ABSTRACT
Geometries and stabilities of various base triplets have been studied using ab initio quantum chemical methods. Their optimized geometries are determined using the STO-3G basis set, and those of Hoogsteen and reverse Hoogsteen base pairs are evaluated with the 4-31G basis set. Moreover, the preferred hydrogen bond patterns of the bases in triple helices are discussed. A cooperative effect for base pairing in triplets is presented, and it can be either positive or negative. Almost all base triplets that contain Watson-Crick G:C base pairs show a positive cooperativity. Conversely, the base triplets with Watson-Crick A:T base pairs mostly display a negative cooperativity. The interaction energies of base triplets are reported and the relative stabilities of base triplets are found as follows: A+.GC > C+.GC(H) > C+.GC(rH) > G.GC(H) > G.GC(rH) > A.GC > T.AT(rH) > U.AU(H) > U.AT(H) > A.AT > G.AT > T.AT(m) > G.TA(2) > G.TA(1) H and rH denote the Hoogsteen and reverse Hoogsteen positions of the third base that would lead to parallel and antiparallel orientations respectively of the third chain with respect to the Watson-Crick paired purine chain. 'm' denotes the middle pairing scheme, in which the third base hydrogen bonds to both bases of Watson-Crick pair.
Subject(s)
Base Composition , Base Sequence , DNA/chemistry , Models, Theoretical , Crystallography, X-Ray , Drug Stability , Hydrogen Bonding , Magnesium , Models, Genetic , Models, Structural , Structure-Activity RelationshipABSTRACT
Oligothymidylate (aminomethyl)phosphonates have been prepared, and their enzymatic and physicochemical properties have been studied. The individual isomers of the protected dimers have been separated, characterized, and incorporated into oligonucleotides in which the backbone consists of alternating (aminomethyl)phosphonate and phosphodiester linkages. One of these net neutral, single isomer oligonucleotides forms a duplex with its complementary sequence which is more stable than the corresponding natural counterpart, whereas the other isomer is considerably less stable. Specificity of hybridization is maintained, as determined by the reduction in melting temperature observed upon the introduction of mismatches into the complementary strand of the duplex. The (aminomethyl)-phosphonate linkage is stable toward enzymatic degradation but can be hydrolyzed in aqueous solution at elevated temperature.
Subject(s)
Oligonucleotides/chemical synthesis , Organophosphorus Compounds/chemical synthesis , Base Sequence , Chemical Phenomena , Chemistry, Physical , Drug Stability , Models, Molecular , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , Oxygen/chemistry , Phosphorus/chemistry , Sensitivity and Specificity , Solutions , Structure-Activity RelationshipABSTRACT
As part of an investigation of the structural requirements for the induction, by phenobarbital-type inducers, of a coordinate pleiotropic response consisting of increases in hepatic cytochrome P450 2B (P450 2B) activity, increases in other phase I and II enzyme activities, and liver hypertrophy, we have examined a series of analogues of phenobarbital in which the ethyl/phenyl substitution at the sp3 carbon of the parent molecule was kept constant while the heterocyclic portion of the molecule was modified. The induction of hepatic P450 2B protein and ethoxy-, pentoxy-, and (benzyloxy)resorufin O-dealkylation activities, and epoxide hydration activity and liver/body weight ratio increase were examined in male F344/NCr rats fed the various congeners for 14 days at doses equimolar to 500 ppm phenobarbital. Increases in the measured parameters were maximal in rats fed phenobarbital or 5-ethyl-5-phenylhydantoin. The responses to primidone or 2-ethyl-2-phenylsuccinimide were approximately 65% of maximal, while glutethimide yielded a response approximately 50% of maximal. Induction of this response in rats fed the ring-opened and decarboxylated analogues, (ethylphenylacetyl)urea and 2-ethyl-2-phenylmalonamide, were < 25% of maximal. 5-Ethyl-5-phenyloxazolidinedione caused minimal increases in the measured end points when administered at a dose equimolar to 500 ppm phenobarbital. The profound differences among the congeners in ability to induce P450 2B protein and associated catalytic activities were not due to differences in food consumption by the various groups of rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Liver/enzymology , Phenobarbital/analogs & derivatives , Phenobarbital/pharmacology , Animals , Body Weight/physiology , Chemical Phenomena , Chemistry, Physical , Cytochrome P-450 CYP1A1 , Cytochrome P-450 Enzyme System/metabolism , Eating/drug effects , Enzyme Induction/drug effects , Epoxide Hydrolases/metabolism , Epoxy Compounds/metabolism , Male , Organ Size/physiology , Oxidoreductases/metabolism , Protein Conformation , Rats , Rats, Inbred F344 , Steroid Hydroxylases/metabolismABSTRACT
