Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay.
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.
Shao
Jimin
J
Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, CA 91010, USA.
Zhou
Bingsen
B
Zhu
Lijun
L
Bilio
Angel J Di
AJ
Su
Leila
L
Yuan
Yate-Ching
YC
Ren
Shijun
S
Lien
Eric J
EJ
Shih
Jennifer
J
Yen
Yun
Y
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
D004791:Enzyme Inhibitors; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D012545:Schiff Bases; D014443:Tyrosine; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea; D004121:Dimethyl Sulfoxide