Heme oxygenase-1 as a target for drug discovery

Page 1 of 57 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. 1 Forum Review Article Heme oxygenase-1 as a target for drug discovery Roberto Motterlini1,2 and Roberta Foresti1,2 1 Université Paris-Est, UMR_S955, UPEC, F-94000, Créteil, 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 Page 2 of 57 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. 2 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. 2 – R. Motterlini and R. Foresti Page 3 of 57 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. 3 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 3 – R. Motterlini and R. Foresti Page 4 of 57 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. 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 4 – R. Motterlini and R. Foresti Page 5 of 57 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. 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 5 – R. Motterlini and R. Foresti Page 6 of 57 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. 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 μmol/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 6 – R. Motterlini and R. Foresti Page 7 of 57 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. 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 90’s 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 µM (126). It was then at the beginning of this century that Otterbein and co-workers 7 – R. Motterlini and R. Foresti Page 8 of 57 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. 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. 8 – R. Motterlini and R. Foresti Page 9 of 57 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. 9 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 9 – R. Motterlini and R. Foresti Page 10 of 57 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. 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 10 – R. Motterlini and R. Foresti Page 11 of 57 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. 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, 11 – R. Motterlini and R. Foresti Page 12 of 57 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. 12 in a model of renal ischemia–reperfusion 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 12 – R. Motterlini and R. Foresti Page 13 of 57 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. 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 13 – R. Motterlini and R. Foresti Page 14 of 57 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. 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) 14 – R. Motterlini and R. Foresti Page 15 of 57 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. 15 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- 15 – R. Motterlini and R. Foresti Page 16 of 57 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. 16 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 µM. 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 CO16 – R. Motterlini and R. Foresti Page 17 of 57 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. 17 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 17 – R. Motterlini and R. Foresti Page 18 of 57 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. 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 18 – R. Motterlini and R. Foresti Page 19 of 57 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. 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. 19 – R. Motterlini and R. Foresti Page 20 of 57 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. 20 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 20 – R. Motterlini and R. Foresti Page 21 of 57 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. 21 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 21 – R. Motterlini and R. Foresti Page 22 of 57 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. 22 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 22 – R. Motterlini and R. Foresti Page 23 of 57 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. 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 Agarwal’s group on renal cells, curcumin was employed either as an inhibitor of the NF-κB 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 µM 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 23 – R. Motterlini and R. Foresti Page 24 of 57 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. 