The work, published in the May 14 issue of Cell, defines another metabolic route to a compound called NAD and suggests that therapeutic approaches for cancer or heart disease may depend on the enzymes discovered.
NAD (nicotinamide adenine dinucleotide) is one of the most well-known small molecules in the cell, said Dr. Charles Brenner, associate professor of genetics and of biochemistry, author of the study with Dr. Pawel Bieganowski, a postdoctoral fellow. It is essential for life in all organisms, from bacteria to humans, and very versatile, working both as a partner that helps enzymes and as an ingredient that other enzymes consume.
NAD is a co-enzyme for hundreds of cellular enzymes. Niacin, or vitamin B3, a mixture of the NAD precursors nicotinic acid and nicotinamide, which were discovered in 1938, prevents pellagra and can help control cholesterol. A class of anti-cancer drugs including tiazofurin and benzamide riboside is converted to toxic NAD analogs. And, more recently, proteins dependent on NAD have been shown to prolong life in experimental systems.
Brenner's laboratory at the Norris Cotton Cancer Center at Dartmouth-Hitchcock Medical Center was studying an enzyme involved in NAD synthesis that was similar to an enzyme implicated in cancer development. Their explorations revealed a novel twist. In yeast without the enzyme, every known NAD biosynthetic pathway was shut down, so in theory, the cells should die; no vitamins or supplements were known to keep the cells alive. However, the researchers discovered that another NAD precursor, nicotinamide riboside, thought to be a vitamin form of NAD only in certain bacteria, served as a vitamin in yeast and could prevent death. The researchers discovered the genes and enzymes in yeast and humans responsible for this vitamin conversion pathway and then they found the vitamin in milk.
Their findings upend some assumptions underlying biosynthetic schemes for NAD that have been in textbooks for decades and refocus the cancer pharmacology of tiazofurin and benzamide riboside. "Cancer drugs that look like nicotinamide riboside are converted to toxic NAD analogs through a pathway that is likely to be the same as our vitamin activation pathway," Brenner said.
The researchers considered nicotinamide riboside as a nutrient through its role in a bizarre bacterium, Haemophilus influenza, that lives in blood. When they determined that the compound also supported the growth of yeast cells, they began a search for the yeast equivalent of the bacterial kinase enzyme that is required to begin turning it into NAD.
They used what Brenner termed a "biochemical genomics approach," taking advantage of shortcuts developed since the entire the yeast genome was sequenced. The team analyzed large yeast gene pools for the enzyme activity and quickly zeroed in on the gene for their novel kinase. Yeast has similarities to mammalian cells, and a gene or enzyme in yeast is likely to have an equivalent in humans.
"We cloned the yeast nicotinamide riboside kinase, and validated it genetically by knocking it out and seeing that the vitamin no longer supported growth," said Brenner. "We then identified two human nicotinamide riboside kinases and showed that all three have the biochemical specificity for nutrient and prodrug [precursor] activation and that all three work in vivo."
To reinforce the vitamin premise, the scientists sought the compound in a food. Testing for it nonfat milk, they separated out the curds from the whey and found it in the whey.
Brenner thinks that nicotinamide riboside may be a useful nutrient for certain metabolic disorders and that kinase screening may benefit certain cancer patients. Niacin, for example, can help lower cholesterol, but it has uncomfortable flushing effects in patients, so nicotinamide riboside supplementation may offer an alternative.
And, while tiazofurin-related drugs have potential against cancer, they are unpredictable. "Certain tumors respond, while others don't, so if we can select the right patients we will have a more effective treatment strategy," he said. "In the future, testing for nicotinamide riboside kinase expression might be used to identify the patients that are likely to respond to this class of drugs."