Interaction of 9-O-(ω-amino) alkyl ether berberine analogs with poly(dT)·poly(dA)*poly(dT) triplex and poly(dA)·poly(dT) duplex: a comparative study

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson–Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.     

Enhanced DNA binding of 9-ω-amino alkyl ether analogs from the plant alkaloid berberine

To understand the structure-activity relationship of isoquinoline alkaloids, absorption, fluorescence, circular dichroism, and thermodynamics were employed to study the interaction of five C-9-ω-amino alkyl ether analogs from the plant alkaloid berberine with double-stranded calf thymus DNA. The C-9 derivatization resulted in dramatic enhancements in the fluorescence emission of these compounds. The most remarkable changes in the spectral and binding properties were in the BC4 and BC5 derivatives. Interactions of these analogs, which have an additional recognition motif with DNA, were evaluated through different spectroscopic and calorimetric titration experiments. The analogs remarkably enhanced the DNA binding affinity and the same was directly dependent on the alkyl chain length. The analog with six alkyl chains enhanced the DNA binding affinity by about 33 times compared with berberine. The binding became more entropically driven with increasing chain length. These results may be of potential use in the design of berberine derivatives and understanding of the structure-activity relationship for improved therapeutic applications.     

Binding properties of Ru(II) polypyridyl complexes with poly(U)·poly(A)*poly(U) triplex: the ancillary ligand effect on third-strand stabilization

The binding properties of [RuL₂(mip)]²⁺ {where L is 1,10-phenanthroline (phen) or 4,7-dimethyl-1,10-phenanthrollne (4,7-dmp) and mip is 2′-(3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]phenanthroline} with regard to the triplex RNA poly(U)·poly(A)*poly(U) were investigated using various biophysical techniques and quantum chemistry calculations. In comparison with [Ru(4,7-dmp)₂(mip)]²⁺, remarkably higher binding affinity of [Ru(phen)₂(mip)]²⁺ for the triplex RNA poly(U)·poly(A)*poly(U) was achieved by changing the ancillary ligands. The stabilization of the Hoogsteen-base-paired third strand was improved by about 10.9 °C by [Ru(phen)₂(mip)]²⁺ against 6.6 °C by [Ru(4,7-dmp)₂(mip)]²⁺. To the best of our knowledge, [Ru(phen)₂(mip)]²⁺ is the first metal complex able to raise the third-strand stabilization of poly(U)·poly(A)*poly(U) from 37.5 to 48.4 °C. The results reveal that the ancillary ligands have an important effect on third-strand stabilization of the triplex RNA poly(U)·poly(A)*poly(U) when metal complexes contain the same intercalative ligands.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

The nature of different influence of Cd<Superscript>2+</Superscript> ions on the conformational equilibrium of triple-stranded polyribonucleotides poly(U)•poly(A)•poly(U) and poly(I)•poly(A)•poly(I) in aqueous solutions

The influence of Cd²⁺ ions on the conformational equilibrium of single-stranded (poly(U), poly(A), poly(I)) and triple-stranded polyribonucleotides (A2I, A2U) in aqueous solutions (0.1 M Na⁺ pH 7) has been investigated using difference UV spectroscopy and thermal denaturation. Analysis of the shape and intensity of the DUV spectra of poly(A), poly(I), and A2I has revealed the presence of two types of complex formed as a result of (i) interaction between Cd²⁺ and the N7 atoms of purines, producing macrochelates; and (ii) binding of Cd²⁺ to the N1 atoms of poly(A) and poly(I). Since Cd²⁺ ions are not bound to heteroatoms of the bases in A2U, the conformation of the structure remains stable up to 0.02 M Cd²⁺. There is a critical Cd²⁺ concentration (～1.5?10^?4 M) above which A2I assumes a new helical conformation with lower thermal stability. It is supposed that, upon the formation of the “metallized” A2I triplex, the Cd²⁺ ions are located inside the triple helix and form bridges between the hypoxanthine and adenine of the homopolynucleotide strands.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Fractionation of functional polystyrenes,poly(ethylene oxide)s and poly(styrene)–b–poly(ethylene oxide) by liquid chromatography at the exclusion–adsorption transition point