24 curcumin-mediated HO-1 induction and performed structural-activity relationship studies revealing that the α,β-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 µM) 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 24 – R. Motterlini and R. Foresti Page 25 of 57 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. 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. 25 – R. Motterlini and R. Foresti Page 26 of 57 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. 26 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). 26 – R. Motterlini and R. Foresti Page 27 of 57 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. 27 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 27 – R. Motterlini and R. Foresti Page 28 of 57 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. 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 28 – R. Motterlini and R. Foresti Page 29 of 57 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. 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. 29 – R. Motterlini and R. Foresti Page 30 of 57 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. 30 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-κB, 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. 30 – R. Motterlini and R. Foresti CORM-A1; Page 31 of 57 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. 31 References 1. Abraham NG, Asija A, Drummond G, and Peterson S. Heme oxygenase -1 gene therapy: recent advances and therapeutic applications. Curr Gene Ther 7: 89108, 2007. 2. Abuarqoub H, Foresti R, Green CJ, and Motterlini R. Heme oxygenase-1 mediates the anti-inflammatory actions of 2'-hydroxychalcone in RAW 264.7 murine macrophages. Am J Physiol Cell Physiol 290: C1092-C1099, 2006. 3. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, and Cook JL. Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 274: 26071-26078, 1999. 4. Alberto R, and Motterlini R. Chemistry and biological activities of CO-releasing molecules (CORMs) and transition metal complexes. Dalton Trans 1651-1660, 2007. 5. Alcaraz MJ, Guillen MI, Ferrandiz ML, Megias J, and Motterlini R. Carbon monoxide-releasing molecules: a pharmacological expedient to counteract inflammation. Curr Pharm Des 14: 465-472, 2008. 6. Almeida SS, Queiroga CS, Sousa MF, Alves PM, and Vieira HL. Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: role of BCL-2. J Biol Chem 287: 10761-10770, 2012. 7. Ashrafian H, Czibik G, Bellahcene M, Aksentijevic D, Smith AC, Mitchell SJ, Dodd MS, Kirwan J, Byrne JJ, Ludwig C, Isackson H, Yavari A, Stottrup NB, Contractor H, Cahill TJ, Sahgal N, Ball DR, Birkler RI, Hargreaves I, Tennant DA, Land J, Lygate CA, Johannsen M, Kharbanda RK, Neubauer S, Redwood C, de Cabo R, Ahmet I, Talan M, Gunther UL, Robinson AJ, Viant MR, Pollard PJ, Tyler DJ, and Watkins H. Fumarate is cardioprotective via activation of the Nrf2 antioxidant pathway. Cell Metab 15: 361-371, 2012. 31 – R. Motterlini and R. Foresti Page 32 of 57 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. 32 8. Bagul A, Hosgood SA, Kaushik M, and Nicholson ML. Carbon monoxide protects against ischemia-reperfusion injury in an experimental model of controlled nonheartbeating donor kidney. Transplantation 85: 576-581, 2008. 9. Baird L, and Dinkova-Kostova AT. The cytoprotective role of the Keap1-Nrf2 pathway. Arch Toxicol 85: 241-272, 2011. 10. Balla G, Jacob HS, Balla J, Rosenberg M, Nath KA, Apple F, Eaton JW, and Vercellotti GM. Ferritin: a cytoprotective antioxidant stratagem of endothelium. J Biol Chem 267: 18148-18153, 1992. 11. Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Jacob HS, Eaton JW, and Balla G. Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal 9: 21192137, 2007. 12. Balogun E, Foresti R, Green CJ, and Motterlini R. Changes in temperature modulate heme oxygenase-1 induction by curcumin in renal epithelial cells. Biochem Biophys Res Commun 308: 950-955, 2003. 13. Balogun E, Hoque M, Gong P, Killeen E, Green CJ, Foresti R, Alam J, and Motterlini R. Curcumin activates the heme oxygenase-1 gene via regulation of Nrf2 and the antioxidant responsive element. Biochem J 371: 887-895, 2003. 14. Bani-Hani MG, Greenstein D, Mann BE, Green CJ, and Motterlini R. Modulation of thrombin-induced neuroinflammation in BV-2 microglia by a carbon monoxide-releasing molecule (CORM-3). J Pharmacol Exp Ther 318: 13151322, 2006. 15. Barbagallo I, Galvano F, Frigiola A, Cappello F, Riccioni G, Murabito P, D'Orazio N, Torella M, Gazzolo D, and Li VG. Potential therapeutic effects of natural heme oxygenase-1 inducers in cardiovascular diseases. Antioxid Redox Signal 18: 507-521, 2013. 16. