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BLOCKING NO SYNTHESIS:
HOW, WHERE AND WHY?
Patrick Vallance and James Leiper
Nitric oxide (NO) is a key physiological mediator, and the association of disordered NO
generation with many pathological conditions has led to much interest in pharmacologically
modulating NO levels. However, the wide range of processes in which NO has been implicated,
and the fact that increases or decreases in NO levels might be therapeutically desirable
depending on the condition or even at different stages of the same condition, pose
considerable challenges for drug development. Here, we focus on the rationale and potential
for approaches that reduce NO synthesis, which have led to the development of several
compounds that will shortly be entering clinical trials.

Centre for Clinical
Pharmacology,
British Heart Foundation
(BHF) Laboratories,
Department of Medicine,
University College London,
5 University Street, London
WC1E 6JJ, UK.
Correspondence to P.V.
e-mail:
patrick.vallance@ucl.ac.uk
doi:10.1038/nrd960
Nitric oxide (NO) is a mediator with protean functions.
It is involved in signalling in the cardiovascular,
gastrointestinal, genitourinary, respiratory and nervous
systems, and disordered NO generation has been
implicated in a wide range of diseases1–3. Therapies
based on increasing NO effects or donating exogenous
NO hold promise, and have been discussed extensively
elsewhere2. However, several companies are develop-
ing compounds that inhibit NO synthesis, and this is
the focus of the present article. What is the rationale for
reducing NO synthesis and which are the approaches
that could yield useful medicines?
Synthesis of NO
NO is synthesized from L-arginine in a reaction that is
catalysed by a family of enzymes — the NO synthases
(NOSs; FIG. 1). Three NOS isoforms have been identified
and named according to the cell type or conditions in
which they were first described: endothelial NOS
(eNOS), neuronal NOS (nNOS) and inducible or
inflammatory NOS (iNOS)1. These enzymes share
~50% sequence homology and catalyse the NADPH-
and O2-dependent oxidation of L-arginine to NO and
citrulline, with N ω-hydroxy-L-arginine formed as an
intermediate3. NOSs are flavohaem enzymes that are
active only as dimers. Each monomer has a carboxy-
terminal diflavin-reductase domain and an amino-
terminal oxygenase domain. Dimerization is thought
to activate the enzyme by sequestering iron, generating
high-affinity binding sites for arginine and the essen-
tial cofactor tetrahydrobiopterin (BH4), and allowing
electron transfer from the reductase-domain flavins
to the oxygenase-domain haem. Activity is also
dependent on bound calmodulin3. In iNOS, calmod-
ulin is tightly bound, whereas in eNOS and nNOS,
calmodulin binding is dependent on calcium, and
enzyme activity is therefore calcium dependent.
Bound calmodulin is thought to enhance the rates of
electron transfer through the reductase domain to the
oxygenase domain.
In addition to regulation of NO synthesis by cofac-
tors and calcium, the activities of NOSs can be altered by
post-translational modifications and by protein–protein
interactions. For example, eNOS activity is increased
by phosphorylation of Ser1179 (REF. 4), and is inhibited
by interaction with the scaffolding domain of the mem-
brane protein caveolin-1. These important areas of reg-
ulation of NO synthesis could be targets for drug
action, but a detailed discussion is outside the scope of
this article (for review, see REF. 5).
Targets for NO
The first described physiological target for NO was
soluble guanylyl cyclase6. Binding of NO to the iron
within the haem moiety of guanylyl cyclase produces a
conformational change that leads to enzyme activation.

