tmpB3D2 TMP

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Antioxidants & Redox Signaling

Heme oxygenase-1 as a target for drug discovery (doi: 10.1089/ars.2013.5658)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Forum Review Article
Heme oxygenase-1 as a target for drug discovery

Roberto Motterlini1,2 and Roberta Foresti1,2
Universit Paris-Est, UMR_S955, UPEC, F-94000, Crteil, France

2
INSERM U955, Equipe 3, F-94000, Creteil, France
Corresponding authors:
Roberto Motterlini and Roberta Foresti
INSERM U955, Faculty of Medicine, University of Paris Est Creteil
94000 Creteil, France
Phone: +33-49813637
Email: roberto.motterlini@inserm.fr and roberta.foresti@inserm.fr
Running head: HO-1 in drug discovery

Word count (excluding references, tables and figure legends): 7965
Reference numbers: 151
Number of grey illustrations: 8
Number of color illustrations: 0
1 R. Motterlini and R. Foresti
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Abstract
Significance: Heme oxygenase enzymes, which exist as constitutive (HO-2) and
inducible (HO-1) isoforms, degrade heme to carbon monoxide (CO) and the bile
pigment biliverdin. In the last two decades substantial scientific evidence has been
collected on the function of HO-1 in cell homeostasis emphasizing these two
important features: 1) HO-1 is a fundamental sensor of cellular stress and directly
contributes to limit or prevent tissue damage; 2) the products of HO-1 activity
dynamically participate in cellular adaptation to stress and are inherently involved in
the mechanisms of defence.
Recent advances: On the bases of its promising cytoprotective features, scientists
have pursued the targeting of HO-1 as an attractive cellular pathway for drug
discovery. Three different pharmacological approaches are currently being
investigated in relation to HO-1, namely the use of CO gas, the development of COreleasing molecules (CO-RMs) and small molecules possessing the ability to upregulate HO-1 in cells and tissues.
Critical issue: Studies on the regulation and amplification of the HO-1/CO pathway
by selective pharmacological approaches may lead to the discovery of novel drugs
for the treatment of a variety of diseases.
Future directions: In this review we will discuss in detail the importance of
pharmacologically manipulating the HO-1 pathway and its products for conferring
protection against a variety of conditions characterized by oxidative stress and
inflammation. We will also evaluate each of the strategic approach being developed
by considering the intrinsic advantages and disadvantages, which may have
implications for their use as therapeutics in specific pathological conditions.
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Introduction
The heme oxygenase pathway is an essential cellular defence system that has been
maintained throughout evolution, being present in algae and progressing in time to
mammalian organisms (143). Its role as an effective antioxidant is quite particular,
since it does not, like the classic antioxidant enzymes catalase or superoxide
dismutase, transform a toxic oxidant (hydrogen peroxide or superoxide anion) into a
molecule harmless for cells. In fact, heme oxygenase exerts its antioxidant function
by removing a pro-oxidant molecule, heme, while simultaneously producing
metabolites that are endowed with unique protective characteristics, i.e. carbon
monoxide (CO) and biliverdin, which is further converted to bilirubin by biliverdin
reductase (BVR) (75, 115, 124) (Note: since biliverdin does not accumulate in
mammalian tissues and is rapidly converted to bilirubin by BVR, throughout the text
we will refer to CO and bilirubin as final products of HO-1). Iron is also released
during heme degradation by heme oxygenase and its increased intracellular levels
lead to up-regulation of ferritin, an iron-storing protein that participates to the
cytoprotective machinery engaged by heme oxygenase to combat stress conditions
(10, 11). Due to the high relevance of heme oxygenase-derived products and their
capacity to modulate many fundamental cellular functions, it is perhaps too restrictive
to label this enzyme as an antioxidant; it is probably more appropriate to consider it
as a regulator of homeostasis. This is exceptionally true for heme oxygenase-1 (HO1), the inducible isoform of this enzyme, which serves a dual purpose as
sensor/effector by sensing cellular stress (oxidative, nitrosative, inflammatory and
metabolic) and damage and efficiently attempting to rescue tissue viability and
functions (35, 87, 103, 132, 137). These properties might also be linked to a recently
identified truncated form of HO-1, which can translocate into the nucleus and activate
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4
oxidant-responsive transcription factors (72). The obligatory role this pathway plays in
the preservation of tissue integrity is crucially exemplified in human and murine HO-1
deficiency, which both exhibit increased oxidative stress, persistent vascular injury
and chronic inflammation (108, 146).
How do heme oxygenase-derived products contribute to this protection? The
work accumulated so far by an increasing number of groups worldwide shows that
CO
possesses
vasorelaxing
properties,
prevents
systemic
and
pulmonary
hypertension and displays remarkable anti-inflammatory and anti-bacterial effects (35,

