DNA-binding Ligands Research Articles

Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide

Cross-talk between nuclear receptors involved in the control of drug metabolism is being increasingly recognised as a source of drug side effects. Omeprazole is a well known activator of the aryl hydrocarbon receptor (AhR). We investigated the regulation of AhR by omeprazole-sulphide, a degradation metabolite of omeprazole, using CYP1A mRNA induction, reporter gene assay, receptor DNA binding, ligand binding, nuclear translocation, trypsin digests, and drug metabolism analysis in mouse Hepa-1c1c7, human HepG2 cells and primary human hepatocytes. Omeprazole-sulphide is a pure antagonist of AhR in Hepa-1c1c7 and HepG2 hepatoma cell lines. In Hepa-1c1c7 cells, omeprazole-sulphide is a ligand of AhR, inhibits AhR activation to a DNA-binding form, induces a specific pattern of AhR trypsin digestion and inhibits AhR nuclear translocation and subsequent degradation in response to 2,3,7,8-tetrachlorodibenzo- p-dioxin. However, in highly differentiated primary human hepatocytes treated with rifampicin an agonist of the pregnane X receptor (PXR), omeprazole-sulphide behaves as an agonist of AhR. Inhibition of drug metabolizing enzymes by ketoconazole restores the antagonist effect of omeprazole-sulphide. Metabolic LC/MS analysis reveals that omeprazole-sulphide (AhR antagonist) is efficiently converted to omeprazole (AhR activator) by cytochrome P450 CYP3A4, a target gene of PXR, in primary human hepatocytes but not in hepatoma cells in which PXR is not expressed. This report provides the first evidence for a cross-talk between PXR/CYP3A4 and AhR. In addition, it clearly shows that conclusions drawn from experiments carried out in cell lines may lead to erroneous in vivo predictions in man.

Molecular characterization of the aryl hydrocarbon receptors (AHR1 and AHR2) from red seabream ( Pagrus major)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs). Bony fishes exposed to PHAHs exhibit a wide range of developmental defects. However, functional roles of fish AHR are not yet fully understood, compared with those of mammalian AHRs. To investigate the potential sensitivity to PHAHs toxic effects, an AHR cDNA was initially cloned and sequenced from red seabream ( Pagrus major), an important fishery resource in Japan. The present study succeeded in identifying two highly divergent red seabream AHR cDNA clones, which shared only 32% identity in full-length amino acid sequence. The phylogenetic analysis revealed that one belonged to AHR1 clade (rsAHR1) and another to AHR2 clade (rsAHR2). The rsAHR1 encoded a 846-residue protein with a predicted molecular mass of 93.2 kDa, and 990 amino acids and 108.9 kDa encoded rsAHR2. In the N-terminal half, both rsAHR genes included bHLH and PAS domains, which participate in ligand binding, AHR/ARNT dimerization and DNA binding. The C-terminal half, which is responsible for transactivation, was poorly conserved between rsAHRs. Quantitative analyses of both rsAHRs mRNAs revealed that their tissue expression profiles were isoform-specific; rsAHR1 mRNA expressed primarily in brain, heart, ovary and spleen, while rsAHR2 mRNA was observed in all tissues examined, indicating distinct roles of each rsAHR. Furthermore, there appeared to be species-differences in the tissue expression profiles of AHR isoforms between red seabream and other fish. These results suggest that there are isoform- and species-specific functions in piscine AHRs.

Steroids and the Scientist

Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor alpha); 2) liver X receptor beta (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor beta, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor beta agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.

