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It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5-triphosphates to 2-deoxynucleoside 5-triphosphates and uses coenzyme B 12 , adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2-deoxy-2-methylenecytidine 5-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.There are three classes of adenosylcobalamin (AdoCbl) 1 (Fig. 1, 1) requiring enzymes (1): those that catalyze reversible carbon skeleton rearrangements (class I), those that catalyze irreversible heteroatom elimination reactions (class II), and those that catalyze rearrangements involving migration of an amino group (class III). All three classes initiate their radicaldependent reactions by catalyzing carbon-cobalt bond homolysis. The mechanism(s) by which these enzymes catalyze C-Co homolysis of 1, the identification of the axial ligand trans to the C-Co bond that is cleaved, and its role in this process have recently been a topic of interest in many laboratories (2). The x-ray structures of methylmalonyl-CoA (MMCoA) mutase (3,4) and EPR studies of cob(II)alamin (2) bound to other class I enzymes (1, 5, 6) revealed that in each case the dimethylbenzimidazole (DMB) moiety of the cofactor is replaced by a protein-derived histidine. On the other hand, in diol dehydrase (DD), typical of the class II enzymes, DMB remains ligated to 2 (7,8). Unique among the AdoCbl-requiring enzymes is ribonucleotide reductase, the only enzyme that does not catalyze a rearrangement reaction and in which C-Co homolysis occurs concomitant with the formation of a protein-based thiyl radical (9 -11). The mechanism of homolysis and the identity of the axial ligand in this enzyme have not previously been addressed and are the subjects of this paper.Our laboratory has long been interested in the ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. In the presence of a nucleoside 5Ј-triphosphate (NTP) substrate, a reducing system, and a 2Ј-deoxynucleoside 5Ј-triphosphate (dNTP) allosteric effector, RTPR catalyzes homolysis of the C-Co bond of 1 to generate 2, 5Ј-deoxyadenosine, and a thiyl radical at Cys-408 of RTPR in a kinetically competent and concerted fashion (9,10,(12)(13)(14). This thiyl radical is then proposed to initiate the nucleotide reduction chemistry by abstraction of the 3Ј-hydrogen atom of the NTP (15). In this paper we report studies with a mechanism-based inhibitor of this protein, 2Ј-deoxy-2Ј-methylenecytidine 5Ј-triphosphate (MdCTP, 3). These studies in conjunction with [U- EXPERIMENTAL PROCEDURESMaterials and Methods-Alkaline phosphatas...

These experiments show a much shorter neuromuscular blocking effect and much-reduced side effects in the case of GW280430A vis-à-vis mivacurium. These results, together with the novel chemical degradation of GW280430A, suggest further evaluation in human subjects.