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

Abstract

Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically-stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α6. Our data validate hRNR as an important target of ClF, provide previously-unreported evidence that in vivo α’s quaternary structure can be perturbed by a non-natural ligand, and suggest small-molecule-promoted, persistent hexamerization as a new strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.