Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis.

LUDI is a program used for de novo structure-based design of ligands and can predict binding of ligands quantitatively using a scoring function. Here we evaluate LUDI in a lead optimisation study with ligands for the antibody MN12H2, that has been raised against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. The ligands were synthetic peptides that are derived from the smallest binding epitope (182)DTNNN(186). LUDI's fragment building rules are used for the proposal of new peptide-ligands for MN12H2 and were focused on replacements of Asp(186) in the epitope. Accordingly, a series of peptides was synthesised with isosteric mutations. The interaction of the peptides with MN12H2 was analysed with a surface plasmon resonance competition assay yielding equilibrium binding constants in solution (K(S)). The binding affinity seems to be largely determined by entropy, and the side chain of Asn(186) is sensitive for charge, inversion, hydrophobicity and size. Head-to-tail cyclisation of the peptide in a nine-amino-acid ring gives little reduction in affinity. It is concluded that the scoring function of LUDI does not help in optimisation of the peptide lead for MN12H2 binding. Other more elaborate molecular mechanics calculations show similar results. This implies that our current knowledge of molecular recognition is insufficient for explaining a case of peptide-protein binding, where the design process requires subtle changes in structure (from lead finding to lead optimisation).

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument.

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.

Mechanism of action of the nonlipophilic antiallergic drug eclazolast (REV 2871) in the inhibition of mediator release in a mast cell model.

OBJECTIVE AND DESIGN: In this study, we compared eclazolast with other lipophilic antiallergic drugs, relating to effects on signal transduction pathways, leading to inhibition of exocytosis in a rat basophilic leukemia cell (RBL-2H3). MATERIALS AND METHODS: Effects of the drugs on mediator release (beta-hexosaminidase, arachidonic acid metabolites) after Fc(epsilon)RI activation in RBL-2H3 cell were quantified. Furthermore, effects of the drugs on cellular signalling (Ca2+ influx, intracellular Ca2+ concentration, inositol 1,4,5-trisphosphate (IP3) concentration) were assayed. Effects of the drugs on bilayer and cell membranes have been recorded. RESULTS: It is shown that eclazolast down-regulates IP3 levels. In contrast to lipophilic drugs, eclazolast does not affect artificial bilayers and erythrocyte membranes, and there is no effect on thapsigargin induced Ca2+ influx. The effect of eclazolast was highly dependent on the antigen concentration with which the cells were triggered. CONCLUSIONS: The mechanism of action of eclazolast is deviant from lipophilic antiallergic agents. It inhibits exocytosis by intracellularly affecting only direct Fc(epsilon)RI linked processes and not through inhibition of Ca2+ influx channels, as found for membrane disturbing lipophilic drugs.

Influence of the antiallergic drug oxatomide and derivatives on membrane structures: relation with inhibition of calcium influx in rat basophilic leukemia cells.

Oxatomide is an H1 antihistaminic drug that also inhibits mediator release from mast cells. From previous studies, it appeared that inhibition of the influx of extracellular calcium is the major cause of this inhibition of exocytosis. Here, we explored the role of drug-membrane interactions in the inhibition of mediator release. We investigated the effects on phase transition and fluidity of artificial membranes. All compounds studied distorted the phase transition in L-alpha-dipalmitoylphosphatidylcholine liposomes, which correlated with the drug-induced increase in membrane fluidity measured by fluorescence anisotropy of the bilayer interacting probe 1-[4-(trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. Erythrocytes were used to study membrane effects on a cellular level. The hypotonic-induced haemolysis of erythrocytes was inhibited by the drugs. Compounds which increased membrane fluidity of liposomes to a greater extent were also more active in decreasing haemolysis. Drug-induced disturbance of the membranes is related to their effect on the activity of store-operated Ca2+ channels. The activity of these channels in rat basophilic leukemia cells, assayed as 45Ca2+ influx, was most effectively inhibited by oxatomide derivatives, thereby inducing a more rigid membrane structure. Small changes in molecular structure affect the activity of the drugs and these structure-activity relations are discussed.

Effects of oxatomide and derivatives on high affinity IgE receptor-activated signal transduction pathways in rat basophilic leukemia cells: role of protein tyrosine hyperphosphorylation and inhibition of extracellular calcium influx.

