of the metallohelices correlates with their efficiency to inhibit DNA-related enzymatic activities, such as those connected with DNA transcription, catalysis of DNA relaxation by DNA topoisomerase I, and cleavage by restriction enzymes.

Introduction : The worldwide incidence of central nervous system (CNS) primary tumors is increasing. Most of the chemotherapeutic agents used for treating these cancer types induce DNA damage, and their activity is affected by the functional status of repair systems involved in the detection or correction of DNA lesions. Unfortunately, treatment of malignant high-grade tumors is still an unmet medical need. Areas covered : We summarize the action mechanisms of the main DNA inhibitors used for the treatment of brain tumors. In addition, studies on new agents or drug combinations investigated for this indication are reviewed, focusing our attention on clinical trials that in the last 3 years have been completed, terminated or are still recruiting patients. Expert opinion : Much still needs to be done to render aggressive CNS tumors curable or at least to transform them from lethal to chronic diseases, as it is possible for other cancer types. Drugs with improved penetration in the CNS, toxicity profile, and activity against primary and recurrent tumors are eagerly needed. Targeted agents with innovative mechanisms of action and ability to harness the cells of the tumor microenvironment against cancer cells represent a promising approach for improving the clinical outcome of CNS tumors.

3,6-Diethynyl-9,10-diethoxyphenanthrene ( 4 ) was synthesized from phenanthrene and employed in the synthesis of the binuclear gold(I) alkynyl complexes (R 3 P)Au(C≡C-3-[C 14 H 6 -9,10-diethoxy]-6-C≡C)Au(PR 3 ) (R = Ph ( 5a ), Cy ( 5b )). The diyne 4 and complexes 5a and 5b were characterized by NMR spectroscopy, mass spectrometry, and elemental analysis. UV-Vis spectroscopy studies of the metal complexes and precursor diyne show strong p à p* transitions in the near UV region that red shift by ca. 50 nm upon coordination at the gold centers. The emission spectrum of 4 shows an intense fluorescence band centered at 420 nm which red shifts, slightly upon coordination of 4 to gold. Binding studies of 4 , 5a, and 5b against calf thymus DNA were carried out, revealing that 4, 5a, and 5b have >40% stronger binding affinities than the commonly used intercalating agent ethidium bromide. The molecular docking scores of 4 , 5a, and 5b with B-DNA suggest a similar trend in behavior to that observed in the DNA-binding study. Unlike the ligand 4 , promising anticancer properties for 5a and 5b were observed against several cell lines; the DNA binding capability of the precursor alkyne was maintained, and its anticancer efficacy enhanced by the gold centers. Such phenanthrenyl complexes could be promising candidates in certain biological applications because the two components (phenanthrenyl bridge and metal centers) can be altered independently to improve the targeting of the complex, as well as the biological and physicochemical properties.

DNA forms not only the canonical duplex structure but also non-canonical structures. Most potential sequences that induce the formation of non-canonical structures are present in disease-related genes. Interestingly, biological reactions are inhibited or dysregulated by non-canonical structure formation in disease-related genes. To control biological reactions, methods for inducing the formation of non-canonical structures have been developed using small molecules and oligonucleotides. In this feature article, we review biological reactions such as replication, transcription, and reverse transcription controlled by non-canonical DNA structures formed by disease-related genes. Furthermore, we discuss recent studies aimed at developing methods for regulating these biological reactions using drugs targeting the DNA structure.

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs' analogs have synthesized by structural changes of nucleoside's susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate ( ap- TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap- TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl - analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti -parallel fashion via reverse Watson-Crick hydrogen bonding and π-π interactions. Hence, ap- TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.

Antisense oligonucleotide (ASO)-induced cellular signaling pathway alteration is evolving as a promising therapeutic strategy for improving antitumor chemotherapy. However, the inherent instability and inefficiency of ASO delivery remain major hurdles for its application. Herein, we developed a self-assembled DNA nanosponge (DNS) for adsorption and clearance of intracellular miR-21. The densely packed DNA nanostructure is able to provide large amounts of repeated ASO copies for efficient capturing of miR-21 and inhibiting the miRNAs function in mammalian cells. The cell apoptosis-related protein expression (Bcl-2, Bax, and cleaved caspase-3/9) can be obviously interrupted with the delivery of DNS. Besides, we have shown that the DNS can efficiently carry Dox for chemotherapy and inducing tumor cell (MCF-7) apoptosis meanwhile has little affect to normal cells (Hs578 Bst). These polymeric DNS systems mimic the natural RNA circle-based miRNA sponges and have potential to be applied for specific and efficient regulation of gene expression in tumor cells for synergistic antitumor chemotherapy.

