Mechanism of action in a 4,5-diarylpyrrole series of selective cyclo-oxygenase-2 inhibitors.

Using semi-empirical AM1 calculation and 6.31G* basis sets, we have calculated the energy of the highest-occupied molecular orbital (E(HOMO)) for anti-inflammatory 4,5-diarylpyrroles which have been shown to have inhibitory activity on cyclooxygenase COX-2, an inducible enzyme expressed during inflammation. We have found a correlation between the E(HOMO) of a molecule and its COX-2 inhibition. However, no correlation was observed between E(HOMO) and the inhibition efficiency of cyclooxygenase-1 (COX-1), the constitutively expressed enzyme, protective to the organism. This result suggests that the inhibitions of the two isoforms follow different molecular mechanisms.

Molecular orbital theory applied to the study of nonsteroidal anti-inflammatory drug efficiency.

Using a simple quantum mechanical method, we calculated the energy of the highest-occupied molecular orbital (E(HOMO)) of three groups of anti-inflammatory compounds, and we have found correlations between E(HOMO) of these molecules and experimental data previously reported on (1) inhibition of sheep-vesicular-gland prostaglandin cyclooxygenase by phenolic compounds, (2) inhibition of prostaglandin cyclooxygenase in mouse macrophages by salicylates, benzoates and phenols, and (3) peroxyl-radical scavenging and radioprotection of a bacterial virus by NSAID drugs, including metiazinic acid, sulindac, D-penicillamine, piroxicam, indomethacin, benoxaprofen, and aspirin. Our correlations using a systematic evaluation of the HOMO energies can be of predictive value in the search for new anti-inflammatory drugs as well as for new radioprotectors.

Fluorescence energy transfer between two triple helix-forming oligonucleotides bound to duplex DNA.

An 11-mer oligopyrimidine was covalently linked via its 5'-phosphate to an acridine derivative (acridine-11-mer), and a 13-mer was covalently linked via its 3'-phosphate to an ethidium derivative (13-mer-ethidium). Each of them formed a triple helix with a 31-bp DNA fragment containing two oligopurine-oligopyrimidine sequences, 11 and 13 bp in length, separated by a variable number of base pairs. When both oligonucleotides were bound to the 31-bp DNA fragment, fluorescence energy transfer (FET) from acridine to ethidium was observed, as revealed by a quenching of acridine fluorescence and a sensitized ethidium emission. FET was temperature-dependent and occurred only when both oligonucleotides were simultaneously bound to the DNA matrix. A single base-pair change in one of the target sequences strongly reduced the energy-transfer efficiency. This method was used to discriminate between a fully complementary and a mismatched target sequence.

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

Characterization of a triple helix-specific ligand. BePI (3-methoxy-7H-8-methyl-11- [(3'-amino)propylamino]-benzo[e]pyrido[4,3-b]indole) intercalates into both double-helical and triple-helical DNA.

A benzo[e]pyridoindole derivative, 3-methoxy-7H-8-methyl-11-[(3'-amino)propylamino] -benzo[e]pyrido[4,3-b]indole (BePI), and its interactions with double and triple-helical DNA have been investigated by a variety of fluorescence, spectrophotometric, hydrodynamic and molecular modeling techniques. Binding to DNA stabilizes the doubly charged (+2) form of BePI, increasing the apparent pKa of the 10-NH proton by approximately 1 pH unit. Binding to DNA also quenches the fluorescence of BePI, with a greater extent of quenching upon binding triplex relative to duplex DNA. BePI preferentially binds (and stabilizes) triple-helical relative to double-helical DNA. This preferential binding is not restricted to triplexes containing solely T x A.T base triplets. In addition, BePI preferentially stabilizes the poly(dA).poly(dT) relative to the poly[d(A-T)].poly[d(A-T)] duplex. Viscosity studies demonstrate that, upon binding, BePI induces the unwinding of negative supercoils in the pBR322 plasmid, and increases the relative contour lengths of double and triple-helical polydeoxynucleotides. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to bound BePI molecules in both BePI/duplex and BePI/triplex complexes. In a BePI/triplex complex, an average of 4.8 bases appear to transfer excitation energy totally to a bound BePI molecule, while in various BePI/duplex complexes an average of only 2.5 bases appear to do so, indicating that energy transfer is more efficient in the former complex. Measurements of fluorescence quenching indicate that BePI is protected from quenching by acrylamide when bound to either double or triple-helical polynucleotides. The viscosity and fluorescence behavior of BePI are fully consistent with the conclusion that BePI intercalates into both double and triple-helical DNA. Molecular modeling studies suggest that stronger stacking interactions between intercalated BePI and adjacent bases in BePI/triplex relative to BePI/duplex complexes may account for the enhanced thermal stability of the former complex.

Photobiological activities of 1,6-dioxapyrene in pro- and eukaryotic cells.