The paper reports the fractionation of functional polystyrenes (PSs) and poly(ethylene oxide)s (PEOs) as well as their block copolymers, by liquid chromatography at the exclusion adsorption transition point (EATP–LC), also called “critical conditions” mode. In this specific elution mode (EATP–LC), the fractionation is only governed by the nature and the number of functions attached to the polymer backbone, independent of the molar mass distribution of the whole sample. Functional polystyrenes (α- and/or α,ω-alcohol-, acetal-, aldehyde- and acidic-PS) could be readily separated from non-functional polystyrenes under various chromatographic conditions. The technique also allowed the fractionation of poly(ethylene oxide)s and PS–PEO block copolymers. In the latter cases, moderately polar columns (grafted silica) and water-based polar eluents were required to obtain a satisfactory fractionation.     

Anionic polymer,poly(methyl vinyl ether–maleic anhydride)-coated beads-based capture of human influenza A and B virus

An anionic magnetic beads-based method was developed for the capture of human influenza A and B viruses from nasal aspirates, allantoic fluid and culture medium. A polymer, poly(methyl vinyl ether–maleic anhydride) [poly(MVE-MA)], was used to endow magnetic beads with a negative charge and bioadhesive properties. After incubation with samples containing human influenza virus, the beads were separated from supernatants by applying a magnetic field. The absorption of the virus by the beads was confirmed by hemagglutinin assay, immunochromatography, Western blotting, egg infection, and cell infection. Successful capture was proved using 5 H1N1 influenza A viruses, 10 H3N2 influenza A viruses, and 6 influenza B viruses. Furthermore, the infectivity in chicken embryonated eggs and Madin–Darby canine kidney (MDCK) cells of the captured human influenza virus was similar to that of the total viral quantity of starting materials. Therefore, this method of capture using magnetic beads coated with poly(MVE-MA) can be broadly used for the recovery of infectious human influenza viruses.     

The structural stabilization of the κ three-way junction by Mg(II) represents the first step in the folding of a group II intron

Folding of group II introns is characterized by a first slow compaction of domain 1 (D1) followed by the rapid docking of other domains to this scaffold. D1 compaction initiates in a small subregion encompassing the κ and ζ elements. These two tertiary elements are also the major interaction sites with domain 5 to form the catalytic core. Here, we provide the first characterization of the structure adopted at an early folding step and show that the folding control element can be narrowed down to the three-way junction with the κ motif. In our nuclear magnetic resonance studies of this substructure derived from the yeast mitochondrial group II intron Sc.ai5γ, we show that a high affinity Mg(II) ion stabilizes the κ element and enables coaxial stacking between helices d′ and d′′, favoring a rigid duplex across the three-way junction. The κ-element folds into a stable GAAA-tetraloop motif and engages in A-minor interactions with helix d′. The addition of cobalt(III)hexammine reveals three distinct binding sites. The Mg(II)-promoted structural rearrangement and rigidification of the D1 core can be identified as the first micro-step of D1 folding.     

Novel approaches towards characterization of the high-affinity nucleotide binding sites on mitochondrial F1-ATPase by the fluorescence probes 3′-O-(1-naphthoyl)adenosine di- and triphosphate

The fluorescence properties of 3′-O(1-naphthoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial F₁-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and ‘ground-state’ quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg²⁺ 120 nM; N-ATP/Mg²⁺ 160 nM; N-ADP/EDTA 560 nM; N-ATP/EDTA 3500 nM. For bound ligand the environment was found to be rather unpolar; the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg²⁺, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg²⁺). After the first fast step (k_on > 1 · 10⁶ M⁻¹ · s⁻¹), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.     

