Chemical and Enzymatic Reductive Activation of Acylfulvene to Isomeric Cytotoxic Reactive Intermediates

Abstract

Acylfulvenes, a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic towards cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capable of direct alkylation, cytosolic reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Data obtained in this study illustrates chemical aspects of the process of AF activation. By tracking reaction mechanisms with stable isotope-labeled reagents, enzymatic versus chemical activation pathways for AF were compared for reactions involving the NADPH-dependent enzyme prostaglandin reductase 1 (PTGR1) or sodium borohydride, respectively. These two processes resulted in isomeric products that appear to give rise to similar patterns of DNA modification. The chemically activated isomer has been newly isolated and chemically characterized in this study, including an assessment of its relative stereochemistry, and stability at varying pH and under bioassay conditions. In mammalian cancer cells, this chemically activated analog was shown to not rely on further cellular activation to significantly enhance cytotoxic potency, in contrast to the requirements of AF. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful chemical probe for evaluating biomolecular interactions independent of enzyme-mediated activation.

Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A

“…After treatment with 2.5 μM SF for 48 h, corresponding to an IC 10 in HT29 cells ( S2 Fig ); we identified 23 differentially abundant proteins ( Fig 2 ; Table 1 ). A preponderance of proteins involved in cellular metabolic and redox processes were found to be enriched, and enzymes characterized previously to mediate the activation of reductive prodrugs, such as AKR1C3, PTGR1, and NQO1 were amongst this group [ 22 , 29 , 42 – 44 ]. The largest fold change was evident for Cyclin-D1-binding protein 1, a negative regulator of transcriptional activation involving the E2F transcription factors [ 45 ], whereas several metabolic and redox-regulating proteins regulated by the transcription factor Nrf2, including AKRs and an aldehyde dehydrogenase, increased by 4.6-fold or higher.…”

Improved Efficacy of Acylfulvene in Colon Cancer Cells When Combined with a Nuclear Excision Repair Inhibitor

“…AF 26,27 , N 3 -AF-adenine (3-AF-Ade) and deuterated- N 3 -AF-adenine (3-AF-Ade-d 3 ) 2 were prepared as previously described. Pure deionized water was obtained from a Milli-Q Integral water purification system (Millipore Corporation, Billerica, MA) .…”

Bioreduction-Mediated Food-Drug Interactions: Opportunities for Oncology Nutrition

“…[41] In recent studies, a stable yet chemically reduced analogue of AF has been synthesized. [44] It was shown that the reaction of this analogue with DNA gives rise to alkylation profiles in DNA that are the same as generated during the AOR-catalyzed reaction, and that the cytotoxicity of the stable reduced form does not depend on AOR levels as it does for AF. [44] Therefore, this ana-could be facilitated by reducing enzymes in cancer cells.…”

“…[44] It was shown that the reaction of this analogue with DNA gives rise to alkylation profiles in DNA that are the same as generated during the AOR-catalyzed reaction, and that the cytotoxicity of the stable reduced form does not depend on AOR levels as it does for AF. [44] Therefore, this ana-could be facilitated by reducing enzymes in cancer cells. It has been suggested that the major cause for activated MMC's antitumor effect are DNA crosslinks that inhibit DNA synthesis.…”

“…[41] Additionally, AFs interact with and inhibit glutathione reductase, consistent with the potential for direct interactions with cytosolic redox-regulating enzyme interactions to be involved in the cellular response to the drugs. [42][43][44] In the presence of AOR, however, the efficiency of DNA alkylation is increased over a hundred-fold and the reaction with DNA instead involves the cyclopropyl group. [41,45] The kinetic parameters for AOR-mediated activation of AF analogues and their cytotoxicities are positively correlated; the highest toxicities were observed in cells engineered to overexpress AOR and for analogues that were efficiently activated by the enzyme.…”