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

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.

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

“…Although the role of hRR inhibition is considered to be secondary to DNA chain termination as the primary mechanism of cytotoxicity by nucleoside drugs where the triphosphate form of the drug is incorporated (22), work reported by the Stubbe laboratory highlighted the importance of hRR inhibition by nucleotide analogs (10,11). The three analogs that are best characterized biochemically are gemcitabine (10), clofarabine (11,13), and cladribine (15). Gemcitabine diphosphate is a substoichiometric mechanism-based inhibitor that targets hRRM1's catalytic site, resulting in the formation of a tight α 6 β 6 holocomplex (10,36).…”

“…The nucleotide analogs of chemotherapeutics such as fludarabine, clofarabine, and cladribine target all three sites of hRRM1 for inhibition (11,14,15) (Fig. 1A).…”

“…In contrast, hydroxyurea (HU) blocks catalysis by targeting the diiron cluster of RR2 (16,17). Gemcitabine diphosphate, clofarabine, and cladribine nucleotides inhibit hRRM1 by stabilizing a form of the α 6 complex (10,11,13,15). Such nucleotide-analog inhibitors of hRR, especially gemcitabine, are effective anticancer agents and can sensitize cancer cells to ionizing radiation and to DNA-damaging drugs (18).…”

Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase

“…17 or using inducible shRNA by Wisitpitthaya et al . 4d to repress RR. The siRNA approach measuring the level of sensitization that a drug renders to RR under knocked-down conditions has been previously utilized to verify the cellular inhibition of RRM1 by gemcitabine and RRM2 by hydroxyurea 17 and to delineate the role of RR oligomerization in the activity of clinically relevant inhibitors 4d .…”

“…3 Recent studies have provided structural insights underlying allosterically driven dNTP regulation by RR. 1d–e, 4, 5c Though previous studies establish the molecular details of multimerization as a basis for development of novel anticancer agents, 5 there has been little focus on identifying non-nucleosidic, reversible and competitive inhibitors of RR.…”