43, 86, 116). For example, important findings from different pre-clinical experimental
models of disease have demonstrated that administration of CO gas at doses that
are well tolerated in animals alleviates inflammatory processes and vascular
disorders and protect tissues against ischemic injury (38, 95, 114, 121, 151). These
unexpected and promising results triggered the idea of utilizing CO as a therapeutic
expedient (114) notwithstanding the notion that inhalation of CO gas and the delivery
of precise amounts of CO gas to living organisms is not an easy task and caution
needs to be taken to avoid undesired toxic effects (41, 46). CO gas is now being
investigated for its therapeutic properties and clinical trials are ongoing to evaluate
whether CO inhalation is beneficial against pulmonary hypertension, organ
transplantation and other pathologies (see below). In the context of CO therapy we
have made the first step into a true translational approach by pioneering the concept
of carbon monoxide-releasing molecules (CO-RMs) and thus synthesizing and
characterizing a novel class of compounds for the controlled delivery of this gas (4, 5,
23, 84, 89, 91, 92). Thus, both CO gas and CO-RMs can be considered as two
parallel pharmacological approaches to deliver CO for therapeutic purposes with the
aim to amplify the action of HO-1 (see Figure 1). The CO-RMs technology is based
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5
on transition metal carbonyls, which can transfer small amounts of CO to cells and
tissues. These compounds have been now extensively used in vitro cell culture
systems to provide evidence that CO regulates a variety of processes ranging from
gene expression, angiogenesis, inflammation, apoptosis and cell survival (47, 62, 88,
138). Their potential therapeutic features have been also confirmed in vivo in rodents
and pigs where the molecules showed efficacy against inflammatory conditions such
as sepsis and rheumatoid arthritis (37, 49, 67, 133, 134) and protected organs from
reperfusion injury (8, 23, 118), bacterial infection (20, 30) and arterial thrombosis (18).
In recent years, CO-RMs have also become a unique tool used by scientists to study
the role of CO in biology (91). Notably, Sigma Aldrich has now started to
commercialize these compounds, corroborating the interest of this innovative
technology in different fields of research.
The other products of HO-1, biliverdin and bilirubin, possess remarkable
antioxidant properties participating in protection of cells and tissues against oxidative
stress (21, 22, 42, 125). In a seminal work by Stocker and colleagues these linear
tetrapyrroles were originally demonstrated to neutralize oxidation of membranes in
vitro with a capacity higher than vitamin E, which is regarded as the best antioxidant
against lipid peroxidation (125). Our group then confirmed these findings by
demonstrating that bilirubin protects vascular smooth muscle cells and cardiac tissue
against oxidative stress and ischemia-reperfusion injury (21, 22, 42). Bile pigments
have also been shown lately to protect against vascular injury and inflammation in
animal disease models of vasculopathy, thrombosis and allograft rejection (65, 101,
132). From a therapeutic perspective, the beneficial properties of bilirubin are
underscored by studies in human subjects reporting a lower prevalence of vascular
complications in diabetic patients with Gilbert syndrome, a condition characterized by
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6
hyperbilirubinemia (59). Notably, these subjects displayed decreased levels of
glycated hemoglobin, reduced markers of oxidative stress and inflammation, and
improved lipid profiles (59). Furthermore, decreased serum bilirubin linked with
polymorphism in the promoter region of the HMOX1 gene has also been associated
with susceptibility to coronary artery disease in patients with type 2 diabetes (19).
Most recently, a large cohort study has confirmed serum bilirubin levels as an
independent risk factor for cardiovascular disease and death in both men and
women (58), showing that patients with bilirubin levels of 5 mol/L (0.3 mg/dL) have a
higher risk of any cardiovascular event, myocardial infarction and death resulting
from any cause. As in the case of CO, the protective mechanism(s) of biliverdin and
bilirubin needs to be further elucidated. However, one great advantage is that these
metabolites are endogenously produced; that is, they are generated by heme
oxygenase in proximity of cellular components where oxidation and damage take
place, thus potentially being more effective in cellular protection compared to
classical antioxidants, which are exogenously introduced with the diet. Therefore, in
parallel to the delivery of CO, an effective pharmacological approach would be to
enhance the endogenous production of HO-1 metabolites (i.e. CO and biliverdin and
consequent formation of bilirubin) based on the design and synthesis of novel
pharmaceuticals acting as either potent inducers of HO-1 protein expression or derepressors of the transcription factor(s) that block HO-1 transcription (see Figure 1).
There exists a growing number of molecules, such as plant-derived polyphenols but
also pharmaceutical compounds like probucol, that possess the ability to up-regulate
HO-1 in tissues (29, 71). These findings are interesting because they highlight that, in
addition to being modulated endogenously in response to stress, HO-1 protein
expression
can be manipulated
by exogenously applied substances thus
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7
representing a pharmacologically-responsive target. Moreover, the existence of such
compounds provides us with a variety of templates and chemical scaffolds that can
be explored for the design of novel drug-like molecules. Modulation of HO-1 can also
be achieved via gene therapy using adenoviral vectors and genetic techniques with
research laboratories having extensively studied these technologies with important
results (readers are referred to the following articles focussing on this aspect (1, 64,
144)). Since this topic is outside our expertise and would require a comprehensive
and detailed overview by itself, here we will review the emerging pharmacological
approaches to exploit the HO-1 pathway as a drug target and evaluate them as
potential therapeutics affording beneficial action in clinical setting of diseases with
underlying oxidative stress and inflammation.
Delivery of CO gas by inhalation
1. Background. The proposition of CO as a possible therapeutic agent

progressively originated in the 90s from studies conducted in different laboratories
on cells and tissues treated with small quantities of CO gas to mimic the effect of
endogenously produced CO by heme oxygenase enzymes. The most representative
of these studies is perhaps the one conducted by Suematsu and colleagues showing
that in isolated perfused livers, submicromolar levels of CO were detectable in the
effluent and that CO production was blocked by zinc protoporphyrin IX, an inhibitor of
heme oxygenase activity. Most importantly, the inhibitor of heme oxygenase elicited
an increase in hepatic perfusion pressure under constant flow conditions, an effect
that was reversed by addition of exogenous CO gas at concentrations as low as 1
M (126). It was then at the beginning of this century that Otterbein and co-workers
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8
made the first attempt to deliver CO gas by inhalation in whole animals to assess its
potential beneficial effects. In two seminal papers they reported that: 1) rats exposed
to low concentrations of CO gas (50-500 ppm) for 72 h exhibited high tolerance to
lethal hyperoxia revealing a significant reduction in lung injury, pulmonary edema and
parenchymal inflammation (16); 2) mice inhaling CO gas for 1 h (250 ppm) prior to
challenge with lipopolysaccharide exhibited a decreased production of proinflammatory cytokines alongside up-regulation of anti-inflammatory mediators.
These unprecedented findings led the way to a series of studies conducted in
different laboratories confirming that non-lethal doses of CO gas delivered by
inhalation can provide therapeutic effects in pre-clinical in vivo models of disease,
the most relevant being summarized as follows: 1) pre-exposure of mice and rats to
250 ppm CO for 1 hour prevents atherosclerotic lesions following carotid balloon
injury, an effect associated with profound inhibition of leukocyte infiltration and
smooth muscle cell proliferation (104) ; 2) inhalation of CO (250 ppm) for 2 h in pigs
protects hearts during reperfusion after cardiopulmonary bypass (69) and ameliorates
hyperacute endotoxic shock (80); 3) in a murine model of chronic colitis, prolonged
exposure to CO (250 ppm) from 8 to 12 weeks resulted in a marked attenuation of
the inflammatory response and tissue damage (52) ; 4) brief daily exposure to CO
gas (250 ppm) for 3 weeks reverses pulmonary arterial hypertension and right
ventricular hypertrophy in mice (151); 5) in an experimental model of cerebral malaria
in mice CO gas inhalation prevented blood-brain barrier disruption, brain
microvasculature congestion and neuroinflammation (105). These and additional set
of data confirm that inhalation of CO gas could be used as a feasible
pharmacological approach to prevent or mitigate pathological disorders characterized
primarily, albeit not exclusively, by vascular dysfunction and inflammation.
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2. CO gas as a therapeutic: advantages and disadvantages. From a mere

