Clofarabine Targets the Large Subunit (α) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers

Clofarabine (CIF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (alpha(2))(m)(beta(2))(n), regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, CIF targets hRNR by converting its alpha-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional alpha and characterization of its altered oligomeric associations in response to CIF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-alpha(6). Our data validate hRNR as an important target of CIF, provide evidence that in vivo alpha's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.