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Haem
H2N NH2 BH4
NH + NADPH, O2 FMN
Zn
H2N COO–
FAD
L-Arginine
CAL
Figure 1 | The NOS pathway. For enzymatic activity, nitric oxide synthase (NOS) enzymes must dimerize and bind the cofactors
tetrahydrobiopterin (BH4), haem, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). On binding calmodulin (CAL),
the active enzyme catalyses the oxidation of L-arginine to citrulline and nitric oxide (NO) and requires molecular oxygen and NADPH
as co-substrates. Each NOS dimer coordinates a single zinc (Zn) atom.
The subsequent rise in cyclic GMP accounts for many of
the physiological effects of NO. However, NO has the
potential to interact directly or indirectly with metals,
thiols and oxides, and affect proteins, nucleic acids, lipids
and sugars7 (TABLE 1). Indeed, because NO is a free radical
(it has an unpaired electron), and the nitrogen can exist
in various oxidation states to generate nitroxyl ions
(NO–), NO free radicals (NO•), nitrosonium cations
(NO+), nitrite ions (NO2–) or nitrate ions (NO3–), the
biological chemistry of NO is complex, and its potential
effects within biological systems are many. There are
probably four main targets for reactions with NO in
cells: metals, reduced thiols, molecular oxygen and other
reactive oxygen species (for example, superoxide (O2–)).
Which reactions are favoured will depend on the con-
centration of the reactants and the reaction rates8.
The fastest reaction rates are with superoxide ions to
form peroxynitrite (ONOO–), a powerful oxidant that
can modify proteins and lipids by nitration9,10. However,
the concentration of superoxide is usually kept low, and
so this reaction is probably not favoured. Reaction rates
with metals are also high, but except for certain haem
proteins, such as soluble guanylyl cyclase, the metal
centres are generally not available for reaction with NO.
Reactions with thiols are important, as S-nitrosation of
specific cysteine residues (an NO+-like reaction) can
affect protein function in a reversible manner. Ion
channels, enzymes, transcription factors and G proteins
can all be S-nitrosated, and structural nitrosation motifs
are being identified10. This means that NO signalling is
intimately bound to the redox state of the cell, and can
affect many signalling pathways. These multiple actions
of NO and dependence on thiol concentration and
redox state probably explain why NO can have both
940 | DECEMBER 2002 | VOLUME 1
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NO•
Haem
BH4
FMN
FAD
H2N O
NH
CAL
H2N COO–
Citrulline
protective and harmful effects. The interaction of NO
with respiratory-chain enzymes illustrates many of these
mechanisms. For example, NO itself can bind reversibly
to the haem enzyme cytochrome c oxidase, NO+-like
reactions lead to reversible S-nitrosation of mitochon-
drial complex I and generation of ONOO– can irre-
versibly inhibit multiple complexes, as well as aconitase.
These effects activate proton leak and the permeability
transition pore, leading to cell death11.
NO in physiology
To understand the potential effects of pharmacological
inhibition of NOS isoforms, it is important to identify
the physiological processes that could be disrupted.
eNOS. In the cardiovascular system, NO generation from
the endothelium is important to maintain the vasculature
in a relaxed state, inhibit the adhesion of platelets and
white cells and suppress the replication of smooth-muscle
cells12. Pharmacological inhibition of NOS causes vaso-
constriction, hypertension and enhanced platelet activa-
tion, and increases atherogenesis in animal models13. In
addition, eNos knockout mice are hypertensive, show
exaggerated smooth-muscle replicative responses to vas-
cular injury, are more prone to atherogenesis and develop
aneurysms14 (FIG. 2, TABLE 2). eNOS is also important to
promote angiogenesis and regulate the expression of
vascular-endothelial growth factor (VEGF). So, prolonged
inhibition of eNOS activity is considered detrimental.
nNOS. NO derived from nNOS in peripheral nerves is
also important in the relaxation of vascular and non-
vascular smooth muscle15. It relaxes sphincters in the gut,
mediates relaxation of the corpus cavernosum and
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Table 1 | Examples of modification of protein function by NO and related species
Target Reactant Example
Metal NO• Fe2+ in haem–guanylyl cyclase
Fe–S cluster in aconitase
Thiol NO+-like reaction Cys249 on DDAH
(S-nitrosation)* Cys3656 on ryanodine receptor
Cys69 on thioredoxin
Tyrosine ONOO– (nitration) Tyrosines in ribonucleotide reductase
NO• Tyrosines in prostaglandin H synthase
*Nitrosation of cysteines is specific for certain cysteine residues held in the three-dimensional protein structure in a responsive state.
DDAH, dimethylarginine dimethylaminohydrolase; NO, nitric oxide.
thereby causes penile erection, relaxes the bladder and
urethra and alters responses in airways. Because splice
variants of nNOS are still expressed in some tissues of
nNos knockout mice16, and highly selective nNOS
inhibitors have not been used extensively, the data from
(non-selective) inhibitor studies and knockout pheno-
types (TABLE 2) are not totally concordant. Thus, owing to
the loss of sphincter relaxation, nNos knockout mice
have dilated bladders and increased urinary frequency,
and show pyloric stenosis with grossly dilated
stomachs17, but they still achieve penile erection, and
strips of corpus cavernosum relax on electrical-field
stimulation18. In the central nervous system (CNS),
nNOS is widely expressed, but it has been less easy to
determine its precise roles. A striking feature of the first
nNos knockout mouse line (in which alternative tran-
scripts can still be expressed) is an increase in aggression
a b
ApoE ko ApoE/eNos ko ApoE/eNos ko
Figure 2 | Loss of eNOS causes vascular disease. a | Intact aorta from a normal apolipoprotein E
(ApoE) knockout (ko) mouse and an ApoE/endothelial nitric oxide synthase (eNos) double
knockout. The double knockout shows enhanced atherosclerosis (white) and an aneurysmal
dilation of the aorta. The inset on the right shows a higher magnification of the aneurysm. The
arrows show the origin of the left renal artery b | An aorta from a male ApoE/eNos double
knockout shows a dissection that extends beyond the renal arteries. The arrows show the start
and end of the dissection. Reproduced from REF. 14 © (2001), with permission from Lippincott,
Williams & Wilkins.
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Effect References
Enzyme activation 1
Enzyme inhibition 92
Enzyme inhibition 91
Sensitizes channel 93
to calcium
Enzyme activation 48
Enzyme inhibition 94
Enzyme activation 95
and inappropriate mounting behaviour, indicating that
NO derived from nNOS might be an important media-
tor of behavioural inhibition — the effects on behaviour
are particularly evident in males16. The mice also show
abnormalities of balance and coordination at night
(when visual cues are reduced), which are consistent
with the high levels of expression of nNOS in
cerebellum16. Despite pharmacological observations
indicating that nNOS might have a role in long-term
potentiation (the process by which neurons ‘remember’),
nNos knockout mice seem to have normal memory and
learning15. A novel nNos knockout that lacks the exon
that encodes the haem-binding domain and therefore
encodes a global nNos knockout shows hypogonadism
and infertility19.
nNOS is highly expressed in skeletal muscle20.
Studies with inhibitors indicate that it might have a role
in regulating muscle contraction, exercise-induced glu-
cose uptake and control of arteriolar tone within the
muscle. The nNOS protein is found in the dystrophin
complex, and a role has been proposed in muscular
dystrophy21. In the heart, nNOS and eNOS seem to have
different and sometimes opposing effects22. NO derived
from nNOS has a positive inotropic response, possibly
secondary to activation of sarcoplasmic-reticulum
calcium release, whereas eNOS activity decreases
β-adrenoceptor-induced contractility through effects
on L-type calcium channels. In the airways, nNOS
activity seems to provide important protection against
ovalbumin-induced airway hyperresponsiveness23.
For both eNOS and nNOS, it is clear that there are
sex differences in susceptibility to the effects of loss of
enzyme function24. This might be because expression of
both isoforms is modulated by sex hormones, and
therefore compensatory mechanisms might be more
evident in one or other sex. It is not clear whether such
gender-specific compensation would also occur with
long-term pharmacological inhibition of NOS. Unlike
eNOS, nNOS activity can also cause significant patho-
physiology (see below), and so inhibition of this iso-
form has the potential for benefit as well as harm.
iNOS. iNOS was first described in macrophages as a
mechanism of macrophage cytotoxicity25. This isoform
is not usually expressed in healthy quiescent cells, but is
rapidly transcriptionally induced in multiple cell types
in response to stimulation with bacterial endotoxins or
pro-inflammatory cytokines. Once expressed, it generates
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large amounts of NO, and its activity is not dependent
on intracellular calcium26. It is becoming clear that
there are species-, tissue- and cell-specific conditions
for inducing the expression of active iNOS. Although
the protein is reasonably well conserved between
species, the promoter region of the iNOS gene varies
greatly27. This difference is of functional importance,
because it is far more difficult to induce expression of
functionally active iNOS in human cells in vitro com-
pared with rodent cells. Furthermore, there are five
copies of the iNOS gene in humans, but it is thought
that only one is transcribed and translated into a func-
tionally active protein27. Therefore, the extrapolation of
studies in rodents to humans must be undertaken with
caution. However, it is clear that expression of iNOS is
important for killing or host defence against certain
protozoa, bacteria, fungi and viruses28. It is also impor-
tant in regulating cytokine production and influencing
T-helper type 1 (TH1)-cell expansion29. But iNOS is also
induced in cells that are not obviously involved in host
defence, and in situations in which no live organism is
present, and it is in these situations that iNOS activity
might be harmful and cause tissue damage. The over-
production of NO in response to infection might also
be an important part of the tissue damage and destruc-
tion caused by the infection.
Many of the effects of increased NO generation
from iNOS are considered in terms of pathology (see
