In a paper published in the Dec. 23 issue of Science, a Johns Hopkins team led by Solomon H. Snyder, M.D., said the iNOS (inducible nitric oxide synthase)-based inflammation pathway has now been found to cross-link with the more well-known COX-2 pathway that is the target of COX-2 inhibitor drugs such as Vioxx. Until now, these two major inflammatory mechanisms were assumed to be unrelated and independent of each other, the researchers say.
"The fundamental significance of this work is that it demonstrates a totally unsuspected connection between the two most important inflammatory systems in the body," says Snyder, professor and director of neuroscience in Johns Hopkins' Institute for Basic Biomedical Sciences. "The therapeutic significance is that drugs which block the binding of iNOS and COX-2 might represent novel anti-inflammatory agents or reduce the dosage needed and side effects of this family of drugs."
COX-2 is an enzyme that makes prostaglandins, molecules that cause inflammation and pain. iNOS is an enzyme that makes NO (nitric oxide), a molecule that acts as a signal for a variety of cellular functions throughout the body, including the triggering of inflammation, dilating of blood vessels and penile erection.
The site on the iNOS protein that binds to COX-2 is close to the active or business end of the iNOS, the researchers found. As a result, it should be possible to design drugs that do double duty by inhibiting iNOS while also blocking iNOS binding to COX-2. This would decrease the formation of both NO and prostaglandins, Snyder said.
In addition to their studies in isolated cells, the team also demonstrated in mice the potential therapeutic role of drugs that block iNOS binding to COX-2. Specifically, they showed that in mice that lacked the gene for iNOS, production of a specific prostaglandin called PGE2 could be reduced by 70 percent.
"Now that we've characterized the iNOS-COX-2 inflammatory system and how to manipulate it, we have a road map for developing new drugs to treat inflammation and pain that permits simultaneous use of reduced dose levels of COX-2 inhibitors," Snyder says. "Our research suggests that the synergism between these two drugs would represent a highly effective and safer form of therapy.
"Blocking iNOS-COX-2 binding might salvage the value of COX-2 inhibitors by permitting the use of lower doses of these drugs, which have been shown to have troublesome potential side effects when used at their originally prescribed levels," Snyder added.
Researchers already knew that COX-2 can produce prostaglandins independently of iNOS. But the Hopkins study showed that iNOS is responsible for about half the total amount of prostaglandins that COX-2 produces in response to stimuli that trigger inflammation. This demonstrated the close connection between these two systems, Snyder says. Therefore, drugs that block iNOS activity could significantly reduce the amount of prostaglandins produced by COX-2 enzymes, he adds.
The Hopkins scientists showed the connection between the iNOS and COX-2 systems in both immune system cells and human embryonic kidney cells by adding substances known to activate these enzymes -- for example, LPS-IFN-gamma, a combination of a component of some infectious bacteria membranes and an immune system protein that targets viruses and tumors. When the investigators broke apart these cells and added antibodies that specifically bind to COX-2, tiny clumps formed that consisted of COX-2 enzymes bound to iNOS. Antibodies that bind specifically to iNOS also formed clumps of COX-2 and iNOS enzymes bound to each other.
The Hopkins team also showed that iNOS first binds to COX-2 and then makes NO. The NO chemically modifies COX-2 by a process called nitrosylation, which stimulates the enzyme to make prostaglandins. In addition, the investigators found that the same part of iNOS that binds to COX-2 also contains the "active site" of the enzyme that makes the NO. Furthermore, when the researchers blocked this dual-purpose section of iNOS, they prevented both iNOS binding to COX-2 and the subsequent activation of COX-2 by NO.
"This tells us that during reactions that cause inflammation, COX-2 and iNOS are attached to each other," Snyder said.
The team also demonstrated that the NO that exists freely in cells cannot stimulate COX-2; rather, only NO generated by iNOS that is bound directly to COX-2 can activate these enzymes to produce prostaglandins.
The other authors of the report include Sangwon Kim and Daniel Huri. This work was support by a U.S. Public Health Service grant, a Research Scientist Award (S.H.S.) and a fellowship from the Canadian Institute of Health Research (S.F.K.).
On the Web: