Since the origins of DNA-based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate-limiting role in de novo deoxynucleoside-triphosphate (dNTP) biosynthesis. Paradoxically, the large subunit, RNR-α, of this obligatory two-component complex in mammals plays a context-specific antiproliferative role. There is little explanation for this dichotomy. Here, we show that RNR-α has a previously unrecognized DNA-replication inhibition function, leading to growth retardation. This underappreciated biological activity functions in the nucleus, where RNR-α interacts with ZRANB3. This process suppresses ZRANB3's function in unstressed cells, which we show to promote DNA synthesis. This nonreductase function of RNR-α is promoted by RNR-α hexamerization-induced by a natural and synthetic nucleotide of dA/ClF/CLA/FLU-which elicits rapid RNR-α nuclear import. The newly discovered nuclear signaling axis is a primary defense against elevated or imbalanced dNTP pools that can exert mutagenic effects irrespective of the cell cycle.
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.
Antibody–drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure–activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.
Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We here developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α covalently labeled with two fluorophores allows direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly driven by the feedback regulator dATP. This information is currently unavailable despite the pharmaceutical relevance of hRNR oligomeric regulation.
Human ribonucleotide reductase (hRNR) is one of the primary targets of nucleotide cancer drugs in clinical use. The nucleotide‐induced oligomeric regulation of hRNR subunit a is increasingly recognized as an innate as well as a drug‐relevant mechanism to enzyme activity modulations. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides [ClFD(T)P], hRNR‐a assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger a dimerization. To date, both knowledge about and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. To fill this void, we developed a fluorimetric assay that can reliably report on the oligomeric state changes of a with high sensitivity. The oligomerization‐directed fluorescence quenching of hRNR‐a covalently labeled with two different fluorophores enables the direct readout of hRNR dimeric and hexameric states. Proof of concept was demonstrated with all three known hexamerization inducers: dATP and ClFD(T)P.We then applied the newly developed platform to reveal the timescales of a self‐assembly pathway driven by the feedback regulator dATP. Dimerization of hRNR‐awas found to be the rate‐limiting step along the oligomerization pathway. (Dimerization rate constant: 6×104 M‐1s‐1) Kinetics of the subsequent steps were estimated to be an order of magnitude faster. The study sheds new light on kinetic understanding of hRNR‐a oligomeric regulation in response to feedback inhibitors.
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