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Ribonucleotide reductase (RR) is an essential multi-subunit enzyme found in all living organisms; it catalyzes the rate-limiting step in dNTP synthesis, namely, the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. As expression levels of human RR (hRR) are high during cell replication, hRR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. While there are many excellent reviews regarding the structure, function, and clinical importance of hRR, recent years have seen an increase in novel approaches to inhibiting hRR that merit an updated discussion of the existing inhibitors and strategies to target this enzyme. In this review, we discuss the mechanisms and clinical applications of classic nucleoside analog inhibitors of hRRM1 (large catalytic subunit), including gemcitabine and clofarabine, as well as inhibitors of the hRRM2 (free radical housing small subunit), including triapine and hydroxyurea. Additionally, we discuss novel approaches to targeting RR and the discovery of new classes of hRR inhibitors.

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-A resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.