Targeting human telomeric DNA quadruplex with novel berberrubine derivatives: insights from spectroscopic and docking studies

Study on bioactive molecules, capable of stabilizing G-Quadruplex structures is considered to be a potential strategy for anticancer drug development. Berberrubine (BER) and two of its analogs bearing alkyl phenyl and biphenyl substitutions at 13-position were studied for targeting human telomeric G-quadruplex DNA sequence. The structures of berberrubine and analogs were optimized by density functional theory (DFT) calculations. Time-dependent DFT (B3LYP) calculations were used to establish and understand the nature of the electronic transitions observed in UV–vis spectra of the alkaloid. The interaction of berberrubine and its analogs with human telomeric G-quadruplex DNA sequence 5′-(GGGTTAGGGTTAGGGTTAGGG)-3′ was investigated by biophysical techniques and molecular docking study. Both the analogs were found to exhibit higher binding affinity than natural precursor berberrrubine. 13-phenylpropyl analog (BER1) showed highest affinity [(1.45 ± 0.03) × 10⁵ M^?1], while the affinity of the 13-diphenyl analog (BER2) was lower at (1.03 ± 0.05) × 10⁵ M^?1, and that of BER was (0.98 ± 0.03) × 10⁵ M^?1. Comparative fluorescence quenching studies gave evidence for a stronger stacking interaction of the analog compared to berberrubine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberrubine. Molecular docking study showed that each alkaloid ligand binds primarily at the G rich regions of hTelo G4 DNA which makes them G specific binder towards hTelo G4 DNA. Isothermal titration calorimetry studies of quadruplex–berberrubine analog interaction revealed an exothermic binding that was favored by both enthalpy and entropy changes in BER in contrast to the analogs where the binding was majorly enthalpy dominated. A 1:1 binding stoichiometry was revealed in all the systems. This study establishes the potentiality of berberrubine analogs as a promising natural product based compounds as G-quadruplex-specific ligands.     

线粒体DNA靶向治疗

线粒体(mitochondrion)是真核生物细胞中的一种非常重要的细胞器,含有独立于细胞核染色体外的遗传物质,通过氧化磷酸化产生ATP,是细胞的能量工厂,与细胞分化、信号转导、代谢稳态等过程密切联系。线粒体功能的紊乱与癌症、神经退行性疾病、糖尿病等许多疾病的发生、发展及治疗息息相关。线粒体在细胞命运中扮演的关键角色,使对线粒体这一特殊细胞器的探索成为生命科学研究热点之一。人线粒体DNA(mitochondrial DNA, mtDNA)是一相对保守且仅16 kb的环状双链DNA分子,只含37个基因,但这些基因都是维持线粒体功能稳定必不可少的部分。随着对线粒体功能认识的不断深入,研究人员发现mtDNA突变,会导致活性氧自由基过量产生,从而引起细胞衰老,甚至引发诸多疾病,例如遗传性视神经病变、线粒体脑肌病伴高乳酸血症和卒中样发作综合征等。但是,目前针对这些线粒体基因疾病尚无非常有效的治疗手段。为了进一步了解这一关键细胞器,研究人员开发了一些有效的方法来突破线粒体的复杂屏障。本文将重点介绍并讨论近几年靶向mtDNA的研究进展,主要从药物修饰、材料递送、基因编辑等方面进行了总结,希望能为推动线粒体的研究提供一些新的思路。     

82 Targeting intramolecular DNA structures with complementary strands

Antisense, antigene, and siRNA strategies are currently used to control the expression of genes. To this end, our laboratory is mimicking the targeting of mRNA by reacting DNA stem-loop motifs with their partially complementary strands. Specifically, we used a combination of isothermal titration (ITC), differential scanning calorimetry (DSC), and temperature-dependent UV spectroscopy to investigate: (1) the unfolding of a pseudoknot and a complex containing joined triplex-duplex motifs (shown below); and (2) the reaction of these compact structures with single strands that are complementary to the bases in the loops and to a portion of their stem.     

