Mutations in the R2 Subunit of Ribonucleotide Reductase That Confer Resistance to Hydroxyurea

Abstract

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

“…2C, K, L, and M), indicating that the nucleoids contained one partially replicated chromosome. HU is thought to have only one target, the aerobic ribonucleotide reductase, such that deoxynucleoside triphosphate (dNTP) pools are depleted and replication forks stall (74,82). Long-term HU treatment leads to hydroxyl radical formation and eventual cell death 2 to 3 h later (83); however, this was not relevant to the division inhibition reported here, which occurred within 65 to 85 min after HU addition.…”

Phase I Study of GTI-2040, an Antisense to Ribonucleotide Reductase, in Combination with High-Dose Cytarabine in Patients with Acute Myeloid Leukemia

“…Although laboratory data support the possible role of hydroxyurea in inhibiting RNR by directly reducing the tyrosyl radical of R2 to a normal tyrosine residue via one-electron transfer from the drug (32,33), the effectiveness of this approach is likely to be limited by the hydroxyurea low affinity for RNR, very high hydrophilicity, short half-life, and early development of mechanisms of resistance (34). The latter is mediated by R2 overexpression via gene amplification and/or alteration in gene transcriptional regulation (35,36), R2 mutations (37), and increased expression of ferritin-encoding genes that leads to increase in iron levels and reactivation of previously druginhibited R2 protein (38).…”

Ribonucleotide Reductase Inhibitors and Future Drug Design

“…The increase of iron could favor the reactivation of R2 after the destruction of the protein by the drug. Mutations in the R2 subunit may also confer resistance to hydroxyurea [256]. Efforts have been made to develop hydroxyurea analogs and other small inhibitors to improve the inhibitory efficiency of hydroxyurea [257][258][259][260].…”