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, and Soares MP. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 192: 1015-1026, 2000. 32 – R. Motterlini and R. Foresti Page 33 of 57 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. 33 17. Caumartin Y, Stephen J, Deng JP, Lian D, Lan Z, Liu W, Garcia B, Jevnikar AM, Wang H, Cepinskas G, and Luke PP. Carbon monoxide-releasing molecules protect against ischemia-reperfusion injury during kidney transplantation. Kidney Int 79: 1080-1089, 2011. 18. Chen B, Guo L, Fan C, Bolisetty S, Joseph R, Wright MM, Agarwal A, and George JF. Carbon monoxide rescues heme oxygenase-1-deficient mice from arterial thrombosis in allogeneic aortic transplantation. Am J Pathol 175: 422429, 2009. 19. Chen YH, Lin SJ, Lin MW, Tsai HL, Kuo SS, Chen JW, Charng MJ, Wu TC, Chen LC, Ding YA, Pan WH, Jou YS, and Chau LY. Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum Genet 111: 1-8, 2002. 20. Chung SW, Liu X, Macias AA, Baron RM, and Perrella MA. Heme oxygenase-1derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J Clin Invest 118: 239-247, 2008. 21. Clark JE, Foresti R, Green CJ, and Motterlini R. Dynamics of haem oxygenase1 expression and bilirubin production in cellular protection against oxidative stress. Biochem J 348: 615-619, 2000. 22. Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ, and Motterlini R. Heme oxygenase-1-derived bilirubin ameliorates post-ischemic myocardial dysfunction. Am J Physiol Heart Circ Physiol 278: H643-H651, 2000. 23. Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R, and Motterlini R. Cardioprotective actions by a water-soluble carbon monoxidereleasing molecule. Circ Res 93: e2-e8, 2003. 24. Clayton CE, Carraway MS, Suliman HB, Thalmann ED, Thalmann KN, Schmechel DE, and Piantadosi CA. Inhaled carbon monoxide and hyperoxic lung injury in rats. Am J Physiol Lung Cell Mol Physiol 281: L949-L957, 2001. 25. Cornblatt BS, Ye L, Dinkova-Kostova AT, Erb M, Fahey JW, Singh NK, Chen MS, Stierer T, Garrett-Mayer E, Argani P, Davidson NE, Talalay P, Kensler TW, 33 – R. Motterlini and R. Foresti Page 34 of 57 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. 34 and Visvanathan K. Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. Carcinogenesis 28: 1485-1490, 2007. 26. Crook SH, Mann BE, Meijer JAHM, Adams H, Sawle P, Scapens D, and Motterlini R. [Mn(CO)4{S2CNMe(CH2CO2H)}], a new water-soluble COreleasing molecule. Dalton Trans 40: 4230-4235, 2011. 27. Davidge KS, Sanguinetti G, Yee CH, Cox AG, McLeod CW, Monk CE, Mann BE, Motterlini R, and Poole RK. Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators. J Biol Chem 284: 45164524, 2009. 28. De Backer O, Elinck E, Blanckaert B, Leybaert L, Motterlini R, and Lefebvre RA. Water-soluble CO-releasing molecules (CO-RMs) reduce the development of postoperative ileus via modulation of MAPK/HO-1 signaling and reduction of oxidative stress. Gut 58: 347-356, 2009. 29. Deng YM, Wu BJ, Witting PK, and Stocker R. Probucol protects against smooth muscle cell proliferation by upregulating heme oxygenase-1. Circulation 110: 1855-1860, 2004. 30. Desmard M, Davidge KS, Bouvet O, Morin D, Roux D, Foresti R, Ricard JD, Denamur E, Poole RK, Montravers P, Motterlini R, and Boczkowski J. A carbon monoxide-releasing molecule (CORM-3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia. FASEB J 23: 1023-1031, 2009. 31. Desmard M, Foresti R, Morin D, Dagoussat M, Berdeaux A, Denamur E, Crook SH, Mann BE, Scapens D, Montravers P, Boczkowski J, and Motterlini R. Differential Antibacterial Activity Against Pseudomonas aeruginosa by Carbon Monoxide-Releasing Molecules. Antioxid Redox Signal 16: 153-163, 2011. 32. Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y, Yamamoto M, and Talalay P. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci U S A 99: 11908-91311, 2002. 34 – R. Motterlini and R. Foresti Page 35 of 57 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. 35 33. Dinkova-Kostova AT, Liby KT, Stephenson KK, Holtzclaw WD, Gao X, Suh N, Williams C, Risingsong R, Honda T, Gribble GW, Sporn MB, and Talalay P. Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress. Proc Natl Acad Sci U S A 102: 4584-4589, 2005. 34. Dordelmann G, Meinhardt T, Sowik T, Krueger A, and Schatzschneider U. CuAAC click functionalization of azide-modified nanodiamond with a photoactivatable CO releasing molecule (PhotoCORM) based on [Mn(CO) 3(tpm)]+. Chem Commun 48: 11528-11530, 2012. 35. Dulak J, Deshane J, Jozkowicz A, and Agarwal A. Heme oxygenase-1 and carbon monoxide in vascular pathobiology: focus on angiogenesis. Circulation 117: 231-241, 2008. 