title: Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay.
abstract: Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [(3)H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The -NH-OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21 > SB-HSC 24 > SB-HSC 2 > hydroxyurea (HU) > SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR.
authors.affiliation: Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: Jimin
authors.initials: J
authors.lastname: Shao
authors.forename: Bingsen
authors.initials: B
authors.lastname: Zhou
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhu
authors.forename: Angel J Di
authors.initials: AJ
authors.lastname: Bilio
authors.forename: Leila
authors.initials: L
authors.lastname: Su
authors.forename: Yate-Ching
authors.initials: YC
authors.lastname: Yuan
authors.forename: Shijun
authors.initials: S
authors.lastname: Ren
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Lien
authors.forename: Jennifer
authors.initials: J
authors.lastname: Shih
authors.forename: Yun
authors.initials: Y
authors.lastname: Yen
mesh_terms: D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020035:Holoenzymes / Q000187:drug effects*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007624:KB Cells; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000187:drug effects; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D012545:Schiff Bases; D014443:Tyrosine / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D012545:Schiff Bases; D014443:Tyrosine; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea; D004121:Dimethyl Sulfoxide