below), but it seems unlikely that the isoform and the
NO it generates in non-immune cells exist solely to
cause harm. Indeed, studies in iNos knockout mice
(TABLE 2) have indicated physiological roles; for example,
in osteoclastic bone resorption30. Furthermore, in the
gut, iNOS can be expressed constitutively, and complete
Table 2 | Some phenotypic features of NOS knockout mice
Knockout Phenotype References
mouse
eNos–/– Elevated systemic and pulmonary blood pressure 96
Increased neointimal thickening after vessel injury 24
Aneurysm formation 14
Increased leukocyte adhesion 97
Increased cardiac ionotropic response 98
Increased tissue damage in experimental models of 99,100
stroke and global cerebral ischaemia
Reduced neurotransmitter release 101
nNos–/– Reduced tissue damage in experimental models of 45,102
stroke and global cerebral ischaemia
Reduced neurotransmitter release 101
Altered airway responsiveness 23,103
Increased leukocyte adhesion 97
Increased stomach size (pyloric stenosis) 104
Gastroparesis, abnormal IJP and NANC relaxation in 105
GI smooth muscle
Urinary bladder urethral sphincter dysfunction 17
Hypogonadism and infertility 19
iNos–/– Increased susceptibility to pathogens 28,106,107
Resistance to sepsis-induced hypotension 106,107,108
Increased susceptibility to tumours 109
Impaired osteoclastic bone resorption 30
Impaired wound healing 33,110
Increased tissue damage in models of lung injury 31
eNos, endothelial nitric oxide synthase; GI, gastrointestinal; IJP, inhibitory-junction potential;
iNos, inducible nitric oxide synthase; NANC, non-adrenergic, non-cholinergic; nNos, neuronal nitric
oxide synthase.
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loss of iNOS is harmful in certain animal models of
inflammation31,32. Expression of iNOS is also impor-
tant for skin wound healing33 and healing of intestinal
mucosa, and it has been implicated as a key mediator
of ischaemic preconditioning in the heart (a mecha-
nism by which the heart is protected against lethal
ischaemia)34. One possibility is that iNOS is not only
involved in the genesis of certain types of inflamma-
tion, but also in the resolution of inflammation. So, it
is probably oversimplistic to assume that eNOS and
nNOS are physiological isoforms and that iNOS is
involved solely with pathophysiology.
Overproduction of NO in pathology
Increased generation of NO, either alone or in the
presence of other free radicals, such as superoxide, has
been implicated in pathophysiological changes in virtu-
ally every organ system. Evidence comes from studies
of NO generation, NOS isoform expression and effects
of NOS inhibitors (usually isoform non-selective).
Induction of iNOS is seen in models of septic shock27,
inflammatory and non-inflammatory pain35,36, arthritis37,
inflammatory bowel disease38, asthma39,40 and in the brain
after ischaemia or trauma, as well as in various models
of neurodegeneration or cerebral inflammation41,42. In
the brain, the precise cellular distribution of iNOS
might vary according to the nature of the insult, and
iNOS expression has been described in neurons as well
as glial cells, astrocytes and infiltrating neutrophils41–43.
There is also evidence from clinical (human) samples
that iNOS is expressed in various pathological tissues
(TABLE 3), but it is interesting to note that in few cases
has functionally active iNOS definitively been shown
to be present. However, the potential importance of
iNOS in contributing to pathophysiology is exempli-
fied by the findings from iNos knockout mice (TABLE 2).
iNOS is not the only the only isoform that can
cause a pathophysiological increase in NO. In particu-
lar, increased nNOS activity is associated with certain
types of neurotoxicity. NMDA (N-methyl-D-aspar-
tate)-induced neurotoxicity is reduced by NOS
inhibitors in some models44, and stroke damage is
reduced in nNos knockout mice45. NOSs can also gen-
erate superoxide through NADPH oxidation that is
uncoupled from NO generation3. nNOS seems to be
the isoform that is most likely to generate superoxide
when arginine or BH4 concentrations are low, or in
the presence of certain arginine-based NOS inhibitors
(see below).
Is overproduction of NO from eNOS ever patho-
logical? Although most interest has focused on the
cardioprotective effects of eNOS, there are some situa-
tions in which eNOS-derived NO might be harmful.
For example, in hyperoxia-induced retinopathy, eNOS
deficiency seems to confer benefit46. Furthermore, in
humans, some of the vasodilatation induced by pro-
inflammatory cytokines might be mediated by
eNOS47, although whether this is protective or harm-
ful is not known. Overall, most data indicate that pro-
longed inhibition of eNOS is likely to do more harm
than good.
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Table 3 | Some human diseases in which iNOS has been detected
Disease
Alzheimer’s disease
Atherosclerosis
Celiac disease
Inflammatory bowel disease
Multiple sclerosis
Arthritis
Parkinson’s disease
Septic shock
Stroke
iNOS, inducible nitric oxide synthase.
APOPTOSIS
Programmed cell death.
CASPASES
A family of intracellular
cysteine proteases that are
responsible for apoptosis.
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References
111
112,113
114
115,116
117
118,119
120
121
122
In addition to issues relating to isoform specificity of
inhibition, it is important to consider long-term effects
of NOS inhibition. Much of the data relating to effects
of NOS inhibitors are based on relatively short-term
studies, and long-term effects of cellular signalling
might be profound and affect several pathways. For
example, thioredoxin, a redox regulator that is impor-
tant in cell growth and APOPTOSIS, is activated by NO48,
and presumably long-term inhibition of NO generation
has the potential to increase cellular oxidative stress
through this mechanism.
Dual effects of NO
A glance at the NO literature reveals many apparently
contradictory papers showing beneficial or harmful
effects of endogenous NO. NO can promote or inhibit
apoptosis49, kill tumours or increase the potential for
metastasis or vascularization50, increase or protect
against damage after stroke51,52 and trigger ischaemic
preconditioning in the heart and mediate late effects of
preconditioning34,53. Some of these dual effects of NO
relate to different isoforms of NOS regulating different
processes. For example, protective effects in stroke
seem to be mediated by eNOS (vascular), whereas
harmful effects are due to nNOS activity (neuronal
toxicity)45,52. Other dual effects might relate simply to
the amount of NO generated or the background redox
state of the cell; at low concentrations, NO seems to be
anti-apoptotic, in part through inhibition of CASPASE
activity by means of nitrosation, whereas at higher
concentrations, it can indirectly activate caspases54.
The dual effects of NO present one of the biggest
challenges for the development of potential inhibitors
— beneficial effects can be offset by harmful effects or
turn into harmful effects, depending on the underlying
rates of NO synthesis.
Inhibition of NO
It seems clear that blocking NO has the potential to
produce therapeutic benefit. The difficulties lie in
achieving isoform specificity, in targeting to specific
cells or tissues and in ensuring that the correct degree
of inhibition is achieved55. As expected, most interest
has focused on iNOS inhibitors, but with substantial
interest also in nNOS inhibitors. A key issue for any
© 2002 Nature Publishing Group
REVIEWS
inhibitor is the degree of selectivity over other NOS
isoforms, and specificity for NOS over other potential
targets. The definition of isoform selectivity has been
discussed extensively elsewhere3, but is important
because some of the apparent contradictions in the
literature relate to differing definitions. Some com-
pounds are apparently selective for one or other iso-
form in vivo, but show no real selectivity at the level of
the enzyme. For example, 7-nitroindazole (7-NI) is
widely quoted as a selective inhibitor of nNOS, but in
reality it is about equipotent as an inhibitor of all three
NOS isozymes (TABLE 4). Its apparent preferential effect
on NO synthesis in the brain in vivo presumably
relates to a kinetic property of the molecule that allows
selective access or metabolism in certain tissues.
Similarly, there are many studies that have used
aminoguanidine as a selective inhibitor of iNOS 56,
although it has at best a tenfold selectivity over eNOS
and perhaps fivefold selectivity over nNOS, and even
this selectivity might be lost if local concentrations of
BH4 are elevated47. Furthermore, aminoguanidine was
initially developed as an inhibitor of advanced glyca-
tion end-product formation and also inhibits diamine
oxidase in a similar concentration range. Because of
these difficulties, some of the claims of therapeutic
effects or potential adverse effects of selective inhibi-
tion of NOS isoforms must be treated with caution.
The industry standard for defining iNOS selectivity
in vivo in rodents has been inhibition of endotoxin-
induced nitrate formation (a stable product of NO)
with no effect on arterial blood pressure. As arterial
blood pressure is well buffered, and inhibition of
eNOS causes changes in vascular resistance (and pre-
sumably effects platelets, white cells and atherogenesis)
at doses far lower than those that affect blood pressure57,
this screen is likely to overestimate the true degree of
selectivity for iNOS.
The selectivity of compounds over other targets is also
a potential problem. Several arginine metabolizing or
transporter proteins might be affected by substrate-based
NOS inhibitors, and there is little information available at
present on the effects on enzymes such as dimethylargi-
nine dimethylaminohydrolase (DDAH), arginine glycine
amidinotransferase, arginase, argininosuccinate synthase
or even peptide arginine deiminases.
Studies in humans. NOS inhibitors have been used in
humans to explore mechanisms of physiology and
pathophysiology, and as potential treatments. In
patients with septic shock, the isoform non-specific
inhibitor NG-monomethyl-L-arginine (L-NMMA)
restores blood pressure and seems to improve haemo-
dynamics58,59 (FIG. 3). However, the largest study so far
showed an adverse effect on outcome. A post hoc
analysis indicated that low doses might have been
beneficial and that the harm occurred when larger
doses were used (and very high circulating concentra-
tions of L-NMMA were achieved). L-NMMA has also
been used to relieve headache 60, block excess NO
exhalation in asthma and reduce skin inflammation
induced by ultraviolet light (UVB)61.
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Table 4 | Relative potencies and selectivity of NOS inhibitors