The photobiological effect of a new pyrene derivative, 1,6-dioxapyrene (1,6-DP), was studied in Salmonella typhimurium (strain TA100) and in the diploid strain D7 of the yeast Saccharomyces cerevisiae. In Salmonella, 1,6-DP shows little mutagenicity in the dark in comparison to benzo[a]pyrene (B[a]P). This mutagenic activity decreases in the presence of liver S9 homogenates from Aroclor induced XVIInc/Z mice. However, in combination with 365 nm (UVA) radiation and in the absence of S9 mix, 1,6-DP behaves as an effective photodynamic compound inducing lethal and mutagenic effects in both organisms. In yeast, its activity, like that of B[a]P, is highly dependent on the presence of oxygen. For the same incident dose of UVA, 1,6-DP is, however, at least 6 times more effective than B[a]P in inducing cytotoxic and mutagenic effects. At equitoxic doses, 1,6-DP is as photomutagenic as B[a]P, suggesting that in both cases mutagenicity is due to similar mechanisms. Spectrophotometric measurements indicate physical interaction of 1,6-DP with DNA in the dark. Laser flash photolysis experiments show that 1,6-DP generates singlet oxygen with a quantum yield of 0.17. In vitro 1,6-DP produces oxidative damage to guanine bases specific for singlet oxygen mediated reactions. Alkaline step elution analysis of 1,6-DP plus UVA treated yeast cells indicates a decrease in average molecular weights in DNA and an induction of single strand breaks (ssb) originating from alkali labile sites. This effect is enhanced by D2O and is thus likely to be due to the production of singlet oxygen. The strand breaks appear to differ from those induced by gamma-rays because little, if any, repair of these ssb occurs during 30 min of post-treatment incubation in complete growth medium. These results suggest that the photobiological effects of 1,6-DP are due to oxidative damage in DNA mostly induced by singlet oxygen.

Correlations between the rate constant of singlet oxygen quenching by imidazole derivatives and anti-inflammatory activity in rats.

The second-order rate constants, k delta, for quenching of molecular singlet oxygen O2 (1 delta g) by nonsteroidal anti-inflammatory imidazole drugs have been determined using time-resolved phosphorescence detection of singlet oxygen. A linear correlation was observed between log k delta (ranging from 7.90 to 8.50) and the anti-inflammatory activity of these compounds (ranging from ED50 = 15 to 300 mg/kg), as measured in rats by Jørgensen and Dyrsting [United States Patent 4,424,229 (1984)]. The correlation between this physico-chemical parameter measured in vitro and a biological activity measured in vivo might be useful in screening other types of candidate anti-inflammatory drugs. The rate constant (k delta) can be considered as a quantitative expression of the electron-donating power of the imidazole drug, as suggested by a correlation of log k deta (ranging from 6.02 to 7.45) with Hammett substituent parameters observed in the case of 2-substituted imidazoles.

Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.

Kinetics and thermodynamics of triple-helix formation: effects of ionic strength and mismatches.

Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C greater than G greater than A much greater than T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62, 383-401; Pörschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].

Triple helix-specific ligands.

A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

Stable triple helices are formed upon binding of RNA oligonucleotides and their 2'-O-methyl derivatives to double-helical DNA.

Pyrimidine oligoribonucleotides bind to the major groove of double-helical DNA at homopurine.homopyrimidine sequences. They recognize Watson-Crick base pairs by forming T.A x U and C.G x C base triplets via Hoogsteen hydrogen bonding. The stability of these triple helices is much higher than that of triple helices formed by oligodeoxyribonucleotides as shown by an increase of the temperature at which half-dissociation of the third strand occurs. When the 2'-hydroxyl group of ribose moieties is replaced by 2'-O-methyl substituent, triple helix stability is further increased.

Deactivation of singlet molecular oxygen by organo-selenium compounds exhibiting glutathione peroxidase activity and by sulfur-containing homologs.

The bimolecular rate constants (k) of quenching of molecular singlet oxygen 1O2 (1 delta g) by organo-selenium compounds exhibiting glutathione peroxidase activity and by sulfur analogs have been determined by time resolved phosphorescence detection of 1O2 in CD3OD and C6D6, with no solvent effect. The rate constants of quenching by the Se-containing compounds were found to be approximately one order of magnitude higher than those of the S-containing homologs. A linear correlation was observed between log k and the Hammett constant omega ortho with p = -0.89, the rate constant being higher for molecules with an electron-donating substituent and lower for those with an electron-withdrawing substituent. This observation is consistent with the involvement of a charge transfer complex in the deactivation of singlet oxygen.

Sequence specificity in triple-helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability.

The specificity of a homopyrimidine oligonucleotide binding to a homopurine-homopyrimidine sequence on double-stranded DNA was investigated by both molecular modeling and thermal dissociation experiments. The presence of a single mismatched triplet at the center of the triplex was shown to destabilize the triple helix, leading to a lower melting temperature and a less favorable energy of interaction. A terminal mismatch was less destabilizing than a central mismatch. The extent of destabilization was shown to be dependent on the nature of the mismatch. Both single base-pair substitution and deletion in the duplex DNA target were investigated. When a homopurine stretch was interrupted by one thymine, guanine was the least destabilizing base on the third strand. However, G in the third strand did not discriminate between a C.G and an A.T base pair. If the stretch of purines was interrupted by a cytosine, the presence of pyrimidines (C or T) in the third strand yielded a less destabilizing effect than purines. This study shows that oligonucleotides forming triple helices can discriminate between duplex DNA sequences that differ by one base pair. It provides a basis for the choice of antigene oligonucleotide sequences targeted to selected sequences on duplex DNA.