The antiallergic drug oxatomide and analogs inhibit mediator release from a rat basophilic leukemia (RBL-2H3) cell line, which is frequently used as a mast cell model. By investigating a series of derivatives of oxatomide with different inhibiting activities on exocytosis, we aimed to evaluate the role of their effects on the early steps of the signal transduction cascade in the inhibition of exocytosis. The active compounds induced hyperphosphorylation of tyrosine residues both in stimulated as well as in resting cells. Furthermore, some elevation of the inositol 1,4,5-trisphosphate (IP3) formation upon antigen activation was observed for the active derivatives. Ca2+ fluxes were also studied. The inhibition of the antigen-induced 45Ca2+ influx correlated with the effects of the drugs on exocytosis. Furthermore, the inhibitory activity on antigen- and thapsigargin-mediated exocytosis correlated well. Adherence of the cells to fibronectin, stimulating cellular integrin receptors, was synergistic to antigen activation of the RBL cells. However, oxatomide did lack any effect on integrin-mediated processes, as the IC50 value for exocytosis was identical for fibronectin-adhered cells and standard cultured cells. We conclude that oxatomide and its analogs inhibit exocytosis, mainly by inhibiting Ca2+ influx over store-operated Ca2+ (SOC) channels. The drugs have a direct effect on the store-operated Ca2+ channels or affect the direct regulation of these channels.

Structure-activity relationships of astemizole derivatives for inhibition of store operated Ca2+ channels and exocytosis.

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme beta-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on Fc epsilonRI receptor-stimulated exocytosis, changes were also observed in Ca2+ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated 45Ca2+ influx. Furthermore, the inhibition of 45Ca2+ influx was correlated with the inhibition of beta-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell beta-hexosaminidase release through inhibition of Ca2+-store-operated Ca2+ channels (SOC). Compounds with high lipophilicity also released Ca2+ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl-benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.

Dual effect of the anti-allergic astemizole on Ca2+ fluxes in rat basophilic leukemia (RBL-2H3) cells: release of Ca2+ from intracellular stores and inhibition of Ca2+ release-activated Ca2+ influx.

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation.

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways: (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene-DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1, 10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1"-n-hydroxymitosen-10"-yl), 2'-deoxyguanosine (n = alpha or beta). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2'-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene.

Effects of the anti-allergics astemizole and norastemizole on Fc epsilon RI receptor-mediated signal transduction processes.

The non-sedating anti-allergic drug astemizole, apart from its potential to antagonise H1 receptors, inhibits the release of inflammation mediators from mast cells. To study the mechanism of this inhibition, we investigated the effects of astemizole and one of its active metabolites, norastemizole, on different phases of Fc epsilon RI (the high affinity receptor for the immunoglobulin IgE) receptor-activated signal transduction in rat basophilic leukemia cells (RBL-2H3), leading to exocytosis. Cells were stimulated either through antigen, or thapsigargin, or synergistic combinations of Fc epsilon RI receptor activation with either adenosine A3 receptors or integrins, activated by fibronectin adherence. The effects of the drugs on mediator release, inositol 1,4,5-trisphosphate formation, tyrosine phosphorylation of cellular proteins and Ca2+ fluxes were investigated. Inositol 1,4,5-trisphosphate levels are not affected. Astemizole increased tyrosine phosphorylation in resting cells, especially a 96-kDa protein band. Particularly tyrosine phosphorylation related to post Ca2+ processes is changed after cell triggering in the presence of astemizole. Both drugs inhibit the influx of 45Ca2+, with similar dose response curves as for the inhibition of exocytosis. Astemizole but not norastemizole, when used in resting cells, released Ca2+ from intracellular stores. Astemizole (> 15 microM) also induced exocytosis in resting cells. It did not induce additional changes in its inhibiting effect when cells were triggered with synergistic combinations of Fc epsilon RI receptor activation with either adenosine A3 receptors or integrins. Effects on haemolysis of erythrocytes and differential scanning calorimetry in liposomes showed clear differences in membrane perturbation between astemizole and norastemizole. The observed differences, and the role of membrane perturbation in the action on Ca2+ fluxes, are discussed.

Relation between effects of a set of anti-allergic drugs on calcium pathways and membrane structure in Fc epsilon RI activated signal transduction.