µM)
IC50 (µ Selectivity (fold)
Inhibitor iNOS nNOS eNOS iNOS vs nNOS iNOS vs eNOS nNOS vs eNOS
L-NNA 3.1 0.29 0.35 0.09 0.11 1.2
L-NMMA 6.6 4.9 3.5 0.7 0.5 0.7
7-NI 9.7 8.3 11.8 0.9 1.2 1.4
ARL 17477 0.33 0.07 1.6 0.2 5.0 23.0
Aminoguanidine 31 170 330 5.5 11 1.9
L-NIL 1.6 37 49 23 9 1.3
PBITU 0.047 9 190
(bis-isothiourea)
vinyl L-NIO 60 0.1 12.0 0.002 5 120
1400W 0.23 7.3 1,000 32 >4,000 >130
GW273629 8.0 630 1,000 78 >125 >1.6
GW274150 1.4 145 466 104 333 3.2
AR-C102222 0.04 >100 2 50 >1,000 >50
The absolute potencies might not be directly comparable because of the experimental conditions under which studies were done.
eNOS, endothelial nitric oxide synthase; IC50, half-maximal inhibitory concentration; iNOS, inflammatory nitric oxide synthase;
7-NI, 7-nitroindazole; L-NIL, N(6)-(1-iminoethyl)lysine; L-NIO, N-iminoethyl-L-ornithine; L-NMMA, NG-monomethyl-L-arginine;
G
L-NNA, N -nitro-L-arginine; nNOS, neuronal nitric oxide synthase; PBITU, S,S′-1,3-phenylene-bis(1,2-ethanediyl)bis-isothiourea.
Data adapted from REF. 2.