Intercalation of ethidium bromide into a triple-stranded oligonucleotide.

We have examined the ability of a cationic planar chromophore, ethidium bromide, to intercalate into a short, defined triple helix. Using UV absorption, fluorescence spectroscopy and a gel retardation assay we demonstrate that ethidium bromide is able to bind to a triple helix with a lower affinity than to the corresponding duplex. Energy transfer from base triplets to ethidium shows that ethidium is intercalated into the triple helix. The spectroscopic characteristics of ethidium intercalated into a triplex are similar to those observed for intercalation into duplex DNA.

Fluorescence energy transfer between dimethyldiazaperopyrenium dication and ethidium intercalated in poly d(A-T).

Dimethyldiazaperopyrenium is one of the largest known DNA intercalators. Fluorescence energy transfer occurred between dimethyldiazaperopyrenium (donor) and ethidium (acceptor) when these dyes were bound to a double-stranded polynucleotide such as poly d(A-T). The addition of increasing amounts of ethidium bromide led to a marked shortening of the fluorescence lifetime of the donor, whereas the excited state of the acceptor was progressively populated via energy transfer from the donor. Critical Förster distance between these two chromophores was calculated to be 3.8 nm. The observed transfer efficiency was lower than that calculated on the basis of this critical distance and a statistical distribution of bound drugs. These results are discussed taking into account the conformational change induced by intercalation of dimethyldiazaperopyrenium in the double-stranded polynucleotide.

Quantum yields of triplet and O2(1 delta g) formation of 4-thiouridine in water and acetonitrile.

The quantum yield of triplet formation, phi T, and that of the photosensitized formation of singlet molecular oxygen, phi delta, were determined for a rare nucleoside, 4-thiouridine (4t-Urd), in water and in acetonitrile, using singlet molecular oxygen phosphorescence, laser-induced optoacoustics and time-resolved thermal lensing. These yields, phi T and phi delta, the latter in aerated solutions, were found to be, respectively, in water: 0.67 +/- 0.17 and 0.18 +/- 0.04 and in acetonitrile: 0.61 +/- 0.15 and 0.50 +/- 0.20. The fraction of the 4t-Urd triplet molecules quenched by oxygen leading to singlet molecular oxygen, S delta, was calculated to be between 0.7 and unity in both solvents, this value being indicative of a pi pi*character for the lowest triplet state of 4t-Urd.

Photophysical properties of 2-nitro-5,10,15,20-tetra-p-tolylporphyrins.

Tetraarylporphyrins substituted with nitro groups at beta-pyrrolic positions are potential candidates for electron-accepting pigments in model systems for photosynthesis. The photophysics of 2-nitro-5,10,15,20-tetra-p-tolylporphyrin and its zinc analog have been studied in order to evaluate this potential. The ground state absorption spectrum, the triplet-triplet absorption spectrum, the fluorescence emission spectrum, and associated photophysical parameters have been determined. The molecules have short singlet lifetimes and anomalous temperature- and solvent-dependent emission spectra which are consistent with the formation of an intramolecular charge transfer state of the type P+.-NO2-. in which the nitro group is twisted about its bond to the porphyrin, relative to the ground state conformation.

Redox properties of phenols, their relationships to singlet oxygen quenching and to their inhibitory effects on benzo(a)pyrene-induced neoplasia.

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al. The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen, k, by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced by benzo(a)pyrene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials.

Singlet molecular oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol.

The rate constants of molecular singlet oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol-membrane critical components - membrane critical components - have been measured by time resolved detection of the 1270 nm phosphorescence of singlet molecular oxygen [O2(1deltag)]. We have determined (i) an increment of 5.7 x 10(2)M(-1)s(-1) per -CH2- in C6D6 and CD3OD for saturated fatty acids between C4 and C20, (ii) an increment of 3 x 10(4)M(-1)s(-1) per non-conjugated cis-double bond for C18 unsaturated fatty acids, identical in C6D6 and DC3OD, (iii) a lower quenching rate constant by a factor of 2.7 for the trans-C16 and trans-C18 as compared to the corresponding cis-monounsaturated fatty acids, (iv) a rate constant of O2x(1deltag) quenching by cholesterol of 5.7 x 10(4)M(-1)s(-1) in benzene. These rate constants are compared to those obtained for other membrane cellular components.

Interactions of the dimethyldiazaperopyrenium dication with nucleic acids. 1. Binding to nucleic acid components and to single-stranded polynucleotides and photocleavage of single-stranded oligonucleotides.

The binding of dimethyldiazaperopyrenium dication (1) with nucleosides, nucleotides, and single-stranded polynucleotides has been studied by photophysical methods. It has been shown that 1 may be a potential selective fluorescent probe for A- and/or T-rich polynucleotides. 1 efficiently cleaves oligonucleotides at guanine sites, under illumination with visible light, and therefore may be used as a sequence-specific artificial photonuclease.