Targeting DNA 5mCpG sites with chimeric endonucleases

Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the ⁵mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C⁵mCGG)—and M.SssI (⁵mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA (⁵mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified λ DNA indicates that cleavage takes place outside the ⁵mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N_4-17⁵mCpG, where / indicates the cleavage site). Such ⁵mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.     

Targeting the hemangioblast with a novel cell type-specific enhancer

Background

Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker.

Results

We report the identification of a hemangioblast-specific enhancer (Hb) located in the cis-regulatory region of chick Cerberus gene (cCer) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis.

Conclusions

We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development.     

Targeting DNA with triplex-forming oligonucleotides to modify gene sequence

Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification.     

Targeting human telomeric G-quadruplex DNA with oxazole-containing macrocyclic compounds

Oxazole-containing macrocycles, which include the natural product telomestatin, represent a promising class of anticancer agents that target G-quadruplex DNA. Two synthetic hexaoxazole-containing macrocyclic compounds (HXDV and HXLV-AC) have been characterized with regard to their cytotoxic activities versus human cancer cells, as well as the mode, thermodynamics, and specificity with which they bind to the intramolecular (3+1) G-quadruplex structural motif formed in the presence of K(+) ions by human telomeric DNA. Both compounds exhibit cytotoxic activities versus human lymphoblast (RPMI 8402) and oral carcinoma (KB3-1) cells, with associated IC(50) values ranging from 0.4 to 0.9muM. The compounds bind solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. Binding to the quadruplex is associated with a stoichiometry of two ligand molecules per DNA molecule, with one ligand molecule binding to each end of the host quadruplex via a nonintercalative "terminal capping" mode of interaction. For both compounds, quadruplex binding is primarily entropy driven, while also being associated with a negative change in heat capacity. These thermodynamic properties reflect contributions from favorable ligand-induced alterations in the loop configurational entropies of the quadruplex, but not from changes in net hydration. The stoichiometry and mode of binding revealed by our studies have profound implications with regard to the number of ligand molecules that can potentially bind the 3-overhang region of human telomeric DNA.     

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.     

Targeting novel folds for structural genomics

The ultimate goal of structural genomics is to obtain the structure of each protein coded by each gene within a genome to determine gene function. Because of cost and time limitations, it remains impractical to solve the structure for every gene product experimentally. Up to a point, reasonably accurate three‐dimensional structures can be deduced for proteins with homologous sequences by using comparative modeling. Beyond this, fold recognition or threading methods can be used for proteins showing little homology to any known fold, although this is relatively time‐consuming and limited by the library of template folds currently available. Therefore, it is appropriate to develop methods that can increase our knowledge base, expanding our fold libraries by earmarking potentially “novel” folds for experimental structure determination. How can we sift through proteomic data rapidly and yet reliably identify novel folds as targets for structural genomics? We have analyzed a number of simple methods that discriminate between “novel” and “known” folds. We propose that simple alignments of secondary structure elements using predicted secondary structure could potentially be a more selective method than both a simple fold recognition method (GenTHREADER) and standard sequence alignment at finding novel folds when sequences show no detectable homology to proteins with known structures. Proteins 2002;48:44–52. © 2002 Wiley‐Liss, Inc.     

Targeting critical regions in genomic DNA with AT-specific anticancer drugs

Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.     

Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.     

Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.     

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P₃). PtdIns(3,4,5)P₃ activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P₃ to PH domain targets. Using an in vitro PtdIns(3,4,5)P₃-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P₃ antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P₃-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P₃ antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P₃/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.     

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.     

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P 3). PtdIns(3,4,5)P 3 activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P 3 to PH domain targets. Using an in vitro PtdIns(3,4,5)P 3-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P 3 antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P 3-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P 3 antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P 3/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.