Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.

Flavonoid-related modulators of multidrug resistance: synthesis, pharmacological activity, and structure-activity relationships.

A series of 28 flavonoid derivatives containing a N-benzylpiperazine chain have been synthesized and tested for their ability to modulate multidrug resistance (MDR) in vitro. At 5 microM, most compounds potentiated doxorubicin cytotoxicity on resistant K562/DOX cells. They were also able to increase the intracellular accumulation of JC-1, a fluorescent molecule recently described as a probe of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity. As in other studies, lipophilicity was shown to influence MDR-modulating activity but was not the only determinant. Diverse di- and trimethoxy substitutions on N-benzyl were examined and found to affect the activity differently. The most active compounds had a 2,3, 4-trimethoxybenzylpiperazine chain attached to either a flavone or a flavanone moiety (13, 19, 33, and 37) and were found to be more potent than verapamil.

A double-blind study of paroxetine, fluoxetine, and placebo in outpatients with major depression.

We report results from a multicenter, placebo-controlled, randomized, double-blind comparison of the efficacy and tolerability of paroxetine and fluoxetine in outpatients with major depression. Across five U.S. sites, 128 outpatients (mean age: 41.3 +/- 12.6; 63 men and 65 women) with moderate to moderately severe major depression without a history of mania or hypomania were recruited between 1993 and 1994. All 128 patients completed a 1-week placebo washout period, and were then randomized to 12 weeks of double-blind treatment with paroxetine up to 50 mg/day (n = 55), fluoxetine up to 80 mg/day (n = 54), or placebo (n = 19). Subjects were evaluated weekly for the first 4 weeks, then at weeks 6, 9, and 12 with the 21-item HAMD and the Covi Anxiety Scale. There were no significant differences among the three treatment groups in baseline and endpoint depression and anxiety severity, as well as in the degree of depression and anxiety improvement. There were no statistically significant differences in rates or mean numbers of adverse events between paroxetine-treated patients and fluoxetine-treated patients. In summary, our results, although limited by the lack of a significant difference from placebo in treatment outcome, suggest that the SSRIs paroxetine and fluoxetine have comparable antidepressant and antianxiety efficacies among depressed outpatients, as well as similar safety and tolerability profiles.

Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe.

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.

Protonophoric activity of ellipticine and isomers across the energy-transducing membrane of mitochondria.

Ellipticine is an antitumor alkaloid capable of uncoupling mitochondrial oxidative phosphorylation. It behaves as a lipophilic weak base with pK = 7.40. We have investigated its molecular mode of action using several of its isomers with pK ranging between 5.8 and 7.7 and ellipticinium, which is a permanent cationic derivative. The effects of these molecules on mitochondrial oxygen uptake and transmembrane potential were compared at different pHs. Ellipticinium exhibited very low effects on both respiratory rate and membrane potential. By contrast, protonable derivatives showed maximal stimulation of oxygen uptake and depolarizing effects when the pH of the medium was close to the drug pK. These effects were lowered when the transmembrane delta pH was dissipated, which indicates that the neutral form of the drug is implicated in the uncoupling mechanism. In addition, protonable derivatives of ellipticine display a linear relationship between oxidation rate and transmembrane potential, which suggests that the uncoupling properties of these molecules result from a protonophoric mechanism. From these results, the following cyclic protonophoric mechanism is proposed for protonable ellipticines: (i) electrophoretical accumulation of the protonated form; (ii) deprotonation at the matrix interface; (iii) diffusion outwards; and (iv) reprotonation at the external interface.

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

Thermodynamics of drug-DNA interactions: entropy-driven intercalation and enthalpy-driven outside binding in the ellipticine series.