pharmaceutical point of view, the advantages of using CO inhalation as a therapeutic
strategy are evident since this gas is rather inexpensive, it can be easily obtained
pure in large quantities and most importantly this approach does not require further
formulation developments as the gas itself is the active principle. Less obvious are
the way to convey CO effectively and specifically to cellular targets without causing
impairment of tissue function and metabolism in vivo, an issue essentially related to
the potential toxicological effects of CO gas on oxygen transport and cellular
respiration (106). The readers should refer to articles previously published (41, 46,
106)
and
to
the
Agency
for
Toxic
Substances
and
(http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=1145&tid=253)
Disease
for
more
Registry
detailed
information on the toxicological profile of CO. Indeed, once inhaled through the lung
into the alveolar compartment, CO rapidly dissolves in plasma and diffuses to red
blood cells where it starts to compete with molecular O2 for the binding to hemoglobin.
Since the binding affinity of ferrous iron (Fe2+) in hemoglobin for CO is much higher
compared to O2 (220-fold), the consequent formation of carbon monoxy hemoglobin
(HbCO) would decrease the O2-carrying capacity of red blood cells and thus,
depending on the amount of CO inhaled, progressively compromise O2 delivery to
tissues (46). Some of the CO dissolved in plasma can also rapidly diffuse to and
penetrate the tissues where it has an important impact on cell metabolism. In fact, at
subcellular levels CO gas is a potent inhibitor of mitochondrial respiration having the
ability to compete with O2 in its binding to cytochrome c oxidase (complex IV), a
highly conserved hemoprotein that couples the reduction of O2 to water with the
production of energy by ATP synthase (complex V) (73). It is clear that both the
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10
amount and time of exposure to CO gas (or CO-RMs, see below) will finally
determine the balance between a therapeutic action and negative effects on O2
transport and cellular respiration. In this context, mitochondria could be seen as the
arbiter of this dual effect since convincing data on controlled delivery of CO to cells or
animals (either via CO gas or CO-RMs) have shown an unexpected increase in
mitochondrial energetic and biogenesis (6, 67, 68, 127, 128) (see Figure 2). The
promising pre-clinical data obtained with the use of CO inhalation reveal that
beneficial effects against a variety of diseases can be achieved by short exposure to
the gas providing that HbCO levels do not increase above a critical threshold (1015%) which may cause cellular apoptosis in the brain (24, 102). From a clinical
perspective, trials on the safety and tolerability of CO gas in kidney transplant
patients have been initiated but recently suspended and no results are available at
present (www.clinicaltrials.gov ).
Similarly, clinical trials on CO gas therapy are
currently planned in patients suffering from pulmonary arterial hypertension,

idiopathic pulmonary fibrosis, intestinal paralysis after colon surgery and sickle cell
disease. It is interesting to note that in all these studies the treatment of patients with
CO gas inhalation is either brief or intermittent as the FDA has set thresholds for
blood HbCO levels at 12-14%. However, relying on the HbCO levels in blood as the
sole biomarker of CO efficacy/toxicity without defining the distribution of CO in tissues
at a given time may pose some restrictions on the use of this approach in humans
(46). Real time measurements of intracellular CO/O2 ratio coupled with mitochondrial
function and bioenergetic in vivo are at present difficult to achieve due to technical
limitations but the development of technologies capable of determining these
parameters will be crucial for assessing the safety doses of CO gas in humans. In
this respect, the optimization of probes that specifically detect the distribution of CO
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11
in cellular compartments will also be advantageous (see section below). Another
important consideration on the clinical side is that the use of CO gas by inhalation
may also be hampered due to the fact that not all individuals respond equally to CO
gas. This is indirectly supported by intriguing case reports on CO intoxication in
humans demonstrating that in spite of equal blood levels of HbCO in subjects
exposed by accident to the same amount of CO gas, the individual symptoms may
totally diverge (48). Thus, so far the studies on the beneficial effects of CO gas
delivery in animal models of disease are of great value as they teach us on the
unique pharmacological properties of this small molecule against oxidative stress and
inflammation. Conversely, whether through this approach a safe and therapeutically
effective threshold of CO can be reached locally in organs and tissues without
impairing energy production and cell metabolism still remains to be determined.
3. Alternative applications of CO gas for therapeutic purposes. Because of

the considerable concerns to administer CO gas in the whole organism by inhalation,
studies on ex vivo treatments have been reported with interesting results. Notably, a
promising application is the preservation of organs for transplantation. The common
procedure for maintaining hearts, kidneys and livers collected from cadaveric donors
viable prior to transplantation consists of preserving the organs in cold storage
solutions. These techniques involve intravascular flushing of the isolated organ using
a hypothermic solution followed by storage at low temperatures for the time required
to transfer the graft to the surgery unit. These solutions can indeed limit but not
completely avoid tissue injury and graft dysfunction in transplanted patients. It has
been shown that flushing the organs with CO gas saturated cold solution can
dramatically improve their function at reperfusion after transplantation. For instance,
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in a model of renal ischemiareperfusion injury following kidney transplantation,
grafts preserved in solutions equilibrated with CO gas had less oxidant-mediated
injury, a reduced production of inflammatory cytokines and improved recipient
survival rate (94). Similarly, intestinal grafts kept in cold storage solutions bubbled
with 5% CO gas displayed significantly less tissue damage at reperfusion occurring
after transplantation, an effect associated with improved intestinal barrier function,
less mucosal denudation and reduced levels of inflammatory mediators (96). Thus,
CO gas can function as an effective adjuvant for preserving tissues and organs ex
vivo and maintaining their viability once transplanted in vivo. This concept is also
supported by data showing that organs such as kidney and liver display an improved
function following their preservation in cold solutions supplemented with CO-RMs
(see below and Figure 3). In line with this promising strategy, one can envision that
CO gas saturated solutions (or solutions containing CO-RMs) could also be applied
locally for topical treatment of injured tissues.
Carbon monoxide-releasing molecules (CO-RMs)
1. Background. The proposition of using CO gas inhalation to prevent or