Here, we report the synthesis and characterization of four new aroyl-hydrazone derivatives L 1 -L 4 , and their structural as well as biological activities have been explored. In addition to docking with bovine serum albumin (BSA) and duplex DNA, the experimental results demonstrate the effective binding of L 1 -L 4 with BSA protein and calf thymus DNA (ct-DNA) which is in agreement with the docking results. Further biological activities of L 1 -L 4 have been examined through molecular docking with different proteins which are involved in the propagation of viral or cancer diseases. L 1 shows best binding affinity with influenza A virus polymerase PB2 subunit (2VY7) with binding energy -11.42 kcal/mol and inhibition constant 4.23 nm, whereas L 2 strongly bind with the hepatitis C virus NS5B polymerase (2WCX) with binding energy -10.47 kcal/mol and inhibition constant 21.06 nm. Ligand L 3 binds strongly with TGF-beta receptor 1 (3FAA) and L 4 with cancer-related EphA2 protein kinases (1MQB) with binding energy -10.61 kcal/mol, -10.02 kcal/mol and inhibition constant 16.67 nm and 45.41 nm, respectively. The binding energies of L 1 -L 4 are comparable with binding energies of their proven inhibitors. L 1 , L 3 and L 4 can be considered as both 3FAA and 1MQB dual targeting anticancer agents, while L 1 and L 3 are both 2VY7 and 2WCX dual targeting antiviral agents. On the other side, L 2 and L 4 target only one virus related target (2WCX). Furthermore, the geometry optimizations of L 1 -L 4 were performed via density functional theory (DFT). Moreover, all four ligands (L 1 -L 4 ) were characterized by NMR, FT-IR, ESI-MS, elemental analysis and their molecular structures were validated by single crystal X-ray diffraction studies., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

CC-115, a triazole-containing compound, is a dual mammalian target of rapamycin (mTOR)/DNA-dependent protein kinase (DNA-PK) inhibitor currently in clinical trials. To develop this compound further, we investigated factors that may affect cellular response to CC-115. Previously, fatty acid synthase (FASN) was shown to upregulate DNA-PK activity and contribute to drug resistance; therefore, we hypothesized that FASN may affect cellular response to CC-115. Instead, however, we showed that CC-115 is a substrate of ATP-binding cassette G2 (ABCG2), a member of the ATP-binding cassette transporter superfamily, and that expression of ABCG2, not FASN, affects the potency of CC-115. ABCG2 overexpression significantly increases resistance to CC-115. Inhibiting ABCG2 function, using small-molecule inhibitors, sensitizes cancer cells to CC-115. We also found that CC-115 may be a substrate of ABCB1, another known ABC protein that contributes to drug resistance. These findings suggest that expression of ABC transporters, including ABCB1 and ABCG2, may affect the outcome in clinical trials testing CC-115. Additionally, the data indicate that ABC transporters may be used as markers for future precision use of CC-115. SIGNIFICANCE STATEMENT: In this article, we report our findings on the potential mechanism of resistance to CC-115, a dual inhibitor of mTOR and DNA-PK currently in clinical trials. We show that CC-115 is a substrate of ABCG2 and can be recognized by ABCB1, which contributes to CC-115 resistance. These findings provide novel information and potential guidance on future clinical testing of CC-115., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Antisense oligonucleotides (ASO), short single-stranded polymers based on DNA or RNA chemistries and synthesized in vitro, regulate gene expression by binding in a sequence-specific manner to an RNA target. The functional activity and selectivity in the action of ASOs largely depends on the combination of nitrogenous bases in a target sequence. This simple and natural property of nucleic acids provides an attractive route by which scientists can create different ASO-based techniques. Over the last 50 years, planned and realized applications in the field of antisense and nucleic acid nanotechnologies have produced astonishing results and posed new challenges for further developments, exemplifying the essence of the post-genomic era. Today the majority of ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake. This review critically analyzes some successful cases using the antisense approach in medicine to address severe diseases, such as Duchenne muscular dystrophy and spinal muscular atrophy, and suggests some prospective directions for future research. We also examine in detail the elaboration of unmodified insect-specific DNA insecticides and RNA preparations in the areas of agriculture and forestry, a relatively new branch of ASO that allows circumvention of the use of non-selective chemical insecticides. When considering the variety of successful ASO modifications with an efficient signal-to-noise ratio of action, coupled with the affordability of in vitro oligonucleotide synthesis and post-synthesis procedures, we predict that the next half-century will produce a fruitful yield of tools created from effective ASO-based end products.

