Finding drugs that stall a critical DNA-synthesis enzyme
June 4, 2012 § Leave a comment
It’s no secret that we need new ways to discover drugs. To address this need in one way, a Swedish duo developed a new approach to screen for drugs against a critical enzyme called ribonucleotide reductase. They describe this new method in a paper appearing in the latest issue of the Proceedings of the National Academy of Sciences, demonstrating its ability to pick out 27 specific inhibitors of the enzyme out of a library of 1,364 small molecules.
Ribonucleotide reductase, called RNR for short, converts the four ribonucleotides into their corresponding deoxyribonucleotides, which are then used to make DNA. RNR is the rate-limiting enzyme for synthesizing DNA. Two anticancer drugs, hydroxyurea and Gemzar, target the enzyme. But, in general, RNR inhibitors are restricted to few chemical types — nucleoside analogs, radical scavengers and metal chelators — explains one of the paper’s authors, Britt-Marie Sjöberg at Stockholm University. The inhibitors have low specificities and are not selective for a particular RNR.
The problem is that it’s hard to quickly search for new RNR inhibitors. The conventional methods for screening for RNR inhibitors are particularly labor-intensive because it’s challenging to tell apart ribonucleotides from deoxyribonucleotides. This slows down the analysis of the enzyme’s activity. So, to take a different tack, Sjöberg and Fredrik Tholander at Stockholm University and the Karolinska Institute used a variation of PCR to measure the activity of ribonucleotide reductase in the presence of inhibitors in a microwell format.
The method works by the researchers adding an excess of three deoxyribonucleotides to the PCR mixture. The fourth one is provided by the RNR reaction. If the enzyme is inhibited by a small molecule, the production of DNA is stalled. The amount of DNA produced by PCR can be monitored by fluoresence. If fluorescence drops in a microwell, it suggests the well contains a molecule that is inhibiting RNR.
As Sjöberg explains, their approach exploits the power of PCR to select for the products of the enzyme, deoxyribonucleotides, instead of the enzyme’s substrates, ribonucleotides. “In retrospect, it is surprising that this powerful assay for measuring the activity of the RNR enzyme hadn’t been developed earlier,” says Sjoberg.
Tholander and Sjöberg demonstrated the utility of the method by screening a library of compounds from the National Cancer Institute for inhibitors of the RNR from the pathogen Pseudomonas aeruginosa. Out of the 1,364 compounds, the investigators found 27 potential inhibitors. Most of the inhibitors don’t work on human cells, including cancerous ones, suggesting they inhibited only bacterial RNR.
Interestingly, four of the 27 compounds not just inhibited the enzyme but also actually stopped P. aeruginosa from growing. Three of them worked just as well as conventional antibacterial agents.
Tholander and Sjöberg pointed out that the assay can be used to find any inhibitors of RNR, no matter what the species or the substrate preferences. They envision using robotics to make the process automated so it can be used for high-throughout screening in early drug discovery.
Sean Ryder at the University of Massachusetts Medical School, an expert in small-molecule screens not involved in the work, explains, “The simple yet robust assay technology should enable identification of new RNR inhibitors, possibly including those that show species specificity and could be useful as antibiotics.”