ameliorate various pathological conditions characterized by oxidative stress and
inflammation coincided with the identification and implementation of CO-releasing
molecules (CO-RMs) (84). Originally, this technology was developed by taking
advantage of the intrinsic chemical properties of transition metal carbonyls, a class
of compounds formed by complexation of transition metals with CO ligands. Although
these chemicals had been essentially used for more than a century as catalysts in
several industrial applications, the presence of a CO group which can dissociate from
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13
the metal under certain conditions suggested to us that they may also function as
carriers of CO in biological systems (54). This was confirmed by our initial studies
showing that addition of the dimethyl sulfoxide-soluble tricarbonyldichloro ruthenium
dimer ([Ru(CO)3Cl2]2) to an aqueous solution containing deoxymyoglobin resulted in
the rapid formation of carboxymyoglobin (MbCO) (84). The CO-mediated
pharmacological effects of [Ru(CO)3Cl2]2, which we subsequently named CORM-2
(90) (see chemical structures and properties in Figure 4), was then corroborated by
showing that this compound promoted dilatation of isolated blood vessels, prevented
coronary vasoconstriction in the ischemic myocardium and reduced systemic acute
hypertension in vivo, thus mimicking the known effects of endogenously generated
CO by heme oxygenase (84). Following these unprecedented findings on the
pharmacological actions of metal carbonyl complexes, the last decade has witnessed
the interesting development of this novel class of molecules both from a chemical
and pharmacological viewpoint. Indeed, different chemical modifications have been
introduced into the CO-RMs scaffold to achieve the following: 1) water-soluble
compounds,
with
CORM-3
(Ru(CO)3-glycinate)
and
CORM-A1
(sodium
boranocarbonates) being the best characterized and mostly used molecules (23, 61,
92) (see chemical structures and properties in Figure 4); 2) different rates of CO
release in vitro which parallel their pharmacologically activities ex-vivo and in vivo (23,
92); 3) a release of CO elicited by diverse physico-chemical stimuli such as light
activation, changes in pH, enzymatic and redox reactions (26, 84, 90, 92, 98, 112); 4)
improved biological compatibility by using metals that are found in human proteins
(i.e. Fe, Mn, Mo) (26, 36, 56, 119, 149) and by coordinating transition metal carbonyls
to physiologically relevant molecules such as vitamin B12 (150). All these chemical
modifications were implemented with the prediction that they will improve the
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14
pharmacological, cytotoxicity and bioavailability profiles of metal-CO-RMs. For
instance, we have recently reported that iron carbonyl-based CO-RMs soluble in
dimethyl sulfoxide exhibit vasodilation and reduce inflammation in vitro but caused a
pronounced toxic effect both in vascular tissue and inflammatory cells. However, cells
and tissues maintained their viability and function when iron-CORMs were rendered
water-soluble (93). On the pharmacological side, the last decade on the research of
CO-RMs revealed that these compounds exhibit anti-ischemic effects in different
organs (17, 23, 118, 122), anti-inflammatory activities in systemic and localized
inflammation (5, 14, 28, 55, 67, 82, 135, 145) and antibacterial actions both in vitro
and in vivo (27, 30, 31, 99). More details on the pharmacological and mechanistic
actions of CO-RMs in vitro and in vivo can be found in recent reviews published by
the authors (88, 91). Here we highlight the most salient studies conducted in different
laboratories on the beneficial effects of the water-soluble CORM-3 in pathological
conditions, which can be summarized as follows: 1) in mice models of myocardial
infarction, ischemia reperfusion following cardiac transplantation or heart failure,
administration of CORM-3 (10-40 mg/kg body weight/day) provides significant
protection against tissue damage and infarct size with consequent amelioration of
cardiac function; (23, 50, 138); 2) CORM-3 administered intraperitoneally (10
mg/kg/day) decreases the inflammatory response and protects against the
degradation of cartilage and bone in arthritic mice (37, 74); 3) intestinal muscularis
inflammation and oxidative stress in response to the development of postoperative
ileus is markedly reduced in animals pre-treated with CORM-3 (40 mg/kg) (28) ; 4) a
single injection of CORM-3 (7.5 mg/kg) decreased bacterial counts in the spleen and
increased survival in immunocompetent and immunosuppressed mice following
Pseudomonas aeruginosa bacteremia (30); 5) administration of CORM-3 (10 mg/kg)
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improves cardiac dysfunction and survival in peritonitis-induced sepsis in mice (67); 6)
decreased inflammation and brain injury are achieved when CORM-3 (4 or 8 mg/kg)
are administered either before or 3 days after in vivo intracerebral hemorrhage (145);
CORM-3 (10 mg/kg twice a day) reduces neuropathic pain and microglia activation in
a model of sciatic nerve injury in mice (55).
2. Importance of metals in delivering CO. At this stage it is clear that CORMs are small active substances that have the potential to be translated into
pharmaceutical agents for the therapeutic delivery of CO. However, since metal
carbonyls and boranocarbonates diverge from classical organic drug-like molecules
that are designed to target specifically a protein or receptor, several aspects related
to the chemical structure and reactivity of CO-RMs as well as their pharmacodynamic
still need to be explored before their effective use as drugs can be materialized. In
general, small active compounds containing transition metals are atypical and
regarded with scepticism in the pharmaceutical field (although some exceptions exist,
i.e. cisplatin) because metal complexes may trigger undesired cytotoxic reactions
within the biological milieu. However, the fact that different CO-RMs evaluated in
models of disease provide significant protection when used at appropriate doses
disagrees with this negative perception and suggests instead that transition metalsbased compounds may open the way to developing a novel class of therapeutic
agents. In fact, circumstantial evidence from the research on CO-RMs reveals that
the metal appears to facilitate and cooperate with CO to exert pharmacological
actions. For example, in mitochondria isolated from kidney and heart, an increased
oxygen consumption in the absence of ADP (uncoupling effect) is observed only after
addition of CORM-2 or CORM-3, which both contain ruthenium, but not with CORM-
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A1, which lacks a transition metal and spontaneously liberates CO (73, 117).
Similarly, transition metal carbonyls containing either ruthenium or manganese
(CORM-371) are more potent anti-bacterial agents than CORM-A1 (31); in addition,
nitrite production by macrophages challenged with lipopolysaccharide is reduced in a
concentration-dependent manner by metal-based CO-RMs but not CORM-A1
(Foresti R and Motterlini R, unpublished observations). This diverse action may relate
to the unique ability of transition metal carbonyls to effectively deliver CO to
intracellular targets, as demonstrated in two recent investigations using novel
fluorescent probes capable of detecting CO with high selectivity both in aqueous
solutions and in living cells (81, 139). Specifically, in these studies the authors
independently showed that the fluorescence intensity was much stronger in cells in
vitro treated with either CORM-2 or CORM-3 compared to CO gas suggesting that
CO is more easily delivered to cells using a metal carbonyl complex than CO gas in
solution. The same authors reported that, unlike CO gas, a very significant
fluorescent response was obtained with CO-RMs at concentrations as low as 1 M.
3. Liberation and delivery of CO from CO-RMs. Although studies in vitro