The antigen induced stimulation of mast cells by aggregation of Fc epsilon RI receptors activates a signal transduction cascade leading to release of mediators of inflammation like histamine, arachidonic acid metabolites and cytokines. In this study we investigated a series of structurally related anti-allergic drugs, containing a common lipophilic diphenylmethyl piperazinyl tail and head groups that differ in lipophilicity. Effects of these drugs on various steps of the signal transduction cascade was investigated to gain insight into the mechanism of action of these drugs. It appeared that addition of the drugs to resting cells induced changes in the tyrosine phosphorylation of cellular proteins. The most active anti-allergics in inhibiting exocytosis, AL3264 and oxatomide, also induced the largest changes in phosphorylation. The effects of the drugs on tyrosine phosphorylation after cell activation was complex. Additionally, Ca2+ fluxes were investigated. Ca2+ efflux from the cells was negligibly influenced by the active drugs. However, the drugs inhibited influx from extracellular Ca2+, which was correlated with the effects of the drugs on inhibition of exocytosis and on membrane stabilization induced by the drugs, measured as haemolysis of erythrocytes. It is concluded that inhibition of Ca2+ influx is the major mechanism with which these drugs inhibit exocytosis and that for this effect drug-membrane interactions, possibly affecting the function of membrane embedded proteins, are of importance. Possible mechanisms including drug-membrane interactions, phosphorylation and inhibition of Ca2+ influx are discussed.

Influence of the anti-allergic drug oxatomide on the signal transduction mechanism in a mast cell model.

In a mast cell model, oxatomide displays inhibition of mediator release which is not related to its histamine H1 receptor antagonistic activity. From a previous study it appeared that especially early steps in the signal transduction leading to exocytosis were influenced by oxatomide. We now studied effects of oxatomide on those early steps in more detail. The antigen- and thapsigargin-mediated exocytosis in rat basophilic leukemia (RBL-2H3) cells were both inhibited by oxatomide. After aggregation of high affinity receptors for immunoglobulin E (Fc epsilon RI), protein tyrosine phosphorylation is induced. Oxatomide caused remarkable changes in the tyrosine phosphorylation pattern in resting cells. Also after antigen and thapsigargin activation, changes in the tyrosine phosphorylation of cellular proteins are observed. In addition, Ca2+ fluxes were studied by means of the net influx of 45Ca2+ and by measuring intracellular free Ca2+ concentrations ([Ca2+]) with the fluorescent probe fura-2. Oxatomide inhibited the 45Ca2+ influx and the increase in [Ca2+]i upon antigen and thapsigargin activation of the cells. Neither the release of Ca2+ from internal stores nor the efflux of Ca2+ over the plasma membrane seems to be affected. The effect of oxatomide on Ca2+ influx was further characterized by studying Ba2+ influx in the absence of extracellular free Ca2+. We conclude that inhibition of mediator release is mainly caused by inhibition of influx of extracellular Ca2+, via plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores. The molecular mechanism with which oxatomide might interfere with these channels is discussed.

Chirality of a 1,10-bisacetoxymitosene compound. Impact on reductive activation, DNA interstrand cross-linking and antitumour activity.

The absolute configuration at the C-1 position of a 1,10-bisacetoxymitosene (WV15) appears to be important for enzymatic reduction, DNA interstrand cross-linking and in vitro antitumour activity of this compound. DNA cross-linking by the (-)-(S)-enantiomer of WV15 upon reduction with sodium dithionite (Na2S2O4) was more efficient than cross-linking by the (+)-(R)-enantiomer. Also, following enzymatic two-electron reduction by DT-diaphorase or one-electron reduction by xanthine oxidase, (-)-(S)-WV15 was more efficient in DNA cross-linking than (+)-(R)-WV15. However, the difference in cross-linking efficiency was less than upon chemical reduction, and in the case of enzymatic reduction that higher amount of DNA cross-links formed by (-)-(S)-WV15 can be explained by more efficient enzymatic activation of this enantiomer as compared to (+)-(R)-WV15. The enantiomeric preference upon chemical reduction can be explained by a second chemical reduction of DNA-bound WV15, which presumably does not occur upon enzymatic reduction. (-)-(S)-WV15 appeared to be more active than its (+)-(R) counterpart in A204 and L1210 tumour cell lines, with (+)-(R)/(-)-(S) toxicity ratios as high as 200 and 68, respectively. In Chinese hamster V79 cell lines, toxicity of the enantiomers was measured under oxic and hypoxic conditions. The oxic/hypoxic toxicity ratios of (+)-(R)-and (-)-(S)-WV15 in the Chinese hamster V79 cell line were 5.5 and 2.4, respectively. These different oxic/hypoxic toxicity ratios may indicate that different reducing enzymes are involved in the activation of the enantiomers. Generally, in biological systems, different activities of (+)-(R)- and (-)-(S)-WV15 appear not to be caused by different intrinsic cross-linking capacities of the enantiomers, but by more efficient enzymatic activation of (-)-(S)-WV15, as compared to (+)-(R)-WV15. The (-)-(S)-enantiomer of WV15 appears to be more active both in in vitro tumour models and in DNA cross-linking assays, and therefore the absolute configuration of mitosenes is indicated to be important for the antitumour activity of these compounds.