The pharmacodynamics of rat hepatic cytochrome P450 2B (P450 2B) induction by phenobarbital (PB) and two structural congeners, dl-5-ethyl-5-phenylhydantoin (EPH) and dl-5-ethyl-5-phenyloxazolidinedione (EPO), were investigated. The in vivo induction of P450 2B was probed in F344/NCr rats by measuring immunoreactive hepatic P450 2B1 protein and by assaying the hepatic 16 beta-hydroxylation of testosterone and O-dealkylation of (benzyloxy)- and pentoxyresorufin. The induction of (benzyloxy)resorufin O-dealkylation activity was also measured in adult rat hepatocyte cultures exposed to the three xenobiotics. The concentration of xenobiotic at the putative active site in the in vivo studies was approximated by measuring serum total xenobiotic levels, while in the hepatocyte culture studies, the nominal xenobiotic concentration in the culture medium was used. Concentration-dependent induction of P450 2B activities was observed in the in vivo and hepatocyte culture studies. The in vivo ED50 values for P450 2B induction were approximately 110, approximately 100, and approximately 3000 dietary ppm (14 days administration) for PB, EPH, and EPO, respectively. The in vivo EC50 values for P450 2B induction were approximately 9, approximately 6, and approximately 130 microM (total serum) for PB, EPH, and EPO, respectively. In cultured rat hepatocytes, the ED50 values for induction of (benzyloxy)resorufin O-dealkylation activity were 14.5, 14.2, and 108 microM for PB, EPH, and EPO, respectively. These data indicate that pharmacodynamic results obtained with cultured hepatocytes represent a good qualitative and quantitative approximation of the in vivo hepatic responses in male rats caused by PB-type inducers.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Anticonvulsants/pharmacology , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Liver/enzymology , Mephenytoin/analogs & derivatives , Oxazoles/pharmacology , Phenobarbital/pharmacology , Animals , Cells, Cultured , Cytochrome P-450 CYP1A1 , Cytochrome P-450 Enzyme System/metabolism , Diet , Enzyme Induction/drug effects , Immunohistochemistry , Liver/drug effects , Male , Mephenytoin/pharmacology , Oxidoreductases/metabolism , Protein Conformation , Proteins/metabolism , Rats , Rats, Inbred F344 , Steroid Hydroxylases/metabolismABSTRACT
To assess the usefulness of computer-assisted modeling of mRNA as an aid in design of antisense DNA, the efficiency of inhibition of translation of rabbit beta-globin mRNA by various antisense sequences was compared with calculated structures of the mRNA. The model obtained by consideration of 30 lowest-energy computer-simulated structures is consistent with the high accessibility of the AUG initiation codon region known from digestion with nucleases and with previous antisense inhibition studies reported in the literature. Additional antisense inhibition data were obtained with 20-mer phosphorothioate oligonucleotides, targeted to regions of beta-globin mRNA differing moderately in their degree of participation in intramolecular folding. The efficiency of translation arrest by the oligonucleotides in cell-free expression systems (wheat germ extract and rabbit reticulocyte lysate) was obtained by measuring incorporation of [35S]methionine into total protein, and corrected for sequence-nonspecific inhibition using brome mosaic virus mRNA. In the presence of RNase H (wheat germ system), the inhibitory activity of the oligonucleotides showed correlation with the calculated secondary structure of mRNA, in particular at low oligonucleotide-to-mRNA ratios (correlation coefficient, 0.95). No correlation was observed in the reticulocyte lysate system, in which the inhibition is mediated by translational arrest.
Subject(s)
DNA, Antisense/pharmacology , Globins/genetics , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/chemistry , Thionucleotides/pharmacology , Animals , Base Sequence , Bromovirus/genetics , Cell-Free System , Codon , Computer Simulation , Molecular Sequence Data , Protein Biosynthesis/drug effects , RNA, Messenger/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Rabbits , Ribonuclease H/metabolismABSTRACT
In order to assess the effects of substitution of one of the non-bridging oxygens in the phosphodiester group with sulfur, molecular dynamics calculations were performed on duplex oligodeoxynucleotides having a complete helical turn, [d(CpGpCpGpApApTpTpCpGpCpG)]2. The calculations were carried out with the unmodified duplex, as well as with duplexes having one phosphorothioate strand with uniform phosphorus stereochemistry (SP or RP). The electrostatic and structural (equilibrium molecular dimensions as well as force constants) parameters of the phosphorothioate group for molecular mechanics and dynamics calculations were derived from ab initio quantum mechanics optimizations of conformers of O,O'-dimethyl phosphorothioate. Molecular dynamics simulations (30 ps) were carried out using the AMBER force field, with explicit inclusion of all hydrogen atoms, counterions, and water. The simulations indicate a significant decrease of stability of the model containing the homochiral RP-phosphorothioate strand, whereas the model containing the homochiral SP-phosphorothioate strand has practically the same energy as the unmodified oligodeoxynucleotide duplex. The energy difference determined indicates some preference for the use of stereochemically pure SP-phosphorothioates as antisense gene inhibitors. Moreover, the significance of these results in connection with use of phosphorodithioates is discussed.