give us important information on the ability of different CO-RMs to liberate CO, very
little is known about their behaviour in vivo. The possibility that metal-based CO-RMs
partially enter the cells cannot be excluded a priori and might explain the observation
reported above. For instance, there is evidence in bacteria that exposure to either
CORM-3 or tetraethylammonium molybdenum pentacarbonyl bromide leads to rapid
endogenous accumulation of their respective transition metals (ruthenium and
molybdenum) (27, 99). These data, however, cannot unequivocally establish whether
CO is released from CO-RMs prior to entering the cells or whether the entire CO-
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RMs molecules are taken up by cells and then liberate CO intracellularly. Another
intriguing possibility is that CO can be transferred directly from one metal to another
(transcarbonylation?) within the biological milieu, although this type of reaction is
chemically unlikely. Irrespective of the mechanim(s) involved, the apparent increased
efficiency of CO-RMs to transfer CO to tissues have also important implications for
limiting the potential toxicity of CO gas in vivo, which as reported in the section above
is ultimately associated with impairment of red blood cell oxygen transport and
delivery. So far no comprehensive studies have been published on the distribution of
CO-RMs in organs following systemic administration of these compounds, but data
on the changes in blood carbonmonoxy hemoglobin (HbCO) levels are rather
instructive. For instance, it has been reported by different groups that while the
percentage of HbCO increases in animals following inhalation of therapeutic doses of
CO gas (102) or after systemic administration of CORM-A1 (113), no changes in
HbCO levels are detected in the case of CORM-3 (28, 50). The fact that CORM-3,
despite being able to cause CO-mediated vasodilatation and hypotension, does not
significantly affect the oxygen carrying capacity of hemoglobin is puzzling and raises
important questions on the mechanism(s) of CO liberation and delivery from metalcontaining CO-RMs in vivo. It is tempting to speculate that once in the circulation the
ruthenium in CORM-3 enables CO to be channelled more directly to tissues either by
releasing more CO locally, favouring its transfer to the putative intracellular target(s)
or both. How this is achieved is unclear at present but experiments utilizing the
recently discovered CO-sensitive fluorescent probes may help us to elucidate this
mechanism (81, 139). It is intriguing that hemoglobin-based oxygen carriers have
been recently proposed as CO-delivery agents; specifically, a type of pegylated
hemoglobin bound to CO (CO-MP4) has been shown to deliver CO into the
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18
circulation and reduce myocardial ischemia/reperfusion injury in rats (136). This data
further support the concept that the undesired effects of CO gas in compromising
oxygen transport by red blood cells and cellular respiration can be better controlled
and mitigated by having CO tightly bound to a transition metal (46). Indeed, from a
mere organometallo chemical perspective, the ferrous (Fe2+)-CO moiety within
hemoglobin is by definition an iron metal carbonyl and thus analogous to the
pharmacologically active metal-containing CO-RMs described so far in the literature
(46, 91).
4. Specificity of CO release and desirable properties for the clinic. In

addition to their efficacy in carrying and delivering CO to tissues, the ideal CO-RM
should release CO with temporal and spatial specificity. Prototypes of CO carriers
possessing such characteristics are CORM-A1, CORM-401, photoinducible CO-RMs
and enzyme-triggered CO-RMs (26, 92, 98, 112) (see Figure 4 and Figure 5 for
chemical structures and properties). CORM-A1 is a water-soluble boranocarbonate
that liberates CO in a pH-dependent manner. At physiological pH, the half-life of
CORM-A1 is in the order of 20 min but the release of CO is markedly augmented by
decreasing the pH to acidic conditions (half-life at pH 5.5 is 2.5 min) (92). Although
CORM-A1 elicits dilatation of blood vessels ex vivo and hypotension in vivo (92), the
pharmacological actions of this compound have not been explored in biological
environments (gastric mucosa) or pathological conditions (rheumatoid arthritis, lactic
acidosis) typified by low pH and where the pH-dependent release of CO could be
aptly exploited. CORM-401 is a recently described water-soluble manganese
tetracarbonyl complex (26). This CO-RM was designed to contain manganese, a
metal present in the human body, and was found to liberate at least 3 moles of CO
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19
with a rate comparable to CORM-A1. This is the first example of a compound
releasing multiple CO groups, a property reflected by increased aortic vasodilatation
when compared to CORM-A1, which releases only 1 mole of CO (Motterlini R. and
Foresti R., unpublished observations). Importantly, we recently found that the rate of
CO release from CORM-401 in vitro is accelerated in the presence of biologically
relevant oxidants such as hydrogen peroxide and hypochlorous acid (Motterlini R.
and Foresti R., unpublished observations). Future experiments will determine
whether CORM-401 is more efficacious in vivo, particularly in pathological conditions
characterized by oxidative stress. Another therapeutically interesting alternative is the
use of photoactivable CO-RMs which can be triggered to release CO by light.
Examples can be found in the literature on stable manganese, tungsten and rhenium
carbonyl complexes which can be incorporated by cells in vitro and release CO upon
irradiation (34, 98, 107, 111). Thus, one can envision administering one of these
molecules in the dark and stimulate with light a local enrichment of CO in a specific
area of the body where a pharmacological action of this gas is needed. Concerning
the possibility of triggering CO release from CO-RMs by enzymatic reaction, recent
studies have described the ability of acyloxydiene-Fe(CO)3 complexes to deliver CO
intracellularly via esterase mediated hydrolysis (112). This approach is in the early
stage of characterization and studies are awaited to demonstrate its feasibility in vivo.
As in the case of photoactivation, this strategy offers us another way of controlling the
delivery of CO although a possible limitation is that esterase is a ubiquitous enzyme
being present in blood and tissues and thus one may expect an uncontrolled CO
release once these molecules are used in vivo. Rendering the CO release from these
CO-RMs dependent on tissue specific enzymes or enzymes highly expressed only in
pathological conditions might be worth considering.
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The CO-RMs described above were chosen to highlight interesting characteristics
that could take the findings of basic chemistry and biology closer to translational and
practical applications. From the elaboration above it is clear that CO-RMs represent a
new class of chemical entities which pharmacological properties can be optimized
only by using approaches that differ from the classical way of designing and
implementing pharmaceutical drugs.
CO-RMs possess a great flexibility to be
modified and adapted to different needs and more in depth studies on the chemical
reactivity of these compounds in vivo will provide crucial information for maximizing
their therapeutic potential.
HO-1 inducers
1. Background. Although it is widely recognized that oxidative stress is
implicated in the pathophysiology of several chronic diseases, therapies based on
antioxidants have consistently failed to provide benefit for cardiovascular and other
conditions (66, 123). The reasons for this negative outcome are unknown at present
and are in contrast to the proven antioxidant activities of vitamins such as vitamin E
or C in vitro (110). The involvement of oxidants and reactive oxygen species (ROS) in
the initiation and progression of disease is nevertheless real and their actual role in
modulating pathologies may originate from the multiple cellular sources responsible
for ROS production and the different oxidant species produced during stress stimuli
(39). The limited knowledge we still have of the delicate balance between oxidative
stress, constitutive and inducible antioxidant systems may partially explain the
difficulty in finding an optimal protocol for the implementation of exogenous
antioxidant therapies. An alternative approach to circumvent this problem would be to
rely on the endogenous antioxidant/protective proteome that cells and tissues
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normally employ to fight the daily insults derived either from the environment,
infection, inflammation or other stimuli. More specifically, the inducible protective
proteome might be the preferential target for a potential pharmacological strategy,
mimicking in this respect the beneficial effect that the pre-conditioning phenomenon
affords in protecting organs against injury. Aiming to control HO-1 protein expression
could constitute a promising start to achieve this goal.
2. The Nrf2/HO-1 axis and beyond. The relationship between the