A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation

A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4′-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential antitumor prodrug. Firstly, according to the structure–activity relationship, drug combination principle and bioisosterism, the –COO– and the –NH– bond substituents at the 4 position of cycloparaffin would be a great modification direction to improve antitumor activity of 4′-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view, the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a series of heterocyclic with pharmacological activity were chosen as module for improving antitumor activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell line HeLa (i.e., the IC₅₀ value of 0.60 ± 0.20 μM), A549 (i.e., the IC₅₀ value of 3.83 ± 0.08 μM), HepG2 (i.e., the IC₅₀ value of 1.21 ± 0.05 μM), and BGC-823 (i.e., the IC₅₀ value of 4.15 ± 1.13 μM) was significantly improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer drug etoposide (i.e., the IC₅₀ values of 15.32 ± 0.10, 59.38 ± 0.77, 67.25 ± 7.05, and 30.74 ± 5.13 μM), respectively. Compound 1 could arrest HeLa cell cycle G₂/M and induce apoptosis by strongly diminishing the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly demonstrated by the bioactivity test.     

In-vitro characterization of the pharmacological effects induced by (-)-α-bisabolol in rat smooth muscle preparations

The present study deals with the pharmacological effects of the sesquiterpene alcohol (-)-α-bisabolol on various smooth-muscle preparations from rats. Under resting tonus, (-)-α-bisabolol (30-300?μmol/L) relaxed duodenal strips, whereas it showed biphasic effects in other preparations, contracting endothelium-intact aortic rings and urinary bladder strips, and relaxing these tissues at higher concentrations (600-1000?μmol/L). In preparations precontracted either electromechanically (by 60?mmol/L K(+)) or pharmacomechanically (by phenylephrine or carbachol), (-)-α-bisabolol showed only relaxing properties. The pharmacological potency of (-)-α-bisabolol was variable, being higher in mesenteric vessels, whereas it exerted relaxing activity with a lesser potency on tracheal or colonic tissues. In tissues possessing spontaneous activity, (-)-α-bisabolol completely decreased spontaneous contractions in duodenum, whereas it increased their amplitude in urinary bladder tissue. Administered in vivo, (-)-α-bisabolol attenuated the increased responses of carbachol in tracheal rings of ovalbumin-sensitized rats challenged with ovalbumin, but was without effect in the decreased responsiveness of urinary bladder strips in mice treated with ifosfamide. In summary, (-)-α-bisabolol is biologically active in smooth muscle. In some tissues, (-)-α-bisabolol preferentially relaxed contractions induced electromechanically, especially in tracheal smooth muscle. The findings from tracheal rings reveal that (-)-α-bisabolol may be an inhibitor of voltage-dependent Ca(2+) channels.     

A polymorphic region defined by pCN2 (the 3′ nontranslated region of N-ras) maps to chromosome 9cen-p12

Summary Abnormalities of chromosome 9p have been reported in human leukemias and lymphomas, and in cell lines lacking the enzyme methylthioadenosine phosphorylase. It has been shown pCN2, the 3 nontranslated region of the N-ras oncogene, crosshybridizes with unknown DNA segments on chromosome 6, 9p, and 22, in addition to the N-ras oncogene itself on chromosome 1p. To use pCN2 to study chromosome 9p abnormalities in malignancies, we undertook to localize the pCN2 crosshybridizing region in chromosome 9p. By analyzing the copy numbers of the pCN2 crosshybridizing bands associated with chromosome 9p among various chromosomally aberrant human cell lines, we mapped the pCN2 hybridizing region to 9cen-p12. Since there is no other available probe in this region, pCN2 should prove very useful in studying abnormalities of chromosome 9p in human malignancies.This is publication number 5969-MEM from the Research Institute of Scripps Clinic, La Jolla, California     

The Synthesis of 5′-O-Triphosphate-4N-(ω-aminoalkyl)deoxycytidine A Useful Precursor to the Generation of Differently Labeled Triphosphates

Abstract

A chemical synthesis is described for 5′-O-triphosphate-4N-[6-(-γ-aminopropylamidosuccinylamido)-hexyl]deoxycytidine (13), the substrate for synthesis of 5′-O-triphosphate deoxycytidine derivatives (17-19) labeled with biotin, fluorescein and photoreactive azide.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (F A) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.