Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by ¹H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)₄ and the c-myc promoter Pu22 sequence. We also performed ¹H and ³¹P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.     

Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U‐turn and a looped‐out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U‐turn fold retained biological activity. An improvement of stacking interactions in the U‐turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U‐turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.     

Peptide Fragments of Odin‐Sam1: Conformational Analysis and Interaction Studies with EphA2‐Sam

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin‐Sam1, binds to the Sam domain of the EphA2 receptor (EphA2‐Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin‐Sam1 associates with EphA2‐Sam by adopting a “mid‐loop/end‐helix” model. In this study three peptide sequences, encompassing the mid‐loop interacting portion of Odin‐Sam1 and its C‐terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2‐Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2‐Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti‐cancer therapeutics as inhibitors of EphA2‐Sam heterotypic interactions.     

Probing the Interaction of Huntingtin Exon-1 Polypeptides with the Chaperonin Nanomachine GroEL

Huntington's disease arises from polyQ expansion within the exon-1 region of huntingtin (htt^ex1), resulting in an aggregation-prone protein that accumulates in neuronal inclusion bodies. We investigate the interaction of various htt^ex1 constructs with the bacterial analog (GroEL) of the human chaperonin Hsp60. Using fluorescence spectroscopy and electron and atomic force microscopy, we show that GroEL inhibits fibril formation. The binding kinetics of htt^ex1 constructs with intact GroEL and a mini-chaperone comprising the apical domain is characterized by relaxation-based NMR measurements. The lifetimes of the complexes range from 100 to 400 μs with equilibrium dissociation constants (K_D) of ∼1–2 mM. The binding interface is formed by the N-terminal amphiphilic region of htt^ex1 (which adopts a partially helical conformation) and the H and I helices of the GroEL apical domain. Sequestration of monomeric htt^ex1 by GroEL likely increases the critical concentration required for fibrillization.     

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1^413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1^413–511. Here, we investigated the interaction of hORC1^413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1^413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1^413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1^413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.     

咪唑喹啉衍生物与小牛胸腺DNA作用机理研究

对于咪唑喹啉衍生物(IQDs)与小牛胸腺脱氧核糖核酸(CT-DNA)的相互作用机理,采用荧光光谱法、黏度法、热变性研究和琼脂糖凝胶电泳法等实验技术进行研究.实验结果显示,咪唑喹啉衍生物Ⅰ以嵌插模式与CT-DNA作用,衍生物Ⅱ以沟槽模式与CT-DNA作用,且衍生物Ⅰ与CT-DNA的作用强于衍生物Ⅱ,而衍生物Ⅲ几乎不与CT...     

Molecular Recognition of Parallel DNA Quadruplex d(TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stoichiometry Leading to Thermal Stabilization and Telomerase Inhibition

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti‐cancer drug with two non‐planar side chains, with heptamer G‐quadruplex d(TTAGGGT)₄, which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔT_m=13–20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side‐chain interaction at the grooves. The inhibition of telomerase (IC₅₀=2 μm), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel‐stranded G‐quadruplex; this might serve as a platform for the rational design of new drugs.     

Unraveling the Interaction between the LPS O‐Antigen of Burkholderia anthina and the 5D8 Monoclonal Antibody by Using a Multidisciplinary Chemical Approach,with Synthesis,NMR, and Molecular Modeling Methods

The interaction between the O‐chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide‐specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8‐bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr‐NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O‐antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O‐chain have been determined.     

Effect of substituting CaO with BaO on the viscosity and structure of CaO‐BaO‐SiO2‐MgO‐Al2O3 slags

In this study, the effect of CaO and BaO substitution on the viscosity and structure of CaO‐BaO‐SiO₂‐MgO‐Al₂O₃ slags was investigated. The results showed that the viscosity increased with an increase in the BaO substitution concentration, which was correlated to an increase in the degree of polymerization (DOP) of the slag structural units as the activation energy increased from 207.9 to 263.8 kJ/mol for viscous flow. Deconvolution and area integration of the Raman spectrum of the slag revealed that the ratio of Q³/Q² (Qⁱ, i is the number of O⁰ in a [SiO₄]‐tetrahedral unit) increased and NBO/Si (nonbridging oxygen per unit silicon atom) decreased with higher BaO content. It was also observed from the ²⁷Al magic angles pinning nuclear magnetic resonance (²⁷Al MAS‐NMR) spectrum that the relative proportion of Al^IV increased, while that of Al^V decreased because of the decrease in the percentage of nonbridging oxygen (O^?), indicating the polymerization of the slag. O_1s X‐ray photoelectron spectroscopy (XPS) was also carried out to semi‐quantitatively analyze the various types of oxygen anions present in the slag. The XPS results correlated well with the results obtained from the analysis of the Raman and ²⁷Al MAS‐NMR spectra of the slags and its viscous behavior.     

Intramolecular Interaction with the E6 Region Stabilizes the Closed Conformation of the N-SH2 Domain and Concurs with the Self-Inhibitory Docking in Downregulating the Activity of the SHP2 Tyrosine Phosphatase: A Molecular Dynamics Study

The localization and activity of the SHP2 tyrosine phosphatase across different cellular compartments to the target substrates are steered by the binding of phosphotyrosine (pY) peptides to the tandem SH2 domains. The most N-terminal domain (N-SH2) can also keep the enzyme inactive by intramolecular occlusion of the catalytic site. Enzyme activity can be recovered by an allosteric disruption of this self-inhibitory docking upon the binding of pY peptides to the N-SH2 domain. Prior to this, the N-SH2 domain must abandon the closed conformation because it impedes the access of pY peptides to the binding cleft. Although it cooperates with the self-inhibitory docking in the negative regulation of the phosphatase activity, the structural determinants of the stability of the closed conformation in the self-inhibited phosphatase are still elusive. To address this issue, a molecular dynamics simulation study is carried out. It is shown that the closed conformation is stabilized by the interaction of the N-SH2 domain with a conserved peptide portion in the region encoded by PTPN11 exon 6 (E6).     

Targeting zinc finger domains with small molecules: solution structure and binding studies of the RanBP2-type zinc finger of RBM5

The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies.