Discovery of antimicrobial ribonucleotide reductase inhibitors by screening in microwell format

Abstract

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 wellestablished 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 PCRbased 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 IC 50 values from ∼200 nM to 30 μM. 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.National Cancer Institute diversity set II | nucleotide metabolism | ribonucleotide reductase assay | high-throughput assay R ibonucleotide reductase (RNR) is an essential enzyme for de novo synthesis of DNA building blocks via reduction of the 2′-hydroxyl of ribonucleotides (1, 2). With its key role in the DNA synthesis pathway, RNR is an absolute requirement for cellular proliferation and a prerequisite for life. RNR is found in all freeliving organisms as well as in some dsDNA viruses. The only known exceptions to this ubiquitous presence are a few parasites and obligate intracellular endosymbionts (3) that rely on the host cell for production of DNA precursors. Thus, RNR is a potential antimicrobial drug target in a wide variety of organisms. RNR enzymes exist in three different classes, each with different cofactor requirements. Eukaryotes generally possess only one class, whereas bacteria possess any combination of RNR classes (3). Different oligomeric states of RNRs are known, and a common active form of the major variants of RNR is a dimer of dimers (α 2 β 2 ) (4). RNR exhibits a radical-based catalytical mechanism that involves redox cycling of cysteine residues, specific metal ion dependencies, essential subunit interactions, and sophisticated allosteric regulation (1). Thus, in addition to conventional competitive inhibition, pharmaceuticals may interfere with subunit interactions, binding to allosteric effector sites, metal chelation, radical formation and transfer, or inhibition of cysteine disulfide exchange. These sites of potential intervention offer a plethora of possibilities for the design of pathogen-specific antibiotics. Because sequence...

Diversity in Overall Activity Regulation of Ribonucleotide Reductase

“…The P. aeruginosa class I RNR is interesting from a drug development viewpoint, as demonstrated in a previous study (35). Some clinically used drugs that target the allosteric and catalytic sites of RNR, such as clofarabine and gemcitabine, have direct effects on oligomerization as well as the affinity between ␣ and ␤ subunits in different RNRs (12,36,37).…”

A ribonucleotide reductase inhibitor with deoxyribonucleoside‐reversible cytotoxicity

“…Glutamine and RPMI medium were from Lonza, RNase A from Macherey‐Nagel, and fetal calf serum (FCS) from Life Technologies. NSC73735 (redoxal, 2‐[(4‐{4‐[(2‐carboxyphenyl)amino]‐3‐methoxyphenyl}‐2‐methoxyphenyl)amino]benzoic acid) were from the Drug synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, USA, and previously found to inhibit a bacterial Ribonucleotide Reductase by a PCR‐based HTS method (Tholander and Sjöberg, 2012). Custom gene synthesis and subcloning into expression vectors were performed by Epoch Lifescience Inc., USA.…”

“…Tholander & Sjöberg previously identified a set of compounds active as inhibitors of the Pseudomonas aeruginosa class I RNR (Tholander and Sjöberg, 2012), of which only a few also possessed activity against P . aeruginosa cells.…”

PubChem applications in drug discovery: a bibliometric analysis

“…Bioactivity profile evaluation of studied compounds for their selectivity, diversity, novelty and cytotoxicity using the PubChem BioAssay data across assays, targets, cell lines, cellular functions, signaling pathways and so on [42–46]. For instance, Vang et al searched the PubChem bioassay datasets among various drug targets and eliminated nonselective inhibitors of lymphoid tyrosine phosphatase [46].…”