Substrate analogues. The first described inhibitors of Although these compounds have some selectivity for
NOS were analogues of L-arginine62. These compounds iNOS over eNOS, their development as drugs was lim-
bind at the arginine-binding site and are competitive ited by toxic effects, including actions on Na+/K+
with arginine. However, some of them are also metabo- ATPase64. Crystal structures of the eNOS haem domain
lized by NOS, and active enzyme and NADPH are with S-alkyl-isothioureas show that the thiourea group
required for inhibition to proceed from weak binding to occupies the same position as the guanidine group of
potent inhibition. The first highly selective iNOS L-arginine. Binding studies with more bulky ligands led
inhibitors described were the bis-isothioureas63. to the suggestion that an isoform-selective inhibitor
would have three characteristics64. First, there should
120
be a structural scaffold that provides a guanidino,
amidino or ureido group that donates hydrogen
SVR 666
110 CO 10.7 bonds to the glutamate residue in the NOS active site.
HR 119 There should also be a small hydrophobic group, such
100
614 as an alkyl or thienyl group, as part of the scaffold to
607 10.9
10.3 573 120
provide further non-polar interaction with the pro-
90
95 575 10.1 tein opposite to the glutamate residue. Second, it
536 122
Mean arterial pressure (mm Hg)

9.7 10.2 should have an isoform-selectivity-conferring func-

110 511
80 9.5
122 tional group bearing hydrogen-bonding capability that
135 519 can reach into the substrate-access channel remote from
487 10.2
70 10.1 123
the active site. Such a group could take advantage of the
123 amino-acid differences in this channel between iso-
367
60 12.2 3
forms. Third, it should have a linker between the scaf-
122 2
fold and the functional group of appropriate length and
391 flexibility to reach isoform-specific regions.
50 8.3 1
105 Despite the high degree of similarity between the
0.30
40
arginine-binding sites of NOS isoforms65, highly selec-
0.16 0.10
0.02 tive iNOS inhibitors have been developed that compete
30
with arginine and presumably bind to this site. For
example, the compound 1400W (FIG. 4) is ~10,000-fold
–5 –4 –3 –2 –1 0 10 20 30 40 50 60 70
selective for iNOS over eNOS and at least 30-fold selec-
Time before first L-NMMA bolus (hr) Time after first L-NMMA bolus (min)
tive for iNOS compared with nNOS66. Once again, this
Figure 3 | Effects of L-NMMA in a patient with severe septic shock. The patient was treated seems to be a ‘mechanism-based’ inhibitor, the action
initially with noradrenaline to try to maintain blood pressure (yellow columns show dose in µg kg–1 of which is dependent on NADPH and develops
min–1). When this approach failed, he was treated with three bolus doses of L-NMMA (1 = 0.3 mg
slowly. Interestingly, whereas the effects of 1400W on
kg–1; 2 = 0.3 mg kg–1 and 3 = 1 mg kg–1). The L-NMMA restored blood pressure, and this was due
to an increase in systemic vascular resistance (SVR), with no significant change in cardiac output iNOS develop slowly and reverse only slowly, the
(CO) or heart rate (HR). L-NMMA, NG-monomethyl-L-arginine. Redrawn from REF. 58 © (1991), with effects on eNOS and nNOS are rapidly reversible by
permission from Elsevier Science. arginine. Although this compound has toxicity that