Subject(s)
DNA, Antisense/chemistry , Models, Molecular , Thionucleotides/chemistry , Drug DesignABSTRACT
A restrained molecular dynamics simulation approach that explicitly includes the effect of the surrounding solvent molecules is applied to the NMR determination of the conformations of the B-loop fragments of human transforming growth factor alpha and epidermal growth factor. Backbone interproton distance restraints are obtained by using two-dimensional rotating frame nuclear Overhauser effect spectroscopy (ROESY). The simulations are carried out both in "vacuum" and in "water." The results are discussed in terms of the energetics, agreement with the NMR distances, and the flexibility of the peptides.
Subject(s)
Epidermal Growth Factor , Transforming Growth Factors , Computer Simulation , Humans , Hydrogen , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Conformation , Solutions , ThermodynamicsABSTRACT
Recently, Vasanthakumar and Ahmed reported (Vasanthakumar, G.; Ahmed, N.K., Cancer Communications 1:225-232; 1989) a complete inhibition of the multiple drug resistance gene (MDR1) in the K562/III erythroleukemia cells, using a 15 bases-long methylphosphonate oligodeoxynucleotide analog. The sequence used, however, contained three mismatches relative to the corresponding fragment of the human MDR1 gene and, hence, the results reported cannot at present be regarded as a classical antisense effect. We have made attempts to inhibit the expression of the MDR1 gene in MCF-7 human breast cancer cells selected for resistance to Adriamycin using phosphorothioate analogs of oligodeoxynucleotides. Studies with model 35S-labeled-phosphorothioates indicated poor uptake of the compounds into the cells; the radioactivity was located mainly in the soluble fraction (cytoplasm), but membranes and the nuclear fraction were also labeled. Unmodified oligodeoxynucleotides were toxic to the cells, whereas the phosphorothioates were not. The MDR1 inhibition with phosphorothioates was studied by measuring their effects on adriamycin toxicity and by immunocytochemical titration of P170. Elevation of adriamycin cytotoxicity consistent with a decreased drug resistance was observed with one antisense sequence, but the immunocytochemical assay indicated only slight inhibition of the synthesis of P170. In the wild type (drug sensitive) MCF-7 cells phosphorothioates decreased adriamycin toxicity in a sequence-independent manner. The results indicate that the effects of antisense oligodeoxynucleotides on cells are complex. Computer simulation of the secondary structure of MDR1 mRNA indicated not only extensive folding but, also, the presence of many regions not involved in intramolecular hybridization, which are of potential interest as targets for antisense oligodeoxynucleotides.
Subject(s)
Breast Neoplasms/genetics , Drug Resistance/genetics , Oligonucleotides/pharmacology , Base Sequence , Cells, Cultured , Chromatography, High Pressure Liquid , Doxorubicin/toxicity , Gene Expression Regulation, Neoplastic , Genes , Humans , In Vitro Techniques , Molecular Sequence Data , Oligonucleotides, Antisense , Thionucleotides/pharmacologyABSTRACT
The induction of cytochrome P-450-mediated alkoxyresorufin O-dealkylase activities by various xenobiotics was examined in liver from a variety of animal species in order to gain insights into the substrate specificities of the induced P-450s. We found that forms of cytochrome P-450 capable of mediating the O-dealkylation of the short-chain phenoxazone ethers methoxy-, ethoxy- and propoxyresorufin were highly induced by 3-methylcholanthrene-type inducers and by Aroclor-1254 in all species tested, although there were species differences in the relative turnover rates for the various substrates. For example, in hamster liver the turnover rates for the short-chain resorufin ethers decreased in the following order: methoxy greater than ethoxy much greater than propoxy, while in the rat liver almost the exact opposite order was observed: ethoxy = propoxy much greater than methoxy. In contrast, the degree of induction by phenobarbital-type inducers of isozymes catalyzing the O-dealkylation of pentoxy- or benzyloxyresorufin was highly species-dependent. Thus, F344/NCr rats, B6C3F1 mice and NZB rabbits showed the greatest (greater than 20-fold) induction of these activities, either by phenobarbital or Aroclor-1254, while Mongolian gerbils showed intermediate levels of induction and Syrian golden hamsters exhibited very low induction. In the Japanese quail, phenobarbital- or DDT-treatment resulted in minimal induction of pentoxy- or benzyloxyresorufin O-dealkylase activity, although significant induction of the latter activity occurred following treatment with 5,6-benzoflavone or with Aroclor-1254. Since substrate specificities of most enzymes can be rationalized based upon differences in the steric requirements at the enzyme active site, we employed molecular modeling techniques to calculate the molecular dimensions of the alkoxyresorufins. Surprisingly, the minimal energy conformations in vacuo of each of the resorufin ethers examined are essentially planar. However, alternative configurations, especially for the pentoxy- and benxyloxy-ethers, having greater three-dimensional bulk are also energetically possible.