369. Targeted Gene Modification Via Triple Helix-Forming Oligonucleotides

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. The resulting triplexes have been shown to constitute a DNA structure that can provoke binding by the damage recognition factors, XPA and RPA, and to stimulate DNA repair via the nucleotide excision repair (NER) pathway. Based on these observations, experiments were undertaken to determine the extent to which triplex structures can stimulate recombination events within genomic DNA in a site-directed manner. Studies in which TFOs were targeted to episomal SV40 genomes in COS cells suggested that intermolecular triplexes could stimulate recombination between tandemly repeated reporter gene sequences in a pathway dependent on XPA and other NER factors. Experiments in mouse Ltk-cells containing a dual TK gene substrate demonstrated that intrachromosomal gene conversion could be provoked by high affinity triplex formation, at frequencies as high as 1-2%, if the TFOs were delivered into the cells by microinjection. Recent work in both COS cells using an episomal target and in human cell free extracts using plasmid substrates has further demonstrated that triplex formation can promote intermolecular recombination. TFOs were shown to induce recombination between a target gene and short DNA fragment, in cases where the fragment is either covalently linked to or is completely separate from the TFO. In the extracts, immunodepletion and complementation with purified proteins demonstrated a requirement for XPA and for the human recombinase, HsRad51, in the reaction. In recent work, the ability of TFOs to stimulate recombination and thereby mediate gene repair in a dose-dependent manner at a chromosomal locus in mammalian cells has been established. Overall, this work raises the possibility that DNA binding ligands such as TFOs and related molecules such as a peptide nucleic acids (PNAs) may be useful in strategies designed to mediate targeted genome modification and genetic therapy by stimulating recombination in site-specific manner.

Contribution of Hydrophobicity to Thermodynamics of Ligand-DNA Binding and DNA Collapse

The importance of understanding the dynamics of DNA condensation is inherent in the biological significance of DNA packaging in cell nuclei, as well as for gene therapy applications. Specifically, the role of ligand hydrophobicity in DNA condensation has received little attention. Considering that only multivalent cations can induce true DNA condensation, previous studies exploring monovalent lipids have been unable to address this question. In this study we have elucidated the contribution of the hydrophobic effect to multivalent cation- and cationic lipid-DNA binding and DNA collapse by studying the thermodynamics of cobalt hexammine-, spermine-, and lipospermine-plasmid DNA binding at different temperatures. Comparable molar heat capacity changes (ΔCp) associated with cobalt hexammine- and spermine-DNA binding (−23.39 cal/mol K and −17.98 cal/mol K, respectively) suggest that upon binding to DNA, there are insignificant changes in the hydration state of the methylene groups in spermine. In contrast, the acyl chain contribution to the ΔCp of lipospermine-DNA binding (ΔCp ϕ=ΔCp lipospermine−ΔCp spermine) is significant (−220.94 cal/mol K). Although lipopermine induces DNA ordering into “tubular” suprastructures, such structures do not assume toroidal dimensions as observed for spermine-DNA complexes. We postulate that a steric barrier posed by the acyl chains in lipospermine precludes packaging of DNA into dimensions comparable to those found in nature.

The Nuclear Receptor Coactivator PGC-1α Exhibits Modes of Interaction with the Estrogen Receptor Distinct From those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1alpha co-regulator (residues 91-408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1alpha to ER. Anisotropy measurements revealed that the affinity of this PGC-1alpha fragment for human ERalpha and beta was fairly strong in the presence of estradiol (approximately 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-191-408 exhibited ligand-independent interactions with ER, particularly with ERbeta (Kd approximately 30 nM). Competition experiments of the complex between ERalpha and fluorescently labelled PGC-1 91-408 with unlabelled SRC-1 570-780 showed that PGC-1 91-408 was an efficient competitor of SRC-1 570-780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERalpha, SRC-1 570-780 and PGC-1 91-408. GST-pull-down experiments with deletion mutants of ERalpha revealed that the constitutive binding of PGC-1 91-408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1alpha confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633-675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.

Molecular dynamics simulations and free energy calculations of netropsin and distamycin binding to an AAAAA DNA binding site

Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex d(CGCGAAAAACGCG)·d(CGCGTTTTTCGCG). The relative free energy of binding of the two non-covalently interacting ligands was calculated using the thermodynamic integration method and reflects the experimental result. From 2 ns simulations of the ligands free in solution and when bound to DNA, the mobility and the hydrogen-bonding patterns of the ligands were studied, as well as their hydration. It is shown that even though distamycin is less hydrated than netropsin, the loss of ligand–solvent interactions is very similar for both ligands. The relative mobilities of the ligands in their bound and free forms indicate a larger entropic penalty for distamycin when binding to the minor groove compared with netropsin, partially explaining the lower binding affinity of the distamycin molecule. The detailed structural and energetic insights obtained from the molecular dynamics simulations allow for a better understanding of the factors determining ligand–DNA binding.