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prevents its administration to humans, it has been use-
ful to define some of the potential for selective iNOS
inhibition in animal models. Other compounds in this
series include GW273629 and GW274150. These com-
pounds have been shown to reduce experimental post-
operative ileus and have some beneficial effects in
models of gastrointestinal damage67.
An alternative chemical approach has been to
develop spiroquinazolone derivatives68. The Astra-
Zeneca compound AR-C102222 has a half-maximal
inhibitory concentration (IC50) of ~0.04 µM, and is
reported to be highly selective for iNOS. In vivo, it is
effective in reducing inflammation in the standard
carageenan paw oedema model and decreases adjuvant-
induced arthritis (including joint destruction) in rats.
A NOS inhibitor that has been used experimentally
for many years is L-N(6)-(1-iminoethyl)lysine69,70
(L-NIL; FIG. 4), and this compound has ~30-fold selectiv-
ity for iNOS over the other two isoforms. A PRODRUG of
70
L-NIL has been developed that again exerts beneficial
effects in carageenan and arthritis models and is reported
to decrease exhaled NO in asthmatic humans. Whether
the degree of isoform selectivity achieved will be suffi-
cient remains to be determined. One thing that has
become clear from the substrate-based inhibitor studies is
that achieving selectivity for iNOS over nNOS is more
difficult than achieving selectivity for iNOS over eNOS.
Dimerization inhibitors. An alternative approach is to
block enzyme dimerization71. Pyrimidineimidazole-
based compounds have been described (FIG. 4) that
coordinate the haem in the iNOS monomer, disrupt
one of the helices and displace a crucial arginine-binding
residue from the active site71,72. Essentially, the effect is

a Substrate-based inhibitors

NH NH NH NH
NH2
CO2H CO2H
N N N N N NH2
N CO2H
H H H H
NH2 NH2 H

L-NMMA ADMA L-NIL 1400W

O
F
H N NH NH
N O NH2
S S CO2H
N N
N N N CO2H
H O H
NH2
F NH2

AR-C102222 GW273629 GW274150

b Dimerization inhibitors

R
N N Cl
O

N O N
N
H N
Cl
N O
N
Cl O
X N N
N
Cl Cl

Pyrimidineimidazole-based Clotrimidazole Miconazole

c Cofactor inhibitors d Isothioureas

O
N S S
PRODRUG N HN NH
A pharmacologically inactive N O
NH2 H2N
compound that is converted to
H2N N N
the active form of the drug by
endogenous enzymes or Aminopteridine 1,4-PBITU
metabolism. It is generally
designed to overcome problems
associated with stability, toxicity, Figure 4 | Inhibitors of NOS. a | Substrate-based inhibitors. b | Dimerization inhibitors. c | Cofactor inhibitors. d | Isothioureas.
lack of specificity or limited ADMA, asymmetric dimethylarginine; L-NIL, L-N(6)-(1-iminoethyl)lysine; L-NMMA, NG-monomethyl-L-arginine; 1,4-PBITU,
(oral) bioavailability. S,S′-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea.