Culturing of bone marrow cells in serum-free RPMI-1640 medium for 24 h was accompanied by a decrease in the rate of [3H]-thymidine incorporation into DNA. Addition of native apolipoprotein A-I (apoA-I) or plasma LDL and HDL to the culture medium increased this parameter. In contrast to native apoA-I, its modified form decelerated DNA synthesis in bone marrow cells. A similar inhibitory effect of modified protein was observed in cultures of human embryonic kidney cells (HEK293) and in rapidly proliferating mouse macrophage cell line ANA-1. The only exclusion was human myeloid cell line U937: neither native nor modified apoA-I affected DNA synthesis in these cells. Thus, the regulatory effects of apoA-I are tissue-specific; this protein can produce either stimulatory or inhibitory effect on DNA biosynthesis in cells depending on its conformation.

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)- N -(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC 50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

The Ugi four-component-reaction (Ugi 4CR) allowed synthesizing bisamide from carboxylic acid, aldehyde, amine, and isocyanide in one-pot operation. However, introducing 2-isocyano glucose into the Ugi 4CR and investigating the inhibitory effects of Ugi adducts against radical-induced oxidation of DNA remained technical challenges. We herein applied 2-isocyano glucose (acetylation of hydroxy groups) to perform a catalyst-free Ugi 4CR at room temperature. The gallic, ferulic, caffeic, or p-hydroxybenzoic acids, aniline (or benzylamine and p-aminophenol), and formaldehyde acted as reagents. In the case of inhibiting DNA oxidations induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), hydroxy radical, and Cu 2+ /glutathione, the Ugi adduct containing glucose moiety exhibited higher antioxidative activities than the structural analog without glucose moiety involved. It was also proved that high antioxidative property was owing to hydroxy groups in glucose moiety. Therefore, sugar-appended Ugi adducts might hold promising inhibitors for DNA oxidation., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

In the creation of double duplex formation with genomic DNA, the probe must be able to invade and hybridize to the duplex DNA. We designed new photoresponsive probes containing CNV K and CN U; these probes have a high double-duplex invasion capability upon photoirradiation.

Three ruthenium(II) phosphine/diimine/picolinate complexes were selected aimed at investigating anticancer activity against several cancer cell lines and the capacity of inhibiting the supercoiled DNA relaxation mediated by human topoisomerase IB (Top 1). The structure-lipophilicity relationship in membrane permeability using the Caco-2 cells have also been evaluated in this study. SCAR 5 was found to present 45 times more cytotoxicity against breast cancer cell when compared to cisplatin. SCAR 4 and 5 were both found to be capable of inhibiting the supercoiled DNA relaxation mediated by Top 1. Interaction studies showed that SCAR 4 and 5 can bind to DNA through electrostatic interactions while SCAR 6 is able to bind covalently to DNA. The complexes SCAR were found to interact differently with bovine serum albumin (BSA) suggesting hydrophobic interactions with albumin. The permeability of all complexes was seen to be dependent on their lipophilicity. SCAR 4 and 5 exhibited high membrane permeability (P app  > 10 × 10 -6 cm·s -1 ) in the presence of BSA. The complexes may pass through Caco-2 monolayer via passive diffusion mechanism and our results suggest that lipophilicity and interaction with BSA may influence the complexes permeation. In conclusion, we demonstrated that complexes have powerful pharmacological activity, with different results for each complex depending on the combination of their ligands.

