Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay.
Biochemical Pharmacology(2005)
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 [3H]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.
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Key words
RR,HU,SC,SB-HSCs,KB-WT,KB-HUR,EPR,QSAR,DTT
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