Molecular Mechanisms and Kinetics between DNA and DNA Binding Ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding α-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on λ-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.

Pregnane X receptor: Predicting and preventing drug interactions

The pregnane X receptor, PXR, is a member of the nuclear receptor family of ligand-activated transcription factors [1]. The hormones that act through the nuclear receptors include the steroid hormones, thyroid hormone, retinoic acid, and vitamin D [2]. They are all small, lipophilic molecules that diffuse into the cell and interact with their cognate receptors. The receptors bind to DNA and regulate gene transcription directly. There are 48 nuclear receptors in the human genome that share structural features, including conserved DNA binding and ligand binding domains.

Heterogeneity in the radiosensitizing effects of the DNA ligand hoechst-33342 in human tumor cell lines.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I & II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 &U-87) and squamous carcinoma (4197 &4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc). Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&7 fold increase in the cell death (loss of clonogenic cell survival) in U-87&BMG-1 and a 3 fold increase in 4197 &4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis.

Potential of Transferrin and Transferrin Conjugates of Liposomes in Drug Delivery and Targeting

Targeted drug delivery has gained recognition in modern therapeutics and attempts are being made to explore the potential of cell biology-related bioevents in the development of specific and target-oriented systems. In connection to modern therapeutic systems, most of the emphasis has been laid upon the bioconjugated drug delivery systems. Bioconjugates involve the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The nature of the linking agent between the pharmacologic agent and the delivery-augmenting moiety dictates the degree of successful delivery and its outcome. The component for the bioconjugated drug delivery includes receptors and ligands, where the receptors act as molecular targets or portals whereas ligands with receptors provide selective and specific trafficking towards the targeting site. Recently, a number of bioconjugated systems have been discovered for the site-specific presentation and delivery of various bioactive substances using biorelevant ligands, including antibodies, glycoprotein, viral proteins, and molecules of endogenous origin. In this review, the potential of transferrin (Tf) and Tf conjugates of liposomes in site-specific drug delivery systems are discussed. Tf is an abundant component of serum with the capacity to bind and transport iron, while Tf receptor (TfR), a dimeric transmembrane glycoprotein, is present on the surface of the most proliferating, higher eukaryotic cells. Tf expression is also found in nonproliferating tissues, such as hepatocytes, tissue macrophages, pituitary cells, pancreatic islet cells, and the endothelium of brain capillaries. Tumor cells frequently carry elevated numbers of TfRs compared with corresponding normal cells, and reduced serum levels of Tf are often observed in patients with tumors. In the past, various strategies have been developed, which include coupling of the liposomal surface with Tf by using various linking agents. Low-molecular weight drugs and proteins as well as liposomes can be linked with Tf. The Tf-coupled vesicular system is physicochemically stable in the bioenvironment and is site-specific. The aim of coupling liposomes with Tf is to improve the physical and biochemical stability of liposomes and make them appropriate for targeting specific organs and cells. Tf may be widely applied either as a carrier or targeting ligand in the active targeting of anticancer agents, proteins, and genes to primarily proliferating malignant cells that overexpress TfRs. Tf has been used as a molecular conjugate to deliver DNA to erythroleukemic, lung, and liver cell lines. Tf can also be modified with the positive charge N-acylurea groups to make them suitable for electrostatic binding of DNA, in order to achieve a well defined DNA-binding ligand for receptor-mediated gene transfer. Association of Tf with lipoplexes, in particular the negatively charged ternary complexes, significantly overcomes the inhibitory effect of serum and facilitates efficient transfection in many cell lines, including HeLa, K-562 cells, and lung carcinoma cells Calu-3 and H-292 cells. Tf-lipoplex has demonstrated high efficiency in tumor-targeted gene delivery and long-term therapeutic accuracy in systemic p53 gene therapy for both human head and neck cancer and prostate cancer. Tf and Tf-coupled liposomal drug delivery systems may prove particularly valuable to enable the use of a drug that seems to be ineffective or toxic if delivered systematically. The delivery of drugs to the brain has been particularly challenging because of the presence of the blood-brain barrier, which restricts the passage of most therapeutic agents into the brain. Therefore, active targeting of the brain is crucial for effective treatment of brain diseases. The anti-TfR antibody, such as OX26, when coupled with therapeutic agents, has shown potential in drug and gene delivery to the brain.

