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ACS Chem Biol. 2016 Jul 15;11(7):2021-32. doi: 10.1021/acschembio.6b00303. Epub 2016 May 20.

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

Author information

1
Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
2
Department of Biomedical Sciences, Cornell University , Ithaca, New York 14853, United States.
3
Department of Biochemistry, Weill Cornell Medicine , New York, New York 10065, United States.

Abstract

The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

PMID:
27159113
PMCID:
PMC4946967
DOI:
10.1021/acschembio.6b00303
[Indexed for MEDLINE]
Free PMC Article

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