The measure of quality in DNA sample processing starts with an effective nucleic acid isolation procedure. Most problems with DNA sample typing can be attributed to low quantity DNA and/or to the presence of inhibitors in the sample. Therefore, establishing which isolation method is best at removing potential inhibitors may help overcome some of the problems analysts encounter by providing useful information in the determination of the optimal approach for any given sample. Direct analysis in real time (DART) mass spectrometry was used in this study to investigate the ability of different extraction methods to remove PCR inhibitors. Methods investigated included both liquid/liquid (phenol-chloroform) and solid phase based robotic procedures, (PrepFiler™ and EZ1 chemistries). Following extraction, samples were analyzed by DART in order to determine the level of remaining inhibitors and then quantified and amplified to determine the effect any remaining inhibitor had on the overall results. The data suggests that organic extraction methods result in detrimental amounts of phenol carryover while automated methods may produce carry-over of bile salts and other chemicals that preferentially bind the solid phase matrix. Both of these effects can have a negative impact in downstream sample processing and genotyping by PCR., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA interstrand crosslinks (ICLs) covalently join the two strands of a DNA duplex and block essential processes such as DNA replication and transcription. Several important anti-tumor drugs such as cisplatin and nitrogen mustards exert their cytotoxicity by forming ICLs. However, multiple complex pathways repair ICLs and these are thought to contribute to the development of resistance towards ICL-inducing agents. While the understanding of many aspects of ICL repair is still rudimentary, studies in recent years have provided significant insights into the pathways of ICL repair. In this perspective we review the recent advances made in elucidating the mechanisms of ICL repair with a focus on the role of TLS polymerases. We describe the emerging models for how these enzymes contribute to and are regulated in ICL repair, discuss the key open questions and examine the implications for this pathway in anti-cancer therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.

Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Cucurbiturils (CB6 and CB7) were shown to inhibit the enzymatically catalyzed restriction of plasmids and linear DNA. This effect can be inverted by supramolecular masking of the macrocycles through competitive complexation with polyamines. These experiments provide supramolecular control of biocatalytic processes.

Objectives: Low blood glucose in neonates predisposes to long term pancreatic damage. We focused on evaluating long term consequences of neonatal hypoglycaemia in pancreatic functions., Methods: Pancreatic function was analysed by measuring DNA/protein synthesis, glucose/ATP uptake in vitro. Gene expression of Pdx1, NeuroD1, Pax4, Bax, caspase 3, Beclin1 were done. Muscarinic receptors were analysed by radio receptor assay., Result: Overall pancreatic efficiency was reduced in one-month-old rats exposed to neonatal hypoglycaemia as indicated by decreased DNA/protein synthesis and glucose/ATP uptake in vitro. Both Pdx1 and Neuro D1 expression were significantly down-regulated whereas Pax4 was up-regulated. Up-regulated Bax, caspase 3 and beclin1 along with reduced muscarinic receptors accounts for activation of cell death pathways., Conclusion: The study revealed a drastic reduction in pancreatic functions along with activation of apoptotic factors in one month old rats exposed to neonatal hypoglycaemia.

We tested the antiproliferative activity and mechanism of the action of several novel aminoacridine derivatives. Six different cancer cell lines were used to evaluate the potential cytotoxic effect of eleven aminoacridine-based molecules. A standard MTT assay was used for cell bioavailability analysis. Additionally, the potential cytotoxic effect of the tested compounds on non-cancer cells was investigated in rat skeletal muscle myotubes (L6) and in bovine aortic smooth muscle cells. In order to investigate whether the DNA binding activity of tested compounds correlated with their cytotoxic effect, circular dichroism (CD) measurement and DNA T4 ligase assay were performed. Finally, the potential mutagenic activity of the lead compound 5 was investigated. The cytotoxic effect of compound 5 in cancer cells was obtained in lower concentrations than the well-known: 9- aminoacridine based drug, amsacrine. The lead compound binds to DNA, but in a different mode than the parent molecules. Additionally, compound 5 was not cytotoxic in the effective range of concentrations in non-cancer cells. In identical concentrations, the parent compound (9-aminoacridine) and amsacrine were extremely toxic for both types of these normal cells. Finally, based on CD measurement and T4 ligase assay, it was confirmed that 5 binds to DNA but in different from the parent compounds manner. Important to mention, that compound 5 might have increased mutagenic activity which must be verified in vivo. Based on these in vitro results, we conclude that 5 is a more potent and more selective antiprolifirative compound than amsacrine. Compound 5 was also more effective in HepG2 and P-12 cells. Thus, 5 is suitable for future in vivo biological evaluation and its structure might be used as a basis for developing novel anticancer drugs.