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© 2002 Nature Publishing Group
REVIEWS
to block the formation of both the arginine- and the
BH4-binding sites and produce an inhibitor–monomer
complex. These effects are probably similar to those pro-
duced by the antifungal imidazoles clotrimidazole and
miconazole (FIG. 4), although these compounds also pre-
vent calmodulin binding to iNOS73,74. Once again, the
problem is to achieve selectivity for iNOS over nNOS.
The dimerization inhibitors show some specific affinity
for iNOS and nNOS monomers and have weak affinity
for eNOS monomers, but the molecular basis for this
effect is not clear. One possibility is that part of the effect
that is seen in vitro is dependent on the stability of the
dimers, and because eNOS dimers are more stable, some
of the selectivity for certain compounds is apparent
rather than real. This class of compound is also known to
inhibit cytochrome P450 enzymes, which could present a
problem in terms of unwanted effects. Dimerization
inhibitors have been shown to have potentially beneficial
effects in the usual screens of endotoxin-induced nitrate
formation and adjuvant arthritis, and also in experimen-
tal allergic encephalomyelitis as a model of demyelinat-
ing disease. The newer compounds seem to be highly
selective over eNOS and nNOS for time courses longer
than the half-life of dimers, but it remains to be deter-
mined whether the apparent isoform selectivity can be
maintained chronically in vivo.
Cofactor blockers. Inhibitors that affect flavoproteins or
calmodulin binding have been developed, but it seems
unlikely that such compounds will have the necessary
selectivity or specificity to be useful as approaches for
drug discovery. A series of inhibitors has been made
that targets the BH4-binding site75. Differences in the
BH4-binding site between NOS isoforms have been
identified, and antipterin compounds have been devel-
oped that show tenfold selectivity for nNOS over the
other two isoforms75.
Protein–protein interactions. Various endogenous
peptides and proteins that bind to NOS isoforms and
modify activity have been identified. For example, the
89-amino-acid protein termed protein inhibitor of
NOS (PIN) binds to the amino-terminal extension of
nNOS and inhibits activity76. Other inhibitory domains
on eNOS and nNOS have been identified, but whether
these can be targets for selective inhibition remains to be
determined. In vivo delivery of the caveolin-1 scaffold-
ing domain inhibits eNOS activity, showing that, in
principle, this approach works.
nNOS inhibitors. There is a rationale for assuming that
nNOS inhibitors might be of value in stroke, in certain
types of neurodegeneration and as analgesics (partic-
ularly, but not exclusively, in the setting of inflammation).
Although the peripheral effects of nNOS inhibition can
include substantial gastrointestinal and genitourinary
effects (see above), the existence of variants of nNOS
raises the possibility of further tissue specificity. Various
dipeptide amines and peptidomimetic inhibitors of
nNOS have been described, but some interfere with
other arginine pathways77. The compound ARL 17477
946 | DECEMBER 2002 | VOLUME 1
© 2002 Nature Publishing Group
inhibits nNOS and seems to offer protection in an
animal model of cerebral ischaemia78. However, this
compound shows only modest selectivity for nNOS
over iNOS, and so some of the effects might also have
been due to inhibition of iNOS. As more selective
inhibitors of nNOS emerge, the full potential for thera-
peutic and unwanted effects should become clearer.
Vinyl N-iminoethyl-L-ornithine (L-NIO)79 has substan-
tial selectivity for nNOS over iNOS and eNOS (TABLE 4),
and should be useful to define the potential for thera-
peutic manipulation of nNOS, as should inhibitors of
nNOS based on the pterin (BH4) site75.
Transcriptional regulation. Inhibition of transcription of
iNOS by glucocorticoids accounts for some of the actions
of these established medicines80. Whether more selective
inhibition of iNOS transcription can be achieved is not
clear. There is interest in transcriptional upregulation of
eNOS, but this is outside the scope of this article.
Alternative molecular targets
Reducing harmful overproduction of NO clearly has
therapeutic potential. Inhibition of iNOS would seem to
have use as an anti-inflammatory strategy in a wide
range of disease states. Inhibition of nNOS also has
potential in CNS disorders associated with neuro-
degeneration and neurotoxicity, as well as in pain models.
However, inhibition of these isoforms might also
cause harm. Inhibition of iNOS might be expected to
increase susceptibility to infections, inhibit wound heal-
ing and exacerbate tissue damage in certain situations.
Inhibition of nNOS might alter gastrointestinal and
genitourinary function, cause spasm of sphincters and
affect higher cerebral functions. For prolonged use, a high
degree of isoform selectivity is essential to avoid the
unwanted effects of inhibiting eNOS, which include
hypertension, enhanced white-cell and platelet activation,
and increased atherogenesis. It is also clear that complete
inhibition of NOS (even if isoform specific) is probably
undesirable, and that partial inhibition and/or tissue or
cell-specific inhibition might be the ultimate goals.
Interestingly, the first described NOS inhibitor,
L-NMMA, is a naturally occurring amino acid that is
generated endogenously81. Arginine residues on proteins
are methylated by the action of protein arginine
methyltransferases (PRMTs). Three methylarginines
are generated: L-NMMA, asymmetric dimethylarginine
(ADMA) and symmetric dimethylarginine (SDMA)82.
The asymmetrically methylated arginine residues
(ADMA and L-NMMA) inhibit all three isoforms of
NOS, whereas SDMA is not an NOS inhibitor.
Normally, the concentration of ADMA and L-NMMA
seems to be kept low by the action of DDAH83,84, which
catalyses their conversion to citrulline, and dimethyl-
amine or monomethylamine, respectively (FIG. 5). This
pathway represents an endogenous mechanism for the
regulation of NO production by competitive inhibition.
Indeed, pharmacological inhibition of DDAH causes a
rise in ADMA levels sufficient to block NO generation85,
and this raises the possibility of a novel approach to
blocking overproduction of NO.
www.nature.com/reviews/drugdisc
 REVIEWS

CIT
a

R
R
R Me
R R
(Me)2 R

R R
R
Me NO•
Me

Me
(Me)2
Competitive
Proteolysis R (Me)2 inhibition
R
Me
(Me)2 R
R
R

DDAH DDAH b
Me Me Me
Me Me

HN NH HN NH HN N H2N O

NH NH NH NH Me Me
Renal
clearance + NH

H2N COO– H2N COO– H2N COO– H2N COO–

SDMA L-NMMA ADMA Citrulline

Figure 5 | The DDAH/NOS pathway. a | Arginine residues (R) within proteins can be post-translationally methylated by a family of
protein arginine methyltransferases (PRMTs). Proteolysis of arginine-methylated proteins releases free methylarginine into the
cytosol. In mammalian cells, three forms of methylarginine are found: symmetric dimethylarginine (SDMA), NG-monomethyl-L-
arginine (L-NMMA) and asymmetric dimethylarginine (ADMA). Of these, only the asymmetrically methylated forms (L-NMMA and
ADMA) are inhibitors of nitric oxide synthase (NOS). These residues also differ in their routes of clearance; whereas SDMA is cleared
by the kidney without further metabolism, L-NMMA and ADMA are hydrolysed to citrulline (CIT) and methylamines by the action of
dimethylarginine dimethylaminohydrolase (DDAH). Accumulation of asymmetrically methylated arginine residues leads to inhibition
of nitric oxide (NO) synthesis, as the residues compete with arginine for the active site of NOS. NO can inhibit DDAH activity by
S-nitrosation91. b | Crystal structure of DDAH with substrate (Protein Data Bank code 1h70).