Oxatomide and derivatives as inhibitors of mediator release from a mast cell model. Structure-activity relationships.

A series of benzimidazolone and benzimidazole analogues of the antiallergic drug oxatomide (1-¿3-[4-(diphenylmethyl)-1-piperazinyl]propyl¿-1,3-dihydro-2H- benzimidazol-2-one, CAS 60607-34-3) [formula: see text], was evaluated for inhibiting the release of the performed mediator beta-hexosaminidase from the rat basophilic leukemia (RBL-2H3) cell line. Activation of the cells was induced by antigen, or by the calcium ionophore A23187 (calcimycin) in combination with or without the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). For the active compounds, inhibition of exocytosis was found with all triggers, with the antigen trigger being somewhat more sensitive. This indicates that the compounds influence several steps in the signal transduction route leading to exocytosis. The activity of the compounds is not totally aspecific as small structural changes strongly affect the inhibiting activity. Introduction of a chlorine substituent at the 6-position of the benzimidazolone group results in loss of activity. There does not seem to be a significant activity difference between the benzimidazolone and benzimidazole analogues. Analogues with n < 3, n > 5 or a branched alkyl chain between the piperazinyl and the benzimidazol(on)e moiety lose inhibitory activity. Secretion of the newly formed mediator arachidonic acid and its metabolites was affected by the compounds comparable to the effect on the release of beta-hexosaminidase. The anti-allergic activity did not correlate with the histamine H1-receptor antagonistic activity.

Indoloquinone EO9: DNA interstrand cross-linking upon reduction by DT-diaphorase or xanthine oxidase.

We report DNA interstrand cross-linking caused by the anti-tumour indoloquinone EO9 following reductive activation with purified rat liver DT-diaphorase or xanthine oxidase. Reduction was a necessary event for cross-linking to occur. DNA cross-link formation by EO9 following DT-diaphorase reduction was completely inhibited by addition 10 microM dicoumarol, whereas only a minor effect of dicoumarol on xanthine oxidase-mediated DNA cross-linking by EO9 was observed. DNA cross-linking was pH dependent, with increasing cross-link formation from pH 5.5 to 7.0 for both DT-diaphorase and xanthine oxidase mediated reactions. Also, conversion of EO9 upon reduction was pH dependent. However, in contrast to DNA cross-linking, conversion rates of EO9 decreased at higher pH. EO9 was shown to be more efficient in DNA cross-linking than mitomycin C under identical conditions, using both DT-diaphorase and xanthine oxidase reductive activation at pH 5.5 and 7.0. This study indicates that the anti-tumour activity of EO9 may be at least partly mediated by interstrand DNA cross-link formation, and that various reducing enzymes may be important for activation of EO9 in vitro and in vivo.

Reduction of antitumour mitosenes in non-aqueous and aqueous environment. An electron spin resonance and cyclic voltammetry study.

Chemical reduction of mitosenes under aerobic conditions in DMSO showed characteristic ESR signals of the mitosene derived semiquinone free radicals. However, these signals diminished strongly upon addition of water to the reaction mixture, indicating a short lifetime of the mitosene semiquinone free radicals under aqueous conditions. In addition, enzymatic one-electron reduction of these mitosenes with either xanthine oxidase or purified NADPH cytochrome P450 reductase under anaerobic conditions showed no signals of the mitosene semiquinone free radicals. Subsequent cyclic voltammetry measurements demonstrated facilitation of the further one-electron reduction of the mitosene semiquinone free radicals in the presence of water in comparison with non-aqueous conditions. The present results strongly suggest that in the presence of water relatively stable hydroquinones are formed upon reduction of mitosenes. Consequently, the steady state concentrations of mitosene semiquinone free radicals will be lowered substantially in aqueous environment. Thus under physiological conditions, two-electron reduction and formation of the mitosene hydroquinone might be important in processes leading to DNA alkylation by these mitosenes.

Inhibition of mediator release in RBL-2H3 cells by some H1-antagonist derived anti-allergic drugs: relation to lipophilicity and membrane effects.