A series of 1,3,6-triphenylpyrazolo[3,4-b]pyridin-4-one derivatives was designed, synthesized and evaluated for cytotoxic activity in A375 human melanoma and human erythroleukemia (HEL) cells. The new pyrazolopyridones displayed comparable activities to the antitumor compound etoposide. The inhibitory effect of compounds 17, 18, 27 and 32 against topoisomerase II-mediated cleavage activities was measured finding good correlation with the results obtained from MTS assay. Docking studies into bacterial topoisomerase II (DNA Gyrase), topoisomerase IIα and topoisomerase IIβ binding sites in the DNA binding interface were performed.

Increasing the affinity of diamidines for AT-rich regions of DNA has long been an important goal of medicinal chemists who wanted to improve the antiparasitic and antifungal properties of that class of derivatives. In recent years it was demonstrated that diamidines could interfere with many other biomolecular targets including ion channels as well as enzymes and modulate some RNA-protein, DNA-protein, and protein-protein interactions. It is therefore not surprising that diamidines now emerge as novel potential drug candidates for the treatment of various diseases, i.a. neurodegenerative disorders, acidosis-related pathological conditions, hypertension, thrombosis, type 2 diabetes, myotonic dystrophy, and cancers. A summary of the most striking results obtained to date in those domains is presented is this review., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: Maximum tolerated dose, safety, pharmacokinetics, and pharmacodynamics were assessed in this phase 1 study of PNT2258, a BCL-2-targeted liposomal formulation of a 24-base DNA oligonucleotide called PNT100., Methods: Patients with malignant solid tumors were assigned sequentially to one of ten dose-escalation cohorts of PNT2258 at 1, 2, 4, 8, 16, 32, 64, 85, 113, and 150 mg/m(2) administered intravenously on days 1 through 5 of each 21-day cycle. Pharmacokinetics were determined on days 1 and 5 of the first cycle. Lymphocyte and platelets concentrations were measured for evidence of BCL2-targeted effect. CT scans were used to identify radiologic evidence of anti-tumor effect., Results: Twenty-two subjects received PNT2258, and the maximum tolerated dose for PNT2258 was not reached. Doses at or above 32 mg/m(2) resulted in exposure to PNT2258 above the exposure level required for anti-tumor activity in preclinical xenograft testing of 22,377 ng h/ml (PK analysis 2012). Fatigue was the most commonly reported adverse event. Dose-limiting toxicity, manifesting as a transient increase in aspartate aminotransferase, occurred at 150 mg/m(2), the highest dose tested. Four subjects, two each with diagnosis of non-small-cell lung cancer and sarcoma, treated at doses of 64 mg/m(2) or higher, remained on study for 5-8 cycles., Conclusions: PNT2258 was safe and well tolerated at the doses tested up to 150 mg/m(2). Exposure to PNT2258 resulted in clinically manageable decreases in lymphocyte and platelet concentrations.

We have shown that polyclonal antibody IgG to native DNA leads to increased levels of apoptosis of moonuclear cells of healthy persons in vitro. Possible biological role of antibodies to DNA is discussed.

The hydroxyl-substituted benzoic acid (as phenyl group A in the product), aniline (as phenyl group B in the product), benzaldehyde (as phenyl group C in the product), and four isocyanides are employed to synthesize bis-amide via an Ugi four-component reaction. The effects of the obtained 20 bis-amides on quenching radicals and inhibiting DNA oxidation are estimated. It is found that the antioxidant effectiveness of bis-amide generated by hydroxyl groups is markedly influenced by the structural feature derived from isocyanide. The phenolic hydroxyl group attaching to phenyl group A plays a major role in scavenging radicals, and the radical-scavenging property is reinforced by the structural moiety introduced from ferrocenylmethyl isocyanide. The same conclusion is also obtained when bis-amides are used to inhibit DNA oxidation. It is still found that the ferrocenylmethyl moiety enhances the antioxidant effect of hydroxyl group at phenyl group A in protecting DNA against the oxidation. Moreover, when the bis-amide is prepared by the same isocyanide, e.g. ethyl isocyanoacetate, it is found that the hydroxyl group at phenyl group C plays the major role in inhibiting DNA oxidation, followed by the hydroxyl groups attaching to phenyl groups B and A.

