Detection of a New Substrate-Derived Radical during Inactivation of Ribonucleotide Reductase from Escherichia coli by Gemcitabine 5‘-Diphosphate

Abstract

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.

Nucleoside analogs: molecular mechanisms signaling cell death

“…Unlike ara-C, gemcitabine has a second mechanism of action that contributes to cytotoxicity. The diphosphate of gemcitabine (dFdCDP) serves as an inhibitory alternative substrate for ribonucleotide reductase and inactivates this key enzyme in a mechanism-based manner, which leads to a decrease in deoxynucleotide pools (Baker et al, 1991;van der Donk et al, 1998;Wang et al, 2007). The change in the dFdCTP:CTP ratio likely leads to enhanced gemcitabine incorporation and further DNA synthesis inhibition, an action known as self-potentiation (Heinemann et al, 1990(Heinemann et al, , 1992.…”

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…The monophosphate of F 2 C (F 2 CMP) is generated by deoxycytidine kinase (7) and is rapidly phosphorylated to the di-and triphosphates (F 2 CDP and F 2 CTP) (8,9). Diphosphorylated F 2 C (F 2 CDP) is an irreversible inhibitor of ribonucleotide reductase (RNR) (10)(11)(12)(13), and F 2 CTP functions as a chain terminator in the DNA polymerase reaction (14,15). Differentially phosphorylated states of gemzar can also interfere with other enzymes involved in nucleotide metabolism.…”

“…A number of years ago, we reported on the inactivation of E. coli RNR by F 2 CDP (11,12). We showed that 1 eq of F 2 CDP per E. coli RNR, presumably ␣ 2 ␤ 2 , in the presence of reductants, thioredoxin (TR) and TR reductase or DTT, is sufficient for enzyme inactivation and that the inactivation is accompanied by release of 2 fluoride ions and one cytosine (12).…”

Biomimetic Modeling of the Nitrogen-centered Radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides.

“…However, no detection of the furanone at 320 nm was observed like most 2'-substituted nucleotides during the inactivation. 39,51 Instead, formation of a new stable substrate derived radical was detected as a triplet EPR signal at 9 GHz during the inhibition of RNR by Gemcitabine. 51 Moreover, unlike other 2'-substituted nucleotides that require the presence of reductants (namely cysteine residues C754 and C759 and NADPH) for the inhibition, the presence or absence of reductants did not affect the inhibitory action of Gemcitabine.…”

“…39,51 Instead, formation of a new stable substrate derived radical was detected as a triplet EPR signal at 9 GHz during the inhibition of RNR by Gemcitabine. 51 Moreover, unlike other 2'-substituted nucleotides that require the presence of reductants (namely cysteine residues C754 and C759 and NADPH) for the inhibition, the presence or absence of reductants did not affect the inhibitory action of Gemcitabine. Inhibition in the presence of the reducing system happened through covalent labeling, which increased interaction between the two subunits.…”