A recently identified path of inflammation once
thought to be wholly independent of other inflammatory
systems has now been linked to another major pathway. The
findings by neuroscientists at Johns Hopkins are likely to
point scientists to novel drugs that significantly reduce
the risks of taking COX-2 inhibitor pain relievers, the
In a paper published in the Dec. 23 issue of
Science, a Johns Hopkins team led by Solomon H.
Snyder 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
"The fundamental significance of this work is that it
demonstrates a totally unsuspected connection between the
two most important inflammatory systems in the body," said
Snyder, Distinguished Service Professor of
Psychiatry 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 nitric oxide, or NO, 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
In addition to their studies in isolated cells, the
team 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 said. "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 said.
Researchers already knew that COX-2 can produce
prostaglandins independently of iNOS. But the Johns 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 said. Therefore, drugs that block iNOS
activity could significantly reduce the amount of
prostaglandins produced by COX-2 enzymes, he added.
The Johns 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 Johns 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,"
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 are 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