Cenderitide, also known as CD-NP, is a designer peptide developed by combining native mammalian c-type natriuretic peptide (CNP) and the C-terminus isolated from the dendroapis natriuretic peptide (DNP) of the venom from the green mamba. In early studies, intravenous and subcutaneous infusion of cenderitide was reported to reduce left ventricular (LV) mass and ameliorate cardiac remodelling. In this work, biodegradable polymeric films encapsulating CD-NP were developed and were investigated for their in vitro release and degradation characteristics. Subsequently, the bioactivity of released peptide and its effects on human cardiac fibroblast (HCF) were explored. We achieved sustained release from three films with low, intermediate and high release profiles for 30 days. Moreover, the bioactivity of released peptide was verified from the elevated production of cyclic guanosine monophospate (cGMP). The CD-NP released from films was able to inhibit the proliferation of hypertrophic HCF as well as suppress DNA synthesis in HCF. Furthermore, the sustained delivery from films showed comparable or superior suppressive actions on hypertrophic HCF compared to daily infusion of CD-NP. The results suggest that these films could be used to inhibit fibrosis and reduce cardiac remodelling via local delivery as cardiac patches.

The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.

Some new psoralen derivatives were synthesized and evaluated as inhibitors of NF-κB/DNA interaction, with the aim to investigate the structural determinants required to inhibit this interaction. Starting from molecular docking studies, several possible protein binding sites were proposed and several three-dimensional quantitative structure-activity relationship (3D-QSAR) models were built using the docked poses of 29 (the most active psoralen in the series) as templates for alignment of the inhibitors. The selected best model was validated through the prediction of the activity of 17 novel compounds. All the experimental data agreed with the computational experiments, supporting the reliability of the computational approach. The hypothesis about the interaction with NF-κB was also supported by surface plasmon resonance based assays using compound 29. All the collected data allowed the identification of compound 29 as a potential candidate for the development of pharmaceutical strategies against the inflammatory phenotype of cystic fibrosis.

The extensive use and misuse of antibiotics in medicine result in the emergence of multidrug-resistant bacteria, creating an urgent need for the development of new chemotherapeutic agents. Nowadays, antimicrobial peptides are widely recognized as a class of promising candidates with activity against multidrug-resistant bacteria. NK-18 is a truncated peptide derived from NK-Lysin, an effector of cytotoxic T cells and natural killer cells. In this study, we studied the antibacterial mechanism of action of NK-18. The results revealed that NK-18 has potent antibacterial activity against Escherichia coli and Staphylococcus aureus. According to our findings, NK-18 is membrane active and its target of action is not only the bacterial membrane but also the DNA in the cytoplasm. The double targets of NK-18 make it difficult for bacteria to generate resistance, which may present a new strategy to defend against multidrug-resistant bacteria and provide a new lead in the design of potent antimicrobial peptides with therapeutic application in the presence of increasing resistance to conventional antibiotics.

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The influence of a number of usnic acid derivatives on auto(polyADP-ribosyl)ation catalyzed by PARP1 and DNA synthesis catalyzed by DNA polymerase β was studied. The derivatives of usnic acid containing aromatic substituents were shown to be moderate inhibitors of PARP1. The presence of both usnic acid tricyclic structure and aromatic substituent at any position of the molecule is a key factor for the inhibitory action. In the case of DNA polymerase β, no relationship between the structure and inhibitory properties has been found with the only exception. Derivatives with modified ring A showed mild activation of DNA synthesis catalyzed by DNA polymerase β.