transcription factor Nrf2 and induction of HO-1 is well proven and is dependent on the
presence of antioxidant response elements in the promoter of the HMOX1 gene (3,
13). Nrf2 is now known as the master regulator of cellular antioxidant defense
systems because it controls, in addition to HO-1, the expression of a battery of
detoxification enzymes, such as NAD(P)H dehydrogenase quinone 1, glutathione Stransferases and peroxiredoxins (9, 32, 60) (see Figure 1). Thus, the protection
exerted by Nrf2 is reliant on these genes and their silencing reverses to a significant
extent the beneficial activities of Nrf2 activation (109). It is not surprising then that
many of the HO-1 inducers that have been described by different authors over the
last decade seem to involve Nrf2 as the upstream factor stimulating this response.
Nrf2 is indeed now the focus for the development of novel treatments for therapeutic
purposes, with specific molecules, such as small synthetic triterpenoids and
dimethylfumarate (7, 131), and broccoli sprout extract rich in sulforaphane, a wellknown Nrf2 activator (83), being investigated in clinical trials following a series of
positive results in pre-clinical studies (25). The example of Nrf2 as a drug target
shows how the field of drug discovery is currently moving from the idea that one
agent should be developed as an agonist (or antagonist) of a particular protein or
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enzyme, thus inhibiting or activating its activity, versus the synthesis of molecules
that affect one pathway (i.e. Nrf2) responsible of multiple pleiotropic and protective
activities dependent on up-regulation of downstream genes. Whether targeting Nrf2
is better than independently targeting one of the downstream genes responsive to
Nrf2 (such as HMOX1) is still unknown and comparison of such two approaches, if
existent, would be very interesting from a pharmacological perspective. That HO-1
expression is regulated by additional nuclear proteins and transcriptional factors
other than Nrf2 should be kept into consideration (114). In fact, it has also been
reported that a repressor, Bach1, controls HO-1 expression (130). Bach1 is a hemebinding nuclear protein which represses the HMOX1 gene in normal physiological
conditions but is displaced to free the HO-1 promoter upon stress and when
intracellular heme levels rise (see Figure 1). The authors of this important discovery
also showed that alleviation of repression by Bach1, rather than activation of Nrf2, is
critical for HO-1 induction as mice lacking Bach1 constitutively expressed high HO-1
levels in most organs (130). Interestingly, examination of the chromatin structure of
HO-1 indicates that it is in a pre-activation state under normal conditions, with Bach1
acting as a repressor but rapidly sensing environmental cues that result in
transcription of HO-1 (129). This phenomenon is quite interesting because it
suggests that cells possess a dynamic, rapid and extremely sensitive system with the
explicit task to respond to endogenous stressful changes and that HO-1 is upregulated as one of the first effector molecules to convey these signals. Therefore, if
manipulation of endogenous HO-1 expression relying on Nrf2/Bach1 is envisaged for
therapeutic approaches, small molecule activators of this axis must, at least, have a
dual effect by de-repressing Bach1 and activating Nrf2. Further understanding of the
regulation of the HMOX1 gene will also help in this developmental strategy. It will be
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23
important to see in the future which other (known and unknown) regulators of the
HMOX1 gene could be viable as pharmacological targets.
3. The discovery of HO-1 inducers. Several compounds, including the

substrate heme, heavy metals like zinc, cadmium and cobalt, have been recognized
since a long time for their ability to induce HO-1 in different cell types and tissues (76,
77, 78). In addition, oxidative and nitrosative stress are also known to up-regulate
HO-1 (44, 87, 97). However, it was only when curcumin, a component of curry found
in the South Asian root Curcuma longa (see chemical structure in Figure 6), was
discovered by serendipity to strongly enhance HO-1 expression in endothelial cells
and renal proximal tubule cells, that the first prototype of a small exogenous and
natural molecule acting as inducer of HO-1 was identified (85). Significantly, both in
our study using endothelial cells and in the study by Anupam Agarwals group on
renal cells, curcumin was employed either as an inhibitor of the NF-B or the AP-1
transcription factors, in an attempt to elucidate the molecular mechanisms affecting
HO-1 induction under hypoxic conditions and inflammation, respectively. Both groups
independently were surprised to discover that, instead of suppressing heme
oxygenase, curcumin was by itself a potent inducer of HO-1 (12, 57, 85). Figure 7
summarizes these results showing that bovine aortic endothelial cells exposed to
hypoxic conditions (95% N2/5%CO2) in the presence of concentrations of curcumin
as low as 5 M exhibit a significantly higher HO-1 mRNA expression and heme
oxygenase activity over time. These interesting findings stimulated a search for novel
molecules with similar properties and we then learned that caffeic acid
phenethylester, rosolic acid, and chalcones could all increase HO-1 to different
extents in various cells (45, 53, 120). We also showed that Nrf2 was fundamental in
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curcumin-mediated HO-1 induction and performed structural-activity relationship
studies revealing that the ,-unsaturated carbonyl functionalities of these chemicals
or compounds containing an electrophilic moiety are necessary for the activation of
the HO-1 and thus for eliciting cytoprotective and anti-inflammatory action (2, 13, 79)
(see chemical structure in Figure 6). In the last few years many new compounds of
natural origin have been reported to similarly affect HO-1 and this list will probably
keep growing (15). However, we have recently performed a screening of compounds
reported in the literature as Nrf2 activators/HO-1 inducers with interesting results. In
this study we have assessed HO-1 protein expression by ELISA in BV2 microglia
cells exposed to low micromolar ranges (5-20 M) of 56 small molecules and found
that the original substances identified as HO-1 inducers, including curcumin and
carnosol (79, 85) (see chemical structure in Figure 6), were still the most potent HO1 activators exhibiting good HO-1 expression/low toxicity profiles (40). These data
suggest that certain chemical scaffold are perhaps unique and evolutionarily
selected to specifically activate this pathway. From a broader biological perspective,
it is fascinating that a group of plant-derived substances, mostly representing
secondary metabolites synthesized during environmental stresses such as lack of
nutrients, disease and infection, also up-regulate HO-1 and Nrf2. That is, the
molecules produced in response to stress and to confer stress tolerance in plants are
also capable to induce pathways that increase resistance to stress in animal tissues,
emphasizing the conservative approach of nature throughout evolution.
4. Aspecificity of HO-1 inducers and the issue of heme availability as a

