Maleimide is a potent inhibitor of topoisomerase II in vitro and in vivo: a new mode of catalytic inhibition

Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase IIalpha, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abolish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these maleimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etoposide-induced DNA damage. 2) In a band depletion assay maleimide reduced etoposide-induced trapping of topoisomerase IIalpha and beta on DNA. 3) In a clonogenic assay maleimide antagonized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase IIalpha content are fully sensitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide demonstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons.     

Cytochrome P450 enzymes contributing to demethylation of maprotiline in man

From case reports of patients treated with the tetracyclic antidepressant drug maprotiline, it appears that this drug is subject to polymorphic metabolism. Thus, we studied formation of the major maprotiline metabolite desmethylmaprotiline to identify the human cytochrome P-450 enzymes (CYP) involved. In incubations with human liver microsomes from two different donors, the substrate maprotiline was used at five different concentrations (5 to 500 microM). For selective inhibition of CYPs, quinidine (0.5-50 microM; CYP2D6), furafylline (0.3-30 microM; CYP1A2), ketoconazole (0.2-20 microM; CYP3A4), mephenytoin (20-200 microM; CYP2C19), chlorzoxazone (1-100 microM; CYP2E1), sulphaphenazole (0.2-100 microM; CYP2C9) and coumarin (0.2-100 microM; CYP2A6) were used. Desmethylmaprotiline concentrations were measured by HPLC, and enzyme kinetic parameters were estimated using extended Michaelis-Menten equations with non-linear regression. Relevant inhibition of the desmethylmaprotiline formation rate was observed in incubations with quinidine, furafylline and ketoconazole only. Formation rates of desmethylmaprotiline were consistent with a two enzyme model with a high (K(M)=71 and 84 microM) and a low (K(M)=531 and 426 microM) affinity site for maprotiline in the two samples, respectively. The high affinity site was competitively inhibited by quinidine (K(i,nc) 0.13 and 0.61 microM), the low-affinity site was non-competitively inhibited by furafylline (K(i,nc) 0.11 and 1.3 microM). Thus it appears that CYP2D6 and CYPIA2 contribute to maprotiline demethylation. Based on the parameters obtained, for plasma concentrations of 1 microM 83% (mean) of desmethylmaprotiline formation in vivo is expected to be mediated by CYP2D6 while 17% only may be attributed to CYPIA2 activity.     

Applicability of a modified Edman procedure for measurement of protein adducts: mechanisms of formation and degradation of phenylthiohydantoins

Adducts to N-terminal valine residues in hemoglobin (Hb) are used for monitoring in vivo doses of electrophiles and are quantitated by means of a modified Edman procedure, the "N-alkyl Edman procedure". In the reaction with pentafluorophenyl isothiocyanate, N-alkylated valines cyclize and detach from the protein as pentafluorophenylthiohydantoins (PFPTHs) much more efficiently than do unsubstituted N-terminal valine residues. The mechanisms of this reaction, and of possible degradation reactions, have been studied with model compounds using phenyl- and pentafluorophenyl isothiocyanate. The rapid cyclization to N-alkylvaline-PTHs occurs as a consequence of the influence of substituents on ring formation. This facilitated cyclization favors a direct attack by the thiocarbamoyl nitrogen atom on valine-C-1, and is also observed to occur slowly at unsubstituted N-terminal valines. Such cyclization is favored in protic solvents. Under alkaline conditions and in the presence of air, hydrolytic and oxidative processes give rise to degradation products. The PTH derivatives of N-alkylvaline are less apt to undergo such reactions than are the corresponding derivatives of unsubstituted valine. We conclude that the presence of an N-substituent exerts a greater influence on the cyclization process than the structure of the amino acid or of the Edman reagent. For adducts of different structures, the method has broad applicability, for which the limits, however, are not yet explored. The knowledge from the studies is valid not only for the N-alkyl Edman procedure, but also, to some extent, for the classical Edman degradation reaction. The oxidative side reaction gave rise to the invention of a novel synthesis route for insertion of nucleophiles at carbon-5 in thiohydantoins. The present investigation provides a basis for the N-alkyl Edman procedure, facilitating new toxicological applications.     

Evidence for environmental influence on CYP2D6-catalysed debrisoquine hydroxylation as demonstrated by phenotyping and genotyping of Ethiopians living in Ethiopia or in Sweden

Black Africans show lower rates of CYP2D6- and CYP2C19-dependent drug metabolism compared to Caucasians of the same apparent genotype. To determine if environmental factors are responsible for this difference, the genotypes and phenotypes of CYP2D6 and CYP2C19 among Ethiopians living in Sweden (n = 70) were assessed and compared to our previously published data from Ethiopians living in Ethiopia (n = 114) and Swedish Caucasians (n = 134). There was no significant difference in CYP2C19 genotype or phenotype as assessed by mephenytoin between Ethiopians in Sweden or in Ethiopia. However, Swedes were significantly more rapid for CYP2C19 activity than both Ethiopian groups (P < 0.01). A comparison of the debrisoquine MR among individuals of the same CYP2D6 genotype revealed that Swedes exhibited the highest rate of debrisoquine metabolism, followed by Ethiopians in Sweden and Ethiopians in Ethiopia. The difference between the Ethiopian groups was significant (P < 0.02 using a univariate test ANOVA) and amounted to approximately 50% of the magnitude of the MR difference between Swedes and Ethiopians in Ethiopia. It is tempting to speculate that inhibitory dietary factors may explain the differences seen between the two Ethiopian groups and that these components in the past might have contributed to dietary stress-mediated selection of duplicated and multiduplicated active CYP2D6 genes, as frequently seen in Ethiopians. In conclusion, the results indicate a significant influence of environmental factors as an explanation for the difference in capacity for CYP2D6, but not CYP2C19 metabolism between Caucasians and Black Africans. Additional factors remain to be elucidated to fully explain the interethnic differences in CYP2D6 activity.     

Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine

Summary. The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5'-nucleotidases. We have analysed dCK and 'high-Km' 5'-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high- and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.     

Expression of cox-2, tie-2 and glycodelin by megakaryocytes in patients with chronic myeloid leukaemia and polycythaemia vera

When evaluating bone marrow sections for markers of neo-angiogenesis, we found that megakaryocytes stained markedly positive for cyclooxygenase-2 (Cox-2), Tie-2 and glycodelin. This apparently novel finding was further evaluated for disease-specific variations. Bone marrow sections from two patient groups, known to be characterized by clonal megakaryocytopoiesis, viz. chronic myeloid leukaemia and polycythaemia vera, stained, however, similarly to healthy marrows for these markers. The biochemical background and clinical significance of Cox-2, Tie-2 and glycodelin remains to be elucidated.