The Reactions of Mitomycin C with Dithiols I. Reductive Activation

We report that the clinically used antitumor drug mitomycin C is reductively activated in vitro by simple thiols; a mechanism for this activation is proposed based on kinetic data and the identification of mitosene metabolites. The biological implications of these findings are discussed. Introduction Mitomycin C (MC, 1) 1 is a natural antitumour antibiotic used in anticancer therapy 2 that requires reductive activation to exert its biological activity. 3 After bioreduction, MC generates a bifunctional electrophile (4, scheme 1) that alkylates cellular nucleophiles, in particular the complementary strands of DNA. 4 A number of enzymes are known to activate MC in mammalian cells. 5 Recent reports indicate that proteins containing a dithiol active site are implicated in the cellular activation of MC. 6 Incongruently, MC was reported to be inert to reductive activation by monothiols (glutathione) and dithiols (DTT), 7,8 We decided to re-examine the reaction of MC with thiols, in particular after recent reports linked the thioredoxin-like domains of GRP58 to the activation of MC in cancer cells, an explicit sign of the involvement of the dithiol group in the cellular activation of MC. The results we present here reconcile this apparent paradox. Figure 1. Structure of mitomycin C and mitomycin A. Results and discussion The UV analysis of a reaction containing MC and a large excess of DTT (0.1 M) at neutral pH clearly showed the gradual formation of mitosene compounds (6, Scheme 1) at the expense of MC (figure 2), the reaction reaching completion in about 120 minutes, as judged by a constant UV spectra. When MC was treated with mercaptoethanol in similar conditions only a marginal conversion of MC to mitosenes was detected during 3 hours. In contrast, MA reacted completely in 2 minutes by treatment using a 500-fold lower DTT concentration. 9a A kinetic analysis of the reaction was then performed. The rate of disappearance of MC by treatment with DTT was measured by the decline of the UV absorbance at 360 nm (figure 3). The decay of MC displayed the shape of a sigmoidal curve, characteristic of an autocatalytic reaction. Dissimilarly, the kinetic study of the reaction of MA with thiols showed a pseudo-first order decay. 9a Figure 2. UV assay of the reduction of mitomycin C by DTT. Reaction times are indicated on top of each spectrum. The autocatalytic activation of MC has been observed before, both during enzymatic 9 and chemical 10 reductions. This mechanism is based on the large redox potential difference between the mitosane (1) and mitosene (4) structures (scheme 1). Thus, the leucomitosene species, e.g. 5 and 6, transfer their electron(s) to unreduced MC, creating an electron transfer chain reaction, provided the reducing agent is slow to reduce excess MC. 7,10,11 Figure 3. (a) Dependence on the concentration of DTT. All reactions contain 30 μM. MC at pH 9.0. Concentrations of DTT were as indicated in the chart. (b) Relationship between t1/2-1 and DTT concentration at pH 9.0.; [MC] = 5 The identity of the mitosenes formed in the reaction of MC with DTT was studied by LCMS. MC was activated with substoichometric DTT at pH 9.5 until all MC is consumed, then quenched with diluted aqueous buffer at neutral pH. LCMS analysis showed the formation of the expected diastereomeric pair of hydroxymitosenes 6. 11 Scheme 1. Reductive activation of MC. NuH represents a nucleophile. 0 0.2 0.4 0.6 0.8 275 300 325 350 375 400