'Proteome Mining' Can Zero in on Drug Targets

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DURHAM, N.C. - Development of drugs to treat a broad array of diseases - including cancer, hypertension, diabetes, inflammation and infectious diseases - could become enormously more effective through the process of "proteome mining," according to a Duke University Medical Center pharmacologist.

Just as the genome is the cell's entire set of genes, the proteome is the set of the cell's proteins. Many of these proteins are the molecular switches called enzymes that catalyze the cell's biological processes. As such, they are prime targets for drugs to manipulate cellular function -- from jamming enzymes that cancer cells need to proliferate, to killing pathological microbes by selectively attacking enzymes they need to live.

According to pharmacologist Tim Haystead, Ph.D., among researchers developing the approach, proteome mining involves isolating the hundreds of enzymes that control key cellular processes and performing mass screening for potential drugs to affect those switches. From this screening -- which is relatively quick and inexpensive -- new drugs to treat disease can be rapidly identified and progressed to animal testing, said Haystead, who is an associate professor of pharmacology and cancer biology.

In a talk delivered Aug. 23, 2004, at the national meeting of the American Chemical Society in Philadelphia, Haystead described the current process of proteome mining. He also explained how he and his colleagues are using the approach to identify new drugs to treat malaria without the serious side effects of current drugs. Malaria affects some 5 to 10 percent of the world's population, with some 300 million people currently infected by the malaria parasite. Malaria kills more than 1 million people -- mostly children -- every year.

"The approach we've been using enables us to mine en masse large combinatorial chemical libraries that contain drug-like molecules for novel target associations," said Haystead in an interview. "And these become starting points for iterative chemistry and improved selectivity, depending on the protein targets."

Haystead and his colleagues concentrate on cellular proteins that bind molecules called purines, because the cell machinery's principal control switches are purine-binding protein enzymes called kinases. Such kinases are activated by the purine adenosine triphosphate (ATP). Thus, reason the pharmacologists, sifting through the many hundreds of purine-binding proteins to find drugs that bind and jam such enzymes constitutes an excellent start to identifying drugs that affect cellular processes.

"The purine-binding proteome represents proteins coded by some 2,000 genes, including all protein kinases," said Haystead. "These proteins represent half or more of the 'druggable genome,' that is, the enzymes that can be targeted by drugs to affect cellular function."

In their proteome mining, Haystead and his colleagues first isolate the collection of purine-binding proteins from specific cells -- including the brain, testes, lung and liver -- that hold a diverse array of such proteins.

To capture these proteins, they create "affinity arrays" -- passing the complex mix of cellular proteins through a column containing plastic beads to which are attached immobilized ATP or similar purines. This column thus captures hundreds of proteins that have a particular affinity for ATP.

Once they have this captured mixture of proteins, the researchers can then do proteome mining by individually treating a series of such columns with each of hundreds of chemical compounds that make up the libraries of drug-like compounds created by chemists.

When a given compound interacts strongly with specific purine-binding proteins, it pulls those proteins from the column, isolating them. The researchers can then analyze this sample to determine the proteins' structure. Thanks to advanced analytical techniques such as "microsequencing" and mass spectrometry, said Haystead, it is now possible to identify any protein in a complex mixture, even with exceedingly small samples.

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With the proteins' structures, the researchers can use database searches to determine whether they play a role in a disease-related cell signaling pathway. The researchers can then refine the structure of the potential drug molecule to make it more selective for one protein and test it on the affinity array. Such studies of "structure-activity relationships" reveal how alterations in the drug's structure changes its effects. This information also enables further searches of drug databases to find existing compounds that might be effective.

Finally, the researchers can perform animal studies of the potential drug's effects to explore its value in treating the target disease or battling a particular pathogenic organism.

Importantly, said Haystead, because a given compound usually pulls more than one protein from the affinity array, the researchers can also easily get a preview of whether a given compound might produce side-effects by interacting with other components in the cell.

To provide such proteome mining services to the pharmaceutical industry, Haystead and his colleagues have founded Serenex, a Durham-based company.

At Duke, Haystead and his colleagues are using proteome mining to identify new antimalarial drugs that do not have the eye-damaging side effects of current drugs such as chloroquine. In these studies, they isolated from proteomes of human cells those proteins that interact with antimalarial drugs. This screen revealed that the drugs' effects on a particular enzyme called aldehyde dehydrogenase appears to be central to the blindness-causing retinopathy that is a side effect of such drugs.

However, the researchers have also discovered that another target of the chloroquine, a human enzyme called quinone reductase 2 found in blood cells appears to play a key role in its antimalarial action. This enzyme appears to be part of a mechanism to protect red blood cells against stress, said Haystead.

"We believe that the drug inhibits the human enzyme, which the malaria parasite is essentially using to protect itself inside the blood cell," said Haystead. "And we believe that if you inhibit the enzyme then the parasite can't survive, so drugs that target it will confer resistance to malaria."

Thus, the researchers are now developing and animal-testing antimalarial drugs that target only this enzyme and not aldehyde dehydrogenase. They are also developing mice that lack the gene for the enzyme and studying malaria-resistant people with mutations in the gene for quinine reductase 2 that give them resistance.

"What's extraordinary about proteome mining is that, even though it begins like many discovery processes as a fishing expedition, it can very quickly and efficiently get down to the level of one protein target that can be iteratively tested against libraries of compounds," concluded Haystead.

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The source of this article is http://www.dukehealth.org

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