New DNA Binding Ligands as a Model of Chromomycin A3.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine–netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine–netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H 2O 2 generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H 2O 2-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using 32P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5′-AG G-3′/3′- TCC-5′ sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.

Alleviation of restriction by DNA condensation and non-specific DNA binding ligands

During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA. In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA. How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme. We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands. The type I restriction enzyme is able to destroy the 'naked' DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA.

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands.

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.

Amplification of anticancer drug-induced DNA damage and apoptosis by DNA-binding compounds.

A number of anticancer drugs exert their effect by causing DNA damage and subsequent apoptosis induction. Most anticancer drugs are known to cause severe side effects. Nontoxic amplification of DNA-cleaving activity of anticancer drugs would enable to reduce drug dose and side effects, leading to development of effective chemotherapy. As a method to approach new cancer chemotherapy, we have investigated the enhancing effects of DNA-binding ligands ("amplifiers"), especially minor groove binders and intercalators, on anticancer drug-induced apoptosis and DNA cleavage, using human cultured cells and(32)P-labeled DNA fragments obtained from the human genes. We have demonstrated as follows: a) DNA-binding molecules (unfused aromatic cations, distamycin A and synthtic triamides) induced amplification of bleomycin-induced DNA cleavage and apoptosis; b) a minor-groove binder distamycin A enhanced duocarmycin A-induced DNA cleavage; c) actinomycin D altered the site specificity of neocarzinostatin-induced DNA cleavage and distamycin A enhanced C1027-induced apoptosis. The mechanism of amplification of DNA cleavage can be explained by assuming that binding of amplifier changes the DNA conformation to allow anticancer drug to interact more appropriately with the specific sequences, resulting in enhancement of anticancer effect. The study on amplifiers of anticancer agents shows a novel approach to the potentially effective anticancer therapy.

New DNA binding ligands as a model of chromomycin A 3

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this study, new DNA-binding ligands were designed to mimic Chromomycin A 3 (CRA 3), which contains a hydroxylated tetrahydroanthracene chromophore substituted with di and trisaccharides. The trisaccharide part of CRA 3 that is supposed to contribute to form the Mg 2+-coordinated dimer was expected to be mimicked by a simple alkyl group attached to the chromophore part as new model compounds. The present study has successfully demonstrated that the new ligands form Mg 2+-coordinated dimer complexes to exhibit DNA-binding affinity.

DNA binding ligands with improved in vitro and in vivo potency against drug-resistant Staphylococcus aureus.

Potent in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA) has been difficult to achieve with previously reported DNA binding antibacterials. Herein, we describe an efficient access to a focused library of new analogues yielding compounds with improved activity in a mouse peritonitis model. The most potent molecules (14 and 19) exhibit efficacy against MRSA at ED50 values of approximately 1 and approximately 5 mg/kg, respectively, and display excellent in vitro activity against vancomycin-resistant S. aureus.

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems.

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.

Biochimie des hormones et leurs mécanismes d'action. D-Récepteurs nucléaires

Nuclear receptors are transcription factors that bind cell permeable hormones (steroid hormones, thyroid hormones) or non hormonal molecules (retinoic acid, vitamin D…). Orphan nuclear receptors do not have obvious naturally occurring ligand. Nuclear receptors display a modular structure with similar characteristics: transactivation domain, ligand binding domain (LBD) and DNA binding domain (DBD). The activity of nuclear receptors is regulated by coactivators and corepressors that play a major role in the control of gene expression by nuclear receptors.