substrate of HO-1 enzymatic activity. A variety of pathways may be affected by
the compounds that we call HO-1 inducers. The very fact that in order to promote
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25
HO-1 induction most of these substances must act on the transcriptional modulators
up-stream of HMOX1 indicates that other genes may well be stimulated/downregulated at the same time. Whether this lack of specificity is seen as a drawback
from a mere pharmaceutical point of view is, however, debatable. Although there is
strong evidence on the protective actions of HO-1, if HO-1 inducers can actually
affect simultaneously other defensive systems and possibly suppress negative or
deleterious cellular signals independently of HO-1 should be advantageous.
Curcumin is a classic example of an HO-1 inducer with multiple molecular targets
that affect cellular processes related to inflammation, tumorigenesis, apoptosis and
possibly many others to be discovered (51). Synthetic triterpenoids are another
example, being small electrophilic molecules that activate Nrf2 and HO-1 and are
under investigation for a variety of inflammatory conditions (33, 63, 131, 148). Yore
and colleagues have reported that exposure of HEK293 and PC-3 cells to the
triterpenoid CDDO-Imidazolide affects 577 proteins involved in hormone and insulin
sensing
and
other
important
signal
transduction
pathways,
exposing
the
multifunctional properties of these compounds which may contribute to their

mechanism(s) of action (147), and also to their side-effects. However, any drug,
whether already commercialized or in development will influence multiple pathways in
addition to the one being developed for; statins are inhibitors of cholesterol synthesis
by blocking HMG-CoA reductase, show pleiotropic activities including antiproliferative and anti-inflammatory actions and, incidentally, also induce HO-1 (70).
Perhaps strategies for the development of novel drugs should be, together with
designing targeted approaches, focussing to reduce as much as possible the
unwanted and health-damaging effects of molecules that show promising features.
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In the case of HO-1 we also have to consider the issue of heme availability.
Do we know whether the increased HO-1 protein following gene induction by an
exogenous stimulator will have access to enough heme to be converted to the
protective products? There would be little use for higher levels of HO-1 if the
substrate is limited, unless HO-1 is beneficial for reasons other than its enzymatic
reaction. We have recently determined whether exposure of BV2 microglia cells to
different inducers results in accumulation of bilirubin in the culture supernatant. We
found that certain substances, including carnosol and curcumin, concomitantly
stimulated HO-1 expression and bilirubin production while others elicited only HO-1
induction (40). In addition, it appeared that the source of heme was external, i.e. the
fetal bovine serum with its residual hemoglobin content (18.5 mg/100 ml according to
the certificate of analysis of the fetal bovine serum used in our cell culture work)
present in the culture medium. These findings are puzzling and we have not
examined if they are particularly relevant for microglia cells or also for other cell types
such as cardiomyocytes, which have high heme content. However, one can envision
inflammatory situations, such as hemorrhagic stroke, in which microglia cells will
have access to abundant heme/hemoglobin and therefore be able to quickly degrade
them if HO-1 is up-regulated. A similar scenario might occur in other pathological
conditions and different tissues. Thus, when working on the potential use of HO-1
inducers for drug discovery, it would seem essential to establish with accurate
methods their capacity to increase also heme oxygenase-derived products in tissues.
Another issue to consider is that whether other pathways related to HO-1, for
instance ferritin expression, would be affected by sustained pharmacological
induction of HO-1 (11).
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5. Pharmacological use of HO-1 inducers versus CO-RMs. We have

described different pharmacological approaches based on the design and synthesis
of new chemical entities that can be utilised for maximizing or amplifying the HO-1
pathway in the attempt to combat oxidative stress and inflammation, the common
denominators of several diseases (see scheme in Figure 8). Assuming we would be
in the ideal situation of having both HO-1 inducers and CO-RMs with excellent
pharmacological profile, which one should we use? Conceptually, we have always
intended CO-RMs as agents to be employed in acute conditions, at times when the
tissue is stressed and a controlled dose of CO could help to redirect cells toward a
healthier phenotype. This thought was also stimulated by the perceived toxicity of the
metal contained in carbonyl complexes and our reasoning was that the delivery of
CO to injured tissue and the cytoprotection afforded by the compounds could
compensate for the potential toxicity due to the metal, if the duration of the treatment
would be restricted. Unfortunately, there are no published articles on the use of CORMs in animal models for prolonged times and we have never administered CO-RMs
for more than 8 days. Our assumption was also that when tissue is damaged then its
ability to induce HO-1 and produce CO would be impaired, compromised or
nevertheless delayed compared to normal tissue and that exogenous administration
of CO-RMs would promptly provide CO exerting homeodynamic action, i.e. the
potential ability of tuning its properties in response to the environment and cellular
milieu that Wegiel and colleagues (141) have recently proposed. In contrast, we view
HO-1 inducers as most interesting agents for use in longer term, acting perhaps as
preventive treatments to be employed at the first signs of disease, whether
inflammatory, metabolic or other. The advantage with this approach, assuming
adequate heme availability, would be the endogenous production of all heme
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28
metabolites that together may synergize in the protection of tissue and in the
restoration of normal physiological conditions.
Biliverdin Reductase: a potential novel target for drug discovery?

It has become apparent in the last few years that biliverdin reductase (BVR),
the enzyme converting biliverdin released by HO-1 into bilirubin, is an important part
of the protective package surrounding the heme oxygenase system and heme
metabolism (142). BVR has also been described as a protein kinase and
transcriptional activator and these differential activities may be involved in the antiinflammatory action, by driving, for example, production of the anti-inflammatory
molecule IL-10 or regulating toll-like receptor (TLR4) expression in macrophages
(140). How we could exploit BVR for therapeutic application is an exciting question
and the answer that comes straight to mind is to administer biliverdin (100). But
should we target simultaneously HO-1 and BVR as both appear to contribute to
protection? After all, if the two enzymes work in coordination in the degradation of
heme, we suspect that their function is tightly regulated and dependent from each
other. Should therapeutic agents aim at increasing BVR expression? It is clear that
the study of this pathway in disease will add much needed information to its
therapeutic potential and we eagerly await such studies.
Conclusions
In summary, in the last decade we have gradually learned on the crucial properties of
the HO-1 pathway and its products in rendering cells and tissues more resistant
against stress-mediated damage thus providing a solid basis for the exploitation of
this enzymatic pathway for therapeutic applications. The use of CO gas inhalation as
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29
a therapeutic agent, the advent of CO-releasing molecules (CO-RMs) as
pharmacological carriers for the delivery of CO and the plethora of natural HO-1
inducers that have been shown to be beneficial against pathological conditions have
confirmed the feasibility of targeting HO-1 for the development of new drugs. More
studies in the future years will enable us to understand more in depth the specific
mechanism(s) of action of CO and the couple biliverdin/bilirubin with the aim to
optimize and implement any of these strategic approaches for the cure of disorders
characterized by persistent oxidative stress and inflammation.
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Acknowledgements
Roberto Motterlini and Roberta Foresti are supported by grants from the AREMCAR
Foundation and the Agence National de la Recherche (ANR Blanc International IIMITO-CO).
Author Disclosure Statement

