Cox-2 inhibitors have turned from an anti-inflammatory gold mine to a cardiovascular quagmire for the pharmaceutical industry.

Vioxx (rofecoxib) was withdrawn by its maker, Whitehouse Station, N.J.-based Merck & Co. Inc., in September 2004 after it was discovered that long-term use increases the risk of heart attacks and strokes. The drug has not returned to the market, despite a decision by the FDA that it could, with restrictions. And while two other Cox-2 inhibitors, Celebrex (celecoxib) and Bextra (valdecoxib), both from New York-based Pfizer Inc., are still available, they have had black-box warnings added to them because they, too, raise the risk of cardiovascular problems. (See BioWorld Today, Feb. 22, 2005.)

Though Cox-2 inhibitors carry this risk, what is not clear is whether this is due to their main effect - preventing Cox-2 from synthesizing inflammatory prostaglandin molecules - or an effect on some other target.

"In fact," scientist Solomon Snyder told BioWorld Today, "there's really no good reason why inhibiting Cox-2 should give you a heart attack." Snyder added that drugs such as aspirin block prostaglandin formation without raising the risk of heart attacks, which adds support to the notion that the Cox-2 inhibitors may be raising cardiovascular risk by acting on some other pathway.

If the risk of cardiovascular problems is indeed a side effect rather than a consequence of the main effect of Cox-2 inhibitors, then inhibiting the Cox-2 pathway indirectly through another pathway might allow patients to reap their anti-inflammatory benefits without their cardiovascular risks.

In the Dec. 23, 2005, issue of Science, Snyder, who is a professor of neuroscience at Johns Hopkins University in Baltimore, and his colleagues provide such an alternate route via inducible nitric oxide synthase, or iNOS.

Nitric oxide is "one of those universal second messenger molecules," Snyder said, influencing physiological systems including circulation, inflammation and neural signaling. But "nobody had ever given any thought to an interaction between nitric oxide and the prostaglandin system. The basic science is very surprising." So much so, in fact, that Snyder described his reaction to the initial findings in his lab that the two might interact as: "Let's do a lot of controls, because things that are too good to be true often aren't even true."

In this case, however, those control experiments supported rather than shot down the initial findings. The scientists first found that Cox-2 and iNOS must bind to each other in cells, as adding antibodies after immunostimulation with a bacterial toxin led to precipitation of both molecules in cell lines. Such close association of nitric oxide synthase has been demonstrated for several of the systems where nitric oxide is a signaling molecule.

"For nitric oxide to do anything in the cell, it has to be delivered, either directly or via a carrier protein," Snyder explained. "If it's just diffusing in the cell, it won't go anywhere" because the cell has other proteins to prevent nitric oxide from turning on second messenger pathways willy-nilly.

The researchers also found that iNOS affects Cox-2 by delivering nitric oxide to one of Cox-2's cysteines, and that this nitrosylation enhances Cox-2's production of prostaglandins.

In a final set of experiments, they determined exactly which amino acid is affected by iNOS. Snyder said that the binding takes place "in the catalytic site, near where [Cox-2] inhibitors work," making the interaction an easier target for drug discovery than would be the case with distant sites.

The discovery by Snyder and his colleagues adds to nitric oxide's status as a near-universal second messenger molecule. It is the first gas that was discovered to be a signaling molecule in the body. Robert Furchgott and Louis Ignarro, who along with Ferid Murad received the 1998 Nobel Prize for their discoveries about nitric oxide, first described nitric oxide's action as a second messenger in 1986.

Manipulating nitric oxide synthase already is a successful clinical strategy; its enhancement is the molecular mechanism behind the erectile dysfunction drugs Viagra (sildenafil), Levitra (vardenafil) and Cialis (tadalafil).

In its citation for the 1998 prize, the Nobel committee noted that Alfred Nobel himself had been prescribed nitroglycerin (which he refused to take), which releases nitric oxide, for heart problems. The molecular mechanism of action was, of course, unknown at the time, but one irony already was apparent to Nobel and his contemporaries: Nobel had made his fortune through the invention of dynamite, which consists of stabilized nitroglycerin.