Purpose: At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR-overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo., Experimental Design: To analyze its mixed EGFR-DNA targeting potential, we performed an enzyme linked immunosorbent assay (ELISA) and western blotting analysis of EGFR phosphorylation in cells stimulated with EGF. DNA damage was analyzed using the comet assay, and apoptosis quantitated by annexin V binding assay. Growth inhibition in vitro was determined by the sulforhodamine B (SRB) assay and in vivo efficacy analyzed in male CD-1 nude mice., Results: The results showed that: Under physiological conditions, JDF12 was hydrolyzed to JDF04R and both agents were capable of inhibiting isolated EGFR tyrosine kinase (TK) and EGFR phosphorylation in EGF-stimulated cells. JDF12 significantly damaged DNA, induced apoptosis in DU145 cells and was up to 2-10-fold more potent than equieffective combinations of MTZ and JDF04R or Iressa in a panel that also included LNCaP and its EGFR and ErbB2 transfectants. In vivo, it induced significant antitumor activity in a DU145 xenograft model., Conclusions: The results suggest that the superior cytotoxicity of JDF12 when compared with MTZ and JDF04R may be imputed to its potent EGFR-DNA targeting properties and confirm the ability of this novel strategy to confer EGFR targeting properties to a classical alkylator., (Copyright © 2012 Wiley Periodicals, Inc.)

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)(PPh(3))] (2), [Ag(Tpms)(PCy(3))] (3), [Ag(PTA)][BF(4)] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh(3) = triphenylphosphane, PCy(3) = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane} have been synthesized and fully characterized by elemental analyses, (1)H, (13)C, and (31)P NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N(3)-facially coordinating mode, while in 2 and 5 a N(2)O-coordination is found, with the SO(3) group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA159), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of Ag complexes shows that the fluorescence quenching of DNA-EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO(3), which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO(3)≫ 4., (© 2011 American Chemical Society)

ZR2003 is a type II of combi-molecule designed to target DNA and the epidermal growth factor receptor (EGFR) without requirement for hydrolysis. In human tumour cell lines cultured as monolayers, it showed 6.5-35 fold greater activity than Iressa. Further evaluation in 3D organ-like multilayer aggregates showed that it could block proliferation at submicromolar level. However, despite the superior potency of ZR2003 over Iressa in vitro, its activity xenograft models was not significantly different from that of Iressa. To rationalize these results, we determined the tumour concentration of both ZR2003 and Iressa in vivo and more importantly in vitro in multicellular aggregates. The results showed that in A431 and 4T1 xenografts, the level of ZR2003 absorbed in the tumours were consistently 2-fold less than those generated by Iressa. Likewise, in the multicellular aggregates model, the penetration of ZR2003 was consistently lower than Iressa. In serum containing media, the level of extractable or free ZR2003 was also inferior to those of Iressa. The results from this bioanalytical study, suggest that the discrepancy between the in vitro and in vivo potency of ZR2003 when compared with Iressa, may be imputed to its significantly lower tumour concentration., (Copyright © 2011. Published by Elsevier B.V.)

Designing proteins or peptides that bind native protein targets can aid the development of novel reagents and/or therapeutics. Rational design also tests our understanding of the principles underlying protein recognition. This article describes several strategies used to design peptides that bind to the basic region leucine zipper (bZIP) domain of the viral transcription factor BZLF1, which is encoded by the Epstein-Barr virus. BZLF1 regulates the transition of the Epstein-Barr virus from a latent state to a lytic state. It shares some properties in common with the more studied human bZIP transcription factors, but also includes novel structural elements that pose interesting challenges to inhibitor design. In designing peptides that bind to BZLF1 by forming a coiled-coil structure, we considered both affinity for BZLF1 and undesired self-association, which can weaken the effectiveness of an inhibitor. Several designed peptides exhibited different degrees of target-binding affinity and self-association. Rationally engineered molecules were more potent inhibitors of DNA binding than a control peptide corresponding to the native BZLF1 dimerization region itself. The most potent inhibitors included both positive and negative design elements and exploited interaction with the coiled-coil and basic DNA-binding regions of BZLF1., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.

Threading intercalators are high affinity DNA binding agents that bind by inserting a chromophore into the duplex and locating one group in each groove. The first threading intercalators that can be conjugated to acids, sulfonic acids and peptides to target them to duplex DNA are described, based upon the well studied acridine-3- or 4-carboxamides. Cellular uptake of the parent acridine is rapid and it can be visualized in the nucleus of cells. Both the parent compounds and their conjugates maintain antitumor activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.