Proteomics yields new drug targets for treating cardiogenic shock

When it comes to cell signaling and bioregulation, it's tough to find a more important molecule than nitric oxide (NO). Dysfunction of NO-mediated signaling is thought to be a culprit in lethal cardiovascular conditions such as angina, and septic, cardiogenic and hemorrhagic shock; it may play a role in many other illnesses as well.

NO is a very unusual cell signaling molecule in that its actions result from chemical bonds made with proteins, rather than simple lock-and-key binding. A predominant reaction of NO with proteins is addition to sulfur in the amino acid cysteine -- a process called "S-nitrosylation."

But because these S-NO bonds are so fragile -- it's been notoriously difficult to spot exactly where, among the thousands of cellular proteins, NO is reacting and having its effects. A comprehensive way of identifying protein S-nitrosylation sites should greatly improve our understanding of how NO acts and hasten new drug discovery.

That "comprehensive way" is now here.

Reporting recently in the Proceedings of the National Academy of Sciences, scientists at the Weill Medical College of Cornell University, New York City, say they've devised the first-ever method of combing through the body's tens of thousands of proteins to inventory sites of S-nitrosylation.

"It's a real breakthrough for both basic science and drug research -- a tool that should significantly accelerate our understanding of nitric oxide signaling," says senior researcher Dr. Steven S. Gross, Professor of Pharmacology at Weill Cornell Medical College.

About 18 years ago, scientists made the remarkable discovery that NO is a product of mammalian cells and not merely an environmental toxin.

"It was surprising because nitric oxide is a reactive 'free radical' molecule -- the kind that's usually thought of as harmful or toxic," Dr. Gross said. "But it turns out that nature has invented a way to use it for physiological signaling via its chemistry with proteins. And this signaling can have broad and important downstream effects -- things like fighting infections, communicating between nerve cells, determining the diameter of blood vessels and the force of a hearts contraction."

In fact, one of the first heart drugs, nitroglycerin, substitutes for nitric oxide to trigger vasodilation (widening of blood vessels), although just how it does so is incompletely understood.

"That's why a better means of spotting S-nitrosylation sites is so important. It will help us better understand how many existing drugs work, and point the way to new targets for drug development," explains study lead author Gang Hao, a postdoctoral researcher in Dr. Gross' lab.

But the instability of the S-NO bond has, in the past, posed a problem for researchers. "You can know that this signaling is going on in tissues, but once you try to define the exact molecular sites, the ease of NO rearrangement can leave you guessing as to which protein sites are real," Dr. Gross says.

The new technique, called SNO Site IDentification (SNOSID), quickly screens the proteome -- the body's thousands of gene-expressed proteins -- to find S-nitrosylation sites. The method relies on state-of-the-art mass spectrometry for sequencing amino acids from hundreds of peptides in protein digests obtained from cells and organs.

SNOSID builds on a technology called "biotin-switch," originally described by Dr. Samie Jaffrey while a postdoc in the lab of Solomon Snyder at Johns Hopkins. (Dr. Jaffrey has since become Assistant Professor of Pharmacology at Weill Cornell.)

Dr. Gross explains: "We know that we can't capture the S-nitrosylated bond itself, but this method uses a kind of surrogate, biotin, to stably replace NO at sites of S-nitrosylation. So the site of biotinylation in a protein becomes a kind of chemical flag showing us where NO used to be and providing us with a chemical strategy to enrich these sites for analysis."

According to the researchers, SNOSID should speed research toward a fuller understanding of the role of NO signaling in healthy cellular functions.

"It should also point the way to protein sites that could be important drug targets in the fight against disease," Hao says.

Phase III Clinical Trial Underway

Dr. Gross and other Weill Cornell scientists anticipate the benefits of new drugs that inhibit NO synthesis. Already, a Phase III clinical trial, based on Weill Cornell technology, is testing whether such a drug can save the lives of patients with cardiogenic shock, an emergency condition that kills more than half of its victims and occurs in 8 to 10 percent of patients with severe heart attacks.

"It's thought that cardiogenic-shock patients suffer from an excess of NO," Dr. Gross explains. "The ongoing study is blinded, so results are not yet available. But there's real hope the drug will be proven to save lives. If so, it would likely become the new standard-of-care for patients stricken by this highly lethal condition."

"This is really only the tip of the iceberg," Dr. Gross adds. "As SNOSID uncovers new and important sites of protein S-nitrosylation, novel drug targets are likely to be revealed. It should open the door to fertile new areas of research."

The study was supported by a grant from the National Institutes of Health.

Co-researchers included Dr. Lei Shi, Dr. Fabien Campagne, and Behrad Derakhshan -- all of the Weill Medical College of Cornell University, New York City.