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Teasing out toxicity Drug toxicity is a major hurdle in the development of novel pharmaceuticals. Drug companies spend huge sums of money to develop a single drug candidate, despite the 30% chance that each would-be pharmaceutical will fail clinical tests because of toxicity. These financial and medical incentives mean that new cost-effective and high-throughput drug toxicity screens are sorely needed. Now, in a paper published in AC (DOI 10.1021/ ac800763r), James Rusling and colleagues at the University of Connecticut describe a two-tiered toxicity screen. “Almost all toxicity testing nowadays is basically biological,” says Rusling. “Not many people use the techniques of analytical chemistry... to find out if things are toxic.” The first component of the toxicity screen is an electrochemiluminescent (ECL) array film built on a pyrolytic graphite block electrode. Each spot on the array contains calf thymus DNA, rat liver microsome—which contains a cocktail of enzymes, including cytochrome P450 and NADPH P450 reductase—and a special light-emitting polymer called ruthenium polyvinylpyridine (ruPVP). The addition of NADPH and substrate to an array spot sets in motion a complex reaction, which is believed to proceed as follows: the reductase catalyzes the donation of electrons from NADPH to cytochrome P450, which, in the presence of oxygen and a toxic substrate, catalyzes the formation of reactive metabolites. These metabolites “damage” the DNA by forming adducts with guanine and other bases. When activated by a power source, the electrode block oxidizes the ruPVP polymer, which in turn oxidizes the guanines in the DNA. The redox reaction excites the polymer, causing it to emit visible light, which the researchers can detect. Although this reaction would occur even in the absence of a toxic agent, DNA damage facilitates oxidation 5674
ANALYTICAL CHEMISTRY /
reaction,” says Rusling. The basic components of the first-tier array are on the particles, but the difference in the second tier is that toxicity is detected by MS instead of by chemiluminescence, he explains. The nanoreactor consists of DNA and the rat liver microsomes affixed to a bead. To determine the rate of DNA adduct production, the reaction was truncated at different time points, and the quantity and structure of the products were determined by LC/MS. When the researchers monitored the rate of tumor formation in rat livers after exposure to N-nitroso compounds, they found that the array and nanoreactor data correlated with the literature toxicity measurements. In addition, the two-tiered screen enabled the researchers to distinguish between highly toxic and moderately toxic compounds. The next phase is to incorporate sequences of human DNA into the screen. By using sequences that are known to be susceptible to damage by toxic compounds, the researchThe toxicity screen’s two-tiered approach. The first tier ers hope their screen will be more relevant to human subjects and utilizes an ECL array, wherein each spot consists of DNA, enzymes, and a light-emitting polymer. The sec- drug development efforts. —Erika Gebel ond tier uses a nanoreactor, which contains DNA and by the polymer, resulting in faster polymer excitation and light emission. To test the array, the researchers used N-nitroso compounds, which are known to cause cancer in vivo. As expected, spots exposed to N-nitroso substrates emitted light more rapidly than the controls did.
enzymes, and LC/MS. The addition of a toxic substrate and NADPH results in DNA damage, which is monitored by ECL or LC/MS.
To decipher the chemical structures of the DNA products from array hits, the researchers developed the screen’s second tier. Knowing the identity and the rate of formation of the damaged DNA could help researchers “design out” toxicity during drug development, according to Rusling. This second step uses nanoreactors and LC/MS. “‘Nanoreactor’ is a buzzword for a little particle that has all the stuff around it that you need for a
AUGUST 1, 2008
10.1021/AC801288K 2008 AMERICAN CHEMICAL SOCIETY
Published on Web 07/31/2008
