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Discovery of antimicrobial ribonucleotide reductase inhibitors by screening in microwell format
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 25, 9798-9803 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) catalyzes reduction of the four different ribonucleotides to their corresponding deoxyribonucleotides and is the rate-limiting enzyme in DNA synthesis. RNR is a well-established target for the antiproliferative drugs Gemzar and Hydrea, for antisense therapy, and in combination chemotherapies. Surprisingly, few novel drugs that target RNR have emerged, partly because RNR activity assays are laboratory-intense and exclude high-throughput methodologies. Here, we present a previously undescribed PCR-based assay for RNR activity measurements in microplate format. We validated the approach by screening a diverse library of 1,364 compounds for inhibitors of class I RNR from the opportunistic pathogen Pseudomonas aeruginosa, and we identified 27 inhibitors with IC50 values from similar to 200 nM to 30 mu M. Interestingly, a majority of the identified inhibitors have been found inactive in human cell lines as well as in anticancer and in vivo tumor tests as reported by the PubChem BioAssay database. Four of the RNR inhibitors inhibited growth of P. aeruginosa, and two were also found to affect the transcription of RNR genes and to decrease the cellular deoxyribonucleotide pools. This unique PCR-based assay works with any RNR enzyme and any substrate nucleotide, and thus opens the door to high-throughput screening for RNR inhibitors in drug discovery.

Place, publisher, year, edition, pages
2012. Vol. 109, no 25, 9798-9803 p.
Keyword [en]
National Cancer Institute diversity set II, nucleotide metabolism, ribonucleotide reductase assay, high-throughput assay
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-80013DOI: 10.1073/pnas.1113051109ISI: 000306061400035OAI: oai:DiVA.org:su-80013DiVA: diva2:552458
Note

AuthorCount:2;

Available from: 2012-09-14 Created: 2012-09-12 Last updated: 2017-12-07Bibliographically approved

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Tholander, FredrikSjöberg, Britt-Marie
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