Roberto Motterlini has financial interests in the CO-RMs technology.
List of abbreviations
AP-1, activator protein 1; BVR, biliverdin reductase; CO, carbon monoxide; carbon
monoxy hemoglobin (HbCO); carbon monoxy myoglobin (MbCO); carbon monoxidereleasing molecules (CO-RMs); FDA, Food and Drug Administration; HO-1, heme
oxygenase-1; [Mn(CO)4{S2CNMeCH2CO2H)}], CORM-401; Nrf2, nuclear factor
(erythroid-derived 2)-like 2; NF-B, nuclear factor kappa B; reactive oxygen species
(ROS);
(Ru(CO)3-glycinate),
CORM-3;
sodium
boranocarbonate,
tricarbonyldichloro ruthenium dimer ([Ru(CO)3Cl2]2), CORM-2.
CORM-A1;
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dicarbonyl CO-releasing molecules on a cobalamin scaffold for biological
application. Dalton Trans 41: 370-378, 2012.
151. Zuckerbraun BS, Chin BY, Wegiel B, Billiar TR, Czsimadia E, Rao J, Shimoda L,
Ifedigbo E, Kanno S, and Otterbein LE. Carbon monoxide reverses established
pulmonary hypertension. J Exp Med 203: 2109-2119, 2006.

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49
Figure Legends
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50
Figure 1. Strategies to exploit pharmacologically the HO-1 system. Three

different pharmacological approaches are currently being investigated to exploit the
HO-1 system for therapeutic applications: 1) the use of CO gas by inhalation, the
development of CO-releasing molecules (CO-RMs) and small molecules possessing
the ability to up-regulate HO-1 protein expression in cells and tissues. The upregulation of HO-1 One interesting class of small molecule activators of this system
could have a dual effect by de-repressing Bach1 and activating the transcription
factor Nrf2. That HO-1 expression is regulated by additional nuclear proteins and
transcriptional factors other than Nrf2 and Bach1 should be kept into consideration
and may indicate novel drug targets for the future. (see text for details).
Page 51 of 57

51
Figure 2. Mitochondria as arbiter of the toxic and beneficial effects of CO. The
amount and time of exposure to CO gas or CO-RMs can determine the balance
between a therapeutic action and negative effects of CO on cellular respiration. In
this context, mitochondria could be the arbiter of this dual effect since convincing
data on controlled delivery of CO to cells or animals have shown an unexpected
increase in mitochondrial energetic and biogenesis (see text for details).
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52
Figure 3. Applications of CO gas and CO-RMs in organ preservation. CO can

function as an effective adjuvant for preserving tissues and organs ex vivo and
maintaining their viability once transplanted in vivo. This concept is supported by data
showing that organs such as kidney and liver display an improved function following
their preservation in cold solutions supplemented with either CO gas or CO-RMs (see
text for details).
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53
Figure 4. Chemical structures and features of the first generation of carbon

monoxide-releasing molecules (CO-RMs). Dimethyl sulfoxide (DMSO)- and watersoluble CO-RMs have been studied for their biochemical and pharmacological
activities. CORM-2, CORM-3 and CORM-A1 represent the best well-characterized
compounds and have been shown to provide protection against a variety of vascular
and inflammatory related diseases (see text for details).
Page 54 of 57

54
Figure 5. Chemical structures and features of the most recently identified

carbon monoxide-releasing molecules (CO-RMs). The release of CO from CORMs can be triggered by different stimuli including light (photoinducible CO-RMs),
enzymatic reactions (enzyme-triggered CO-RMs) or oxidants (redox-sensitive CORMs). This can increase the amount of CO liberated as well as their temporal and
spatial specificity (see text for details).
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55
Figure 6. Chemical characteristics of HO-1 inducers. A chemical feature shared

by many natural compounds that increase HO-1 transcription is the electrophilic -
unsaturated carbonyl group or the presence of an electrophilic moiety. This is present
in curcumin found in curry, carnosol derived from rosemary and caffeic acid
phenethyl ester which is a component of honey (see text for details).
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56
Figure 7. Curcumin: an HO-1 inducer discovered by serendipity. Curcumin, a

component of curry found in the root Curcuma longa, was discovered by serendipity
to strongly enhance HO-1 expression in endothelial cells and renal proximal tubule
cells (see text for details). The results presented here show that bovine aortic
endothelial cells exposed to hypoxic conditions (95% N2/5%CO2) in the presence of
concentrations of curcumin as low as 5 M exhibit a significantly higher HO-1 mRNA
expression and heme oxygenase activity over time compared to cells exposed to
hypoxia alone (adapted from reference 85).
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57
Figure 8. Targeting HO-1 for drug discovery. Different pharmacological

approaches based on the design and synthesis of new chemical entities can be
utilised for maximizing or amplifying the HO-1 pathway in the attempt to combat
oxidative stress and inflammation, the common denominators of several diseases.

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Page 1 of 57

Antioxidants & Redox Signaling

Forum Review Article

Heme oxygenase-1 as a target for drug discovery

Universit Paris-Est, UMR_S955, UPEC, F-94000, Crteil, France

INSERM U955, Equipe 3, F-94000, Creteil, France

Running head: HO-1 in drug discovery

1 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

2 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

3 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

hypertension and displays remarkable anti-inflammatory and anti-bacterial effects (35,

4 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

5 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

by exogenously applied substances thus

6 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

Delivery of CO gas by inhalation

1. Background. The proposition of CO as a possible therapeutic agent

Antioxidants & Redox Signaling

8 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

2. CO gas as a therapeutic: advantages and disadvantages. From a mere

9 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

Similarly, clinical trials on CO gas therapy are

currently planned in patients suffering from pulmonary arterial hypertension,

10 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

3. Alternative applications of CO gas for therapeutic purposes. Because of

11 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

Carbon monoxide-releasing molecules (CO-RMs)

1. Background. The proposition of using CO gas inhalation to prevent or

Antioxidants & Redox Signaling

13 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

14 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

15 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

3. Liberation and delivery of CO from CO-RMs. Although studies in vitro

16 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

17 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

4. Specificity of CO release and desirable properties for the clinic. In

18 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

19 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

CO-RMs possess a great flexibility to be

Antioxidants & Redox Signaling

2. The Nrf2/HO-1 axis and beyond. The relationship between the

Antioxidants & Redox Signaling

22 R. Motterlini and R. Foresti

Antioxidants & Redox Signaling

3. The discovery of HO-1 inducers. Several compounds, including the