Viscosimetric and kinetic results allow one to characterize three modes of DNA binding in the ellipticine series: (1) Ellipticine and its 9 methoxy derivative, which present maximal DNA lengthening properties and bind DNA through a single step mechanism, can be considered as pure intercalators. (2) Ellipticinium derivatives and short-chain substituted oxazolopyridocarbazoles, which present intermediate DNA lengthening properties, bind DNA through a two-step mechanism, one being intercalation. (3) Long-chain substituted oxazolopyridocarbazole derivatives, which display the smallest DNA lengthening properties, bind DNA through a single-step mechanism, probably resulting from an outside binding mode. The viscosimetric and kinetic results are compared with the thermodynamic results obtained from the temperature dependence of the binding constants. It appears that drugs binding on the outside of the DNA double helix tend to have large enthalpy and small entropy contributions, whereas pure intercalating drugs have contributions from both enthalpy and entropy, with entropy dominating by about 2:1. Drugs showing two binding modes exhibit a continuum between the aforementioned extremes, with no breaks in behavior. From this comparison, a correlation between thermodynamic data and DNA binding modes is proposed. Possible molecular implications of both enthalpy and entropy to DNA binding free energy are discussed.

Ditercalinium, a nucleic acid binder, inhibits the respiratory chain of isolated mammalian mitochondria.

Ditercalinium (a 7H-pyridocarbazole dimer) has been designed to bisintercalate into double-stranded DNA with high affinity. In this paper we provide evidence for inhibitory interactions of ditercalinium with electron transport in isolated rat liver mitochondria. It is shown that ditercalinium probably inhibits the electron transfer between membrane cytochrome c and oxygen (cytochrome c oxidase activity) and the electron transfer between the matrix side of inner membrane (Complexes II and III) and membrane cytochrome c. The level of inhibition of the last oxidation step of the respiratory chain appears to be highly dependent on the drug/membrane diphosphatidylglycerol ratio. It is suggested that the mechanism of cytochrome c oxidase inhibition by ditercalinium could be due to the complexation with the diphosphatidylglycerol environment essential for its activity rather than to a drug-enzyme direct interaction. This hypothesis is strengthened by experiments with pure cytochrome oxidase. Therefore, the interaction of ditercalinium with diphosphatidylglycerol may be envisaged as one factor, among others, responsible for its hepatotoxicity.

Acid-base properties of ellipticine bound to DNA, micelles and liposomes.

We have determined the acid-base properties of the alkaloid ellipticine, bound to DNA and to micelles and liposomes, taken as models for membranes, in the prospect of characterizing the actual structure of the bound ligand, this being relevant to the mode of action of the drug. The acid-base properties of ellipticine bound to sonicated calf thymus DNA and SDS micelles are similar as regards their pK values and their dependence on NaCl concentrations. This observation is satisfactorily understood in terms of sodium ion condensation around the negative phosphate and sulphate groups. The slope of pK vs log(Na+) is -1, a value predicted by Friedman theory. The pK of ellipticine bound to cationic (CTAB, DDTAB) or to neutral (Triton X100) micelles and to neutral liposomes (PC) is significantly lower than water (7.4), and, in contrast to the situation in DNA and SDS micelles, does not vary with addition of NaCl. Interestingly, this result is good evidence for ellipticine having a specific pK when bound to a hydrophobic structure. This view is likely to hold for ellipticine bound to DNA.

Kinetic and thermodynamic studies on drug-DNA interactions in the ellipticine series.

The temperature-jump (T-jump) method has been used to investigate the binding mechanism to calf-thymus DNA of ellipticine and some of its derivatives. The results show that the plant alkaloid, ellipticine, interacts with DNA at a unique intercalation site whereas most of its synthetic derivatives, such as ellipticinium, 9-hydroxy-ellipticinium and related alkyl-oxazolopyridocarbazoles recognize two distinct DNA sites. Parallel analysis of kinetic data and DNA lengthening abilities of these derivatives suggests that only one of these two DNA sites is an intercalation site. Owing to the determination of the genuine number of drug-DNA complexes (inferred from T-jump experiments) and with the results of thermodynamic investigations (Van't Hoff plots), further characterization of the molecular interactions involved in the binding process was proposed. Thus, the formation of the unique intercalation complex of ellipticine was found to be entropy driven whereas binding of drugs which recognize the second class of binding sites was essentially enthalpy driven. These different thermodynamic behaviors suggest that intercalation essentially results from hydrophobic solvent structure effects in contrast to the second binding mode which principally arises from hydrogen bonding interactions through DNA grooves.

Effect of intercalative binding compared to external binding on Z/B equilibrium of poly d(GMe5C) using fluorescent oxazolopyridocarbazoles as probes.

