Inactivation of Ribonucleotide Reductase by (E)-2‘-Fluoromethylene-2‘-deoxycytidine 5‘-Diphosphate:  A Paradigm for Nucleotide Mechanism-Based Inhibitors

Abstract

Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [3H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations.

An Increase in the Expression of Ribonucleotide Reductase Large Subunit 1 Is Associated with Gemcitabine Resistance in Non-Small Cell Lung Cancer Cell Lines

“…By contrast, previous studies with bacterial ribonucleotide reductase have shown that 2Ј-substituted diphosphate nucleosides may directly interfere with the RRM1 subunit. In fact, a 2Ј-fluoro-methylenyl derivative appears to form a covalent interaction with RRM1 from Escherichia coli that permanently inactivates that subunit (21). The physical relationship between gemcitabine and mammalian ribonucleotide reductase has not been well characterized, however, data support the hypothesis that the RRM1 subunit is the most likely intracellular target of gemcitabine diphosphate (22).…”

Synthesis and Anticancer Evaluation of Benzyloxyurea Derivatives

“…Crucial for rapidly dividing cells, RR is an attractive therapeutic target of cancer, and over the past years, many chemotherapeutics inhibiting different subunits of RR have been developed and tested clinically for their anticancer activities and anti-HIV activities. [10][11][12][13] HU is the first RR inhibitor applied to the clinic, and is utilized for the therapeutic of neoplasms because of its influences on the DNA replication of cancer cells. 1,14) To overcome the drawbacks of HU, numerous medicinal chemistry efforts have been made to design and synthesize novel HU derivatives.…”

Action of (E)-2′-Deoxy-2′-(fluoromethylene)cytidine on DNA Metabolism: Incorporation, Excision, and Cellular Response

“…Early studies demonstrated that the diphosphate of this compound affects ribonucleotide reductase through a mechanism-based inhibition (van der Donk et al, 1996) similar to that of gemcitabine. However, unlike gemcitabine and 1-␤-D-arabinofuranosylcytosine, FMdC is relatively resistant to deamination by cytidine deaminase .…”

“…The inhibition of ribonucleotide reductase by FMdC diphosphate has been well characterized (van der Donk et al, 1996;Kanazawa et al, 1998). In vitro studies suggest that the triphosphate of FMdC may inhibit human DNA polymerase (pol) ␣ and cause a pause in further DNA synthesis (Yonetani and Mizukami, 1996).…”