Anthracycline antibiotic reduction by spinach ferredoxin-NADP+ reductase and ferredoxin

Abstract

Spinach NADPH:ferredoxin oxidoreductase (EC 1.6.7.1) catalyzes the NADPH-dependent reduction of the anthracyclines daunomycin, aclacinomycin A, and nogalamycin and their respective 7-deoxyanthracyclinones. Under anaerobic conditions, the endogenous rate of O2 reduction by NADPH catalyzed by ferredoxin reductase (0.12 s-1 at pH 7.4) is augmented by the anthracyclines and 7-deoxyanthracyclinones. The catalytic constants are approximately equivalent for this augmentation for all substrates (approximate V of 2 s-1 and KM of 75 microM). Both O2- and H2O2 are made. Under anaerobic conditions, anthracycline reduction catalyzed by ferredoxin reductase results in the elimination of the C-7 substituent to provide a quinone methide intermediate. Following tautomerization by C-7 protonation, 7-deoxyanthracyclinones are obtained. Under appropriate conditions these may be further reduced to the 7-deoxyanthracyclinone hydroquinones. For daunomycin, the quinone methide is formed rapidly after reduction and is easily monitored at 600 nm. It may react with electrophiles other than H+, as demonstrated by its competitive trapping by p-carboxybenzaldehyde. It may also react with nucleophiles, as demonstrated by its competitive trapping by N-acetylcysteine. For aclacinomycin, quinone methide formation is also rapid although no distinct transient near 600 nm occurs. In addition to protonation, it reacts with itself providing the 7,7'-dimer. With ethyl xanthate as a thiolate nucleophile, adducts derived from addition to C-7 are obtained. For nogalamycin, quinone methide formation is not rapid. Nogalamycin is reduced to its hydroquinone, which slowly converts in a first-order process [k = (1.2 +/- 0.2) X 10(-3) s-1, pH 8.0, 30 degrees C] to the quinone methide, which is then quenched by protonation. Spinach ferredoxin in its reduced form is chemically competent for anthracycline reduction. Its effect on both the aerobic and anaerobic reactions catalyzed by ferredoxin reductase is to increase severalfold the overall velocity for anthracycline reduction. In conclusion, the aerobic reaction pathways for the anthracyclines as mediated by ferredoxin reductase are remarkably similar, while the anaerobic reactions are remarkably different. If these anthracyclines exert their antitumor activity by a common anaerobic pathway, it is most likely that the pathway is determined by the properties of the anthracycline as complexed to its in vivo target. The behavior of ferredoxin further suggests that not only low-potential flavin centers but also iron-sulfur centers should be regarded as important loci for anthracycline reductive activation.

DNA sequence‐specific adducts of adriamycin and mitomycin C

“…Reductive activation of adriamycin did not yield any transcriptional blockages in addition to those observed under non-reducing conditions, even though the adriamycin was clearly being reduced, as shown by an increasing amount of adriamycinone precipitating from the reaction [26]. The reason that adducts previously observed by others under reducing conditions [12,13] were not observed in the transcriptional analysis is presumably due to the 1000-fold lower DNA con-centration employed in the present work which is based on nanomolar levels of promoter-containing DNA fragments.…”

A chemical perspective on the anthracycline antitumor antibiotics.

“…However menogarol, being epimeric at C-7, has the pseudoaxial position available (25). Yet it reacts only somewhat faster than nogalamycin (18). Since within this family leaving group stability does not influence the reaction rate, it must be concluded that stereoelectronics alone is insufficient to account for the slow rate of quinone methide formation within the nogalamycin family.…”

“…This is a uniquely suitable reducing agent for quinone methide production in that it is stable in the absence of an oxidant, a strong and rapid reducing agent in the presence of an oxidant, and possesses complete regiochemistry for quinone reduction over C-13 carbonyl reduction. Daunomycin reduction in aqueous solution is most conveniently done with NAD(P)H as the penultimate reducing agent and an enzyme catalyst, NADP:ferredoxin oxidoreductase from spinach (18), as the ultimate reducing agent. In both cases, spectroscopic examination reveals the essentially immediate formation of the quinone methide from the daunomycin hydroquinone, observing at the X__.…”

“…The one functional group found to qualify thus far is the aldehyde (15,16,18). Quinone methide trapping at the re face is followed by intramolecular hemiacetal formation at the C-13 carbonyl.…”

“…Nor is NADH dehydrogenase the only candidate in the mitochondrion for this reaction. Attention is drawn to this enzyme both by its classification as a low potential flavoenzyme (43) and by Doroshow's studies (72,73); however, the observation that low potential iron-sulfur centers are chemically competent anthracycline reductants (18) opens the range of possibilities to other enzymes. While it is possible that these may be minor contributors to the overall reduced oxygen flux, the inhibition of these enzymes if specific and potent, may contribute to cytotoxicity.…”