In a model for mucosal mast cells (RBL-2H3 cells) a set H1-antagonist derived anti-allergic drugs containing a diphenylmethyl piperazinyl moiety was examined for their ability to inhibit release of the mediator beta-hexosaminidase. Cells were activated with antigen or the calcium ionophore A23187, whether or not in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Oxatomide, hydroxyzine and cetirizine inhibit the antigen induced beta-hexosaminidase release. The release triggered by A23187, whether or not in combination with TPA is hardly influenced by the compounds. A biphasic dependence of the inhibition of exocytosis in RBL cells on lipophilicity is observed with the optimum at log P is 5-6. The extremely lipophilic compounds meclozine and buclizine are not active in this model. pH dependence of the effect of the drugs shows that especially the uncharged species are active in inhibiting exocytosis. The investigated compounds show an effect on phase transitions in L-alpha-phosphatidylcholine dipalmitoyl liposomes as assayed with differential scanning calorimetry (DSC). For the less extremely lipophilic compounds the induced changes in the phospholipid membranes increased with lipophilicity. The relation between structural features of the drug and the interaction with phospholipids is discussed in view of the DSC results. We conclude that location of the active drugs at the membrane or the membrane/protein interface is important for the inhibiting activity on exocytosis. This could affect several membrane related processes, which are abundant in the early phases of the IgE-mediated signal transduction process.

Potential antitumour mitosenes: relationship between in vitro DNA interstrand cross-link formation and DNA damage in Escherichia coli K-12 strains.

This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds. Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5. DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0. Without reduction or under aerobic conditions no cross-link formation was detected. The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E. coli K-12 strain. A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E. coli strain was found. The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells. In these cells, mitosenes appeared to be very active. Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions. The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system. Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

Kinetic evaluation of the oxidation of phenothiazine derivatives by methemoglobin and horseradish peroxidase in the presence of hydrogen peroxide. Implications for the reaction mechanisms.

The oxidation of ten 2-substituted 10-(3-(dimethylamino)propyl) phenothiazines (PHs) by methemoglobin (metHb) and horseradish peroxidase (HRP) in the presence of H2O2 was kinetically analysed based on an enzymic-chemical second-order reaction with substrate regeneration: PHs are oxidized enzymatically to their radical cations (PH+) which subsequently, in a second order reaction, react further to parent compound and PH-sulfoxide (PHSO). The enzymic reaction rate can be obtained from the accumulation curves of both radical cation formation and sulfoxide formation. In the case of chlorpromazine and promazine both methods gave similar reaction rates. The rate constant of PH+. decay could also be determined from the radical concentrations of their radicals. The rate constant of reaction of PHs with HRP compound II was also analysed. The logarithm of this rate constant correlated well with the Hammett sigma para and the Swain and Lupton F and R substituent constants, whereas no correlation with hydrophobic and steric parameters was found. This indicates that the interaction of PH with the porphyrin ring, which is the active site of HRP, is predominantly under electronic control. In the case of catalysis by hemoglobin (Hb), the formation of the reactive Hb form, ferry1Hb with a protein radical, appeared to be rate limiting in the oxidation of PHs by metHb-H2O2. Differences in the conversion rates of various PHs can be explained by a competition between their electron transfer reaction to the protein radical and the denaturation reaction(s) involving the protein radical. Our results confirm our earlier observation that the mechanism of oxidation by metHb-H2O2 differs from that of the classical peroxidases. In the former case, electron transfer from PH occurs most likely to a tyrosine residue on the globin part, whilst in the latter case electron transfer to the porphyrin moiety takes place.

Reductive activation of potential antitumor mitosene compounds.

The reductive activation of mitosene compounds was studied with cyclic voltammetry and HPLC analysis. Reduction of mitosenes, possessing good leaving groups at C-1 and C-10, was shown to result in loss of these groups at pH 7.0 and pH 6.0. The loss of leaving groups from mitosenes occurred faster at lower pH. Mitosenes without good leaving groups were found to be stable upon reduction. In the presence of acetoxy groups at C-1 and C-10, the C-10 site is the most reactive site upon reductive activation. This is opposite to the case of mitomycin C, where the C-1 site is the first to react upon reduction. At pH 6.0 without reduction, acid degradation also caused the loss of leaving groups of mitosenes, although at a very slow rate. In contrast to reductive activation, upon acid degradation of a diacetoxymitosene the C-1 group appeared to be lost faster. Electrochemical as well as dithionite reduction of a bifunctional (diacetoxy) mitosene compound in the presence of calf thymus DNA at pH 5.5 resulted in the formation of DNA interstrand cross-links. Depending on activation method, this diacetoxymitosene was at least as efficient in DNA cross-linking as mitomycin C under comparable conditions.

Redox cycling of potential antitumor aziridinyl quinones.

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.