Two isoforms of DDAH have been identified84, one
with a predominantly neuronal distribution (DDAH1)
and the other with a more widespread and vascular dis-
tribution (DDAH2). Isoform-selective inhibition of
DDAH might provide a different profile of NOS inhibi-
tion to direct inhibition of NOS isozymes. Clearly, inhi-
bition of DDAH would not provide isoform-selective
inhibition of NOS, as ADMA and L-NMMA block all
three isoforms. It is also unlikely that endogenous
ADMA or L-NMMA would ever increase to levels suffi-
cient to inhibit NOS by more than 30%85. However, inhi-
bition of DDAH might provide tissue-specific partial
inhibition of NOS. It is conceivable that this could be of
value to limit excess production of NO without the dan-
ger inherent in using direct NOS inhibitors of causing
near total inhibition of NO. Specifically, inhibition of
DDAH1 would be expected to be of value in neuro-
degeneration and possibly in pain. Consistent with this,
DDAH1 expression is increased in models of neuronal
injury86, and ADMA levels have been reported to be
reduced in patients with Alzheimer’s disease87. More
generalized inhibition of DDAH might allow L-NMMA
and ADMA levels to rise sufficiently to inhibit partially
the excess NO generation seen in septic shock (or
other types of inflammation), a condition in which
low doses of L-NMMA might be beneficial, whereas
larger doses are detrimental. The ADMA/L-NMMA
binding site of DDAH is sufficiently different to that of
NOSs for a selective inhibitor to be developed88.
The notion of preferentially inhibiting excess NO
generation while leaving physiological NO production
untouched also underlies the approach of targeting
enzymes that allow regeneration of arginine within
cells. Arginosuccinate-synthase activity seems to be
required for high-output NO synthesis, at least in some
situations, and therefore might provide a target for drug
action89. An alternative approach that could emerge is to
target individual proteins on which NO acts to inhibit
specific adverse effects of NO while leaving other NO
signalling pathways intact.

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© 2002 Nature Publishing Group
REVIEWS

Into the clinic?
The identification of NO as a biological mediator has
led to vast numbers of publications — in the order of
3,000 per year at present. There can be little doubt that
NOS activity is of considerable importance physiologi-
cally, or that overproduction of NO or increased NOS
activity has the potential to cause harm. However, part
of the problem in turning scientific discovery into useful
therapeutics has been the sheer range of processes in
which NO has been implicated, and the opposing effects
of NO even within a single disease. A good example is
provided in the field of cancer biology. High-output NO
generation from macrophages or from tumour cells
themselves can be tumoricidal and prevent metastasis.
However, iNOS is expressed constitutively in some
tumour cells, where it promotes tumour growth, neo-
vascularization and invasiveness through induction of
mutations to the tumour-suppressor gene TP53 and
upregulation of VEGF expression28. Furthermore, in
other tumours, it is eNOS expression that correlates
with tumour malignancy and vascularity90. How can
selective benefit be gained without a welter of unwanted
effects, and which is the single therapeutic target at
which the pharmaceutical industry should aim?
The field of NO therapeutics is now entering a cru-
cial and exciting stage. Selective inhibitors of iNOS have
been identified and will enter clinical trials shortly.
Companies are considering, or have considered, asthma,
arthritis, inflammatory bowel disease, inflammatory
skin diseases and other indications, and it will be inter-
esting to see which are chosen. Selective inhibitors of
nNOS are also in development, and will help to answer
important questions about potential therapeutic appli-
cations of nNOS blockade. In summary, the current
position seems to be as follows. First, partial inhibition
of NO generation is probably preferable to complete
inhibition, whichever isoform is targeted. Second, the
desired and undesired effects of selective iNOS inhibi-
tion might change with time depending on the stage of
inflammation — initiation, chronic or resolving. Third,
the range of effects (desired and undesired) of iNOS or
nNOS inhibitors is likely to be broad, and the idea that
iNOS inhibitors will be ‘glucocorticoids without the side
effects’ seems misplaced. Fourth, the full range of dis-
eases for which NOS inhibition might be beneficial is
unlikely to become clear until, and unless, one or more
compounds have been in clinical use for some years and
clinical researchers have had a chance to explore their
therapeutic effect. And finally, novel approaches to
achieving isoform, cell or tissue selectivity of NOS inhi-
bition might lead to more specific therapies.
Note added in proof
Long-term treatment with N ω-nitro-L-arginine methyl
ester enhances atherosclerosis in eNos knockout mice,
indicating that some substrate-based NOS inhibitors
might have further detrimental effects independent of
eNOS inhibition123.

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© 2002 Nature Publishing Group
Acknowledgements
Work in the laboratory of P. V. is generously supported by grants
from the British Heart Foundation, the Wellcome Trust and the
Medical Research Council. The authors are grateful to R. Knowles,
N. Boughton-Smith, and C. S. Raman for information provided,
and to A. Hobbs for critical reading of the manuscript. Figure 3 was
prepared by S. Rossiter. From the work of the authors, University
College London (UCL) holds patents in relation to DDAH as a target
for drug action.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
aconitase | ApoE | arginase | arginine deiminases | arginine glycine
amidinotransferase | argininosuccinate synthase | calmodulin |
caveolin-1 | DDAH | diamine oxidase | dystrophin | eNOS | eNos |
iNOS | iNos | nNOS | nNos | PIN | soluble guanylyl cyclase |
thioredoxin | TP53 | VEGF
OMIM: http://www.ncbi.nlm.nih.gov/Omim/
Alzheimer’s disease | Celiac disease | multiple sclerosis |
Parkinson’s disease
FURTHER INFORMATION
Encyclopedia of Life Sciences: http://www.els.net/
nitric oxide: role in human disease | nitric oxide: synthesis and action
Protein Data Bank: http://www.rcsb.org/pdb/
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