Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay

Jimin Shao, Bingsen Zhou, Lijun Zhu, Angel J Di Bilio, Leila Su, Yate Ching Yuan, Shijun Ren, Eric J. Lien, Jennifer Shih, Yun Yen

Research output: Contribution to journalArticle

31 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)627-634
Number of pages8
JournalBiochemical Pharmacology
Volume69
Issue number4
DOIs
Publication statusPublished - Feb 15 2005
Externally publishedYes

Fingerprint

Ribonucleotide Reductases
Holoenzymes
Schiff Bases
Assays
Hydroxyurea
Cytidine Diphosphate
In Vitro Techniques
Reducing Agents
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Molecules

Keywords

  • In vitro assay
  • Potency and subunit-selectivity
  • Recombinant subunit proteins
  • Ribonucleotide reductase inhibitors
  • Two forms of holoenzyme

ASJC Scopus subject areas

  • Pharmacology

Cite this

Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay. / Shao, Jimin; Zhou, Bingsen; Zhu, Lijun; Bilio, Angel J Di; Su, Leila; Yuan, Yate Ching; Ren, Shijun; Lien, Eric J.; Shih, Jennifer; Yen, Yun.

In: Biochemical Pharmacology, Vol. 69, No. 4, 15.02.2005, p. 627-634.

Research output: Contribution to journalArticle

Shao, Jimin ; Zhou, Bingsen ; Zhu, Lijun ; Bilio, Angel J Di ; Su, Leila ; Yuan, Yate Ching ; Ren, Shijun ; Lien, Eric J. ; Shih, Jennifer ; Yen, Yun. / Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay. In: Biochemical Pharmacology. 2005 ; Vol. 69, No. 4. pp. 627-634.
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T1 - Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay

AU - Shao, Jimin

AU - Zhou, Bingsen

AU - Zhu, Lijun

AU - Bilio, Angel J Di

AU - Su, Leila

AU - Yuan, Yate Ching

AU - Ren, Shijun

AU - Lien, Eric J.

AU - Shih, Jennifer

AU - Yen, Yun

PY - 2005/2/15

Y1 - 2005/2/15

N2 - 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.

AB - 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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KW - Potency and subunit-selectivity

KW - Recombinant subunit proteins

KW - Ribonucleotide reductase inhibitors

KW - Two forms of holoenzyme

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