Using the fluorimetric determination of the binding isotherms in combination with circular dichroism, we have investigated the effect of the binding of the intercalating chromophore oxazolopyridocarbazole (OPC) to poly d(GMe5C) on B/Z equilibrium, compared to the effect of the external binder OPC derivative pentyl-2-OPC. The intercalating OPC appears to be very efficient in reversing left-handed poly d(GMe5C) into the right-handed conformation, according to a cooperative mode. For each OPC molecule intercalated into the B form, 7 base pairs were switched from the Z to B conformation. In contrast, the binding of the external binder pentyl-OPC resulted in a limited Z to B transition, involving the switch of 1.4 base pairs from the Z to B conformation. Moreover, OPC appears much more efficient than pentyl-OPC in inhibiting both the extent and kinetics of the salt-induced B/Z transition. At low drug to DNA ratio (D/P = 1/50), a 7-fold and 1.5-fold inhibition of the B/Z transition kinetics occurs in the presence of OPC and pentyl-OPC, respectively. These features are discussed in terms of the difference existing between the entropic contribution in the DNA binding of intercalating agents, compared to external binders.

The G.C base-pair preference of 2-N-methyl 9-hydroxyellipticinium.

Among the DNA-intercalating drugs in the ellipticinium series, 9-hydroxy derivatives elicit the highest antitumoral properties. In water these drugs display a very low fluorescence quantum yield. Replacement of H2O by D2O partially restores the fluorescence of the ellipticinium chromophore. The possibility that such a proton-exchange mechanism could be involved in a base-recognizing process at the DNA level (and therefore be responsible for some base preference) was examined by direct fluorescence titration in deuterated buffer and DNA/drug fluorescence energy transfer. These experimental approaches provide mutually consistent results showing that the 9-hydroxylated drug recognizes specific DNA sites that are not recognized by the non-hydroxylated drug. When compared to 2-N-methyl ellipticinium, the 2-N-methyl 9-hydroxyellipticinium presents: (1) higher binding constants for each DNA studied; (2) a base dependence of the fluorescence properties of the bound form (fluorescence increment upon DNA binding varying over 5-11); (3) a base dependence of its DNA affinity constants (1.1-3.3 x 10(6) M-1) and of its site size (exclusion parameters varying over 3.0-4.4); (4) a base dependence of its energy transfer from DNA bases. Analysis of the binding data suggests that the 9-hydroxyl group of 2-N-methyl ellipticinium is responsible for a G.C base-pair preference, the preferred binding site being a doublet sequence of two adjacent G.C which could be flanked either by a additional G.C base pair or by an A.T base pair.

Dynamics of drug-DNA interactions: a comparative temperature jump study of ellipticinium and 9-hydroxy ellipticinium.

The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNA's of different AT/GC composition. The relaxation signals, analyzed by the Padé-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range. In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens). Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition. The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites). Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA. They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies. The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation. The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNA's and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study.

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).

Relationships between physicochemical and biological properties in a series of oxazolopyridocarbazole derivatives (OPCd); comparison with related anti-tumor agents.

Oxazolopyridocarbazole derivatives (OPCd) are intercalating polycyclic molecules related to the anti-tumor drug 9-hydroxyellipticinium (Celiptium). From a pharmacological point of view, OPCd compounds are highly cytotoxic to malignant cultured cells but inactive or only weakly active against experimental tumors in vivo. Extensive physicochemical and biological investigations have been performed in this series including the determination of hydrophobic properties, interaction parameters with DNA and polynucleotides, interaction with DNA topoisomerase II in vitro, diffusion through cell membranes, accessibility to genomic DNA in cells and in chromatin preparations and finally, cytotoxic and anti-tumor activities. Establishment of relationships between physicochemical data and biological properties have been attempted. The results show that all the OPCd compounds display favorable parameters in terms of association constant values to DNA, accessibility to DNA in chromatin structure and permeation through cellular membranes. However, in contrast with intercalating drugs such as m-AMSA, adriamycin and 9-hydroxyellipticinium, OPCd compounds are not able to generate cleavable complexes in DNA through the interaction with topoisomerase II. With respect to design of anti-tumor drugs, these findings indicate that a high association constant value to DNA, the ability to intercalate between DNA base pairs without causing physical damage and an efficient diffusion through cell membranes are not by themselves sufficient for the expression of anti-tumor activity.