Ribonucleotide reductase is essential to maintain the cellular pools of dNTPs, and its activity is controlled allosterically by ATP (activator) and dATP (inhibitor). Now crystal and EM structures of human and yeast ribonucleotide reductase 1 in complex with different nucleotides, together with mutagenesis and functional analysis, reveal how dATP binding induces hexamerization and consequence inhibition of the enzyme. Ribonucleotide reductase (RR) is an αnβn (RR1–RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP–GDP, TTP–ATP, and TTP–dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1–dATP hexamer and used single-particle electron microscopy to visualize the α6–ββ′–dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

Amyloid aggregates of the amyloid-β (Aβ) peptide are implicated in the pathology of Alzheimer's disease. Anti-Aβ monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-Aβ mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize Aβ monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the Aβ(1–8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to Aβ(1–8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target Aβ with improved specificity and higher affinity.

Significance: There is immense difficulty in mapping out the complete details of an enzyme’s mechanism, especially those that catalyze an acid-base reaction, owing to the simple fact that hydrogen atom positions are rarely known with any confidence. Ultrahigh-resolution X-ray and, better still, neutron crystallography can provide this crucial layer of information. We paired these techniques to reveal the catalytic mechanism of dihydrofolate reductase (DHFR), an enzyme necessary for nucleotide biosynthesis and a classical drug target. In a complex that closely resembles the catalytically active state, DHFR stabilizes a particular substrate conformer and likely elevates the p K _a of the substrate atom that is protonated. This protonation occurs directly via water, with its access to the substrate regulated by structural fluctuation of the enzyme.

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.

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-Å 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.