36. Fairlamb IJS, Duhme-Klair AK, Lynam JM, Moulton BE, O'Brien CT, Whitwood AC, Sawle P, Hammad J, and Motterlini R. -Pyrone iron(0) carbonyl complexes as effective CO-releasing molecules (CO-RMs). Bioorg Med Chem Lett 16: 995-998, 2006. 37. Ferrandiz ML, Maicas N, Garcia-Arnandis I, Terencio MC, Motterlini R, Devesa I, Joosten LA, van den Berg WB, and Alcaraz MJ. Treatment with a CO-releasing molecule (CORM-3) reduces joint inflammation and erosion in murine collageninduced arthritis. Ann Rheum Dis 67: 1211-1217, 2008. 38. Ferreira A, Marguti I, Bechmann I, Jeney V, Chora A, Palha NR, Rebelo S, Henri A, Beuzard Y, and Soares MP. Sickle hemoglobin confers tolerance to Plasmodium infection. Cell 145: 398-409, 2011. 39. Finkel T, and Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 408: 239-247, 2000. 40. Foresti R, Bains SK, Pitchumony TS, Castro Bras LE, Drago F, Dubois-Rande JL, Bucolo C, and Motterlini R. Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells. Pharmacol Res 76:132-148, 2013. 35 – R. Motterlini and R. Foresti Page 36 of 57 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. 36 41. Foresti R, Bani-Hani MG, and Motterlini R. Use of carbon monoxide as a therapeutic agent: promises and challenges. Intensive Care Med 34: 649-658, 2008. 42. Foresti R, Goatly H, Green CJ, and Motterlini R. Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection. Am J Physiol Heart Circ Physiol 281: H1976-H1984, 2001. 43. Foresti R, Hammad J, Clark JE, Johnson RA, Mann BE, Friebe A, Green CJ, and Motterlini R. Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule. Br J Pharmacol 142: 453-460, 2004. 44. Foresti R, Hoque M, Bains S, Green CJ, and Motterlini R. Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway. Biochem J 372: 381-390, 2003. 45. Foresti R, Hoque M, Monti D, Green CJ, and Motterlini R. Differential activation of heme oxygenase-1 by chalcones and rosolic acid in endothelial cells. J Pharmacol Exp Ther 312: 686-693, 2005. 46. Foresti R, and Motterlini R. Interaction of carbon monoxide with transition metals: evolutionary insights into drug target discovery. Curr Drug Targets 11: 15951604, 2010. 47. Garcia-Arnandis I, Guillen MI, Gomar F, Castejon MA, and Alcaraz MJ. Control of cell migration and inflammatory mediators production by CORM-2 in osteoarthritic synoviocytes. PLoS One 6: e24591, 2011. 48. Grieb G, Groger A, Bozkurt A, Stoffels I, Piatkowski A, and Pallua N. The diversity of carbon monoxide intoxication: medical courses can differ extremely A case report. Inhal Toxicol 20: 911-915, 2008. 49. Guillen MI, Megias J, Clerigues V, Gomar F, and Alcaraz MJ. The CO-releasing molecule CORM-2 is a novel regulator of the inflammatory process in osteoarthritic chondrocytes. Rheumatology (Oxford) 47: 1323-1328, 2008. 36 – R. Motterlini and R. Foresti Page 37 of 57 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. 37 50. Guo Y, Stein AB, Wu WJ, Tan W, Zhu X, Li QH, Dawn B, Motterlini R, and Bolli R. Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol 286: H1649-H1653, 2004. 51. Gupta SC, Prasad S, Kim JH, Patchva S, Webb LJ, Priyadarsini IK, and Aggarwal BB. Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep 28: 1937-1955, 2011. 52. Hegazi RA, Rao KN, Mayle A, Sepulveda AR, Otterbein LE, and Plevy SE. Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway. J Exp Med 202: 1703-1713, 2005. 53. Herencia F, Ferrandiz ML, Ubeda A, Guillen I, Dominguez JN, Charris JE, Lobo GM, and Alcaraz MJ. 4-dimethylamino-3',4'-dimethoxychalcone downregulates iNOS expression and exerts anti-inflammatory effects. Free Radic Biol Med 30: 43-50, 2001. 54. Herrmann WA. 100 Years of metal carbonyls. A serendipitous chemical discovery of major scientific and industrial impact. J Organomet Chem 383: 2144, 1990. 55. Hervera A, Leanez S, Negrete R, Motterlini R, and Pol O. Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice. PLoS One 7: e43693, 2012. 56. Hewison L, Crook SH, Johnson TR, Mann BE, Adams H, Plant SE, Sawle P, and Motterlini R. Iron indenyl carbonyl compounds: CO-releasing molecules. Dalton Trans 39: 8967-8975, 2010. 57. Hill-Kapturczak N, Thamilselvan V, Liu F, Nick HS, and Agarwal A. Mechanism of heme oxygenase-1 gene induction by curcumin in human renal proximal tubule cells. Am J Physiol Renal Physiol 281: F851-F859, 2001. 58. Horsfall LJ, Nazareth I, and Petersen I. Cardiovascular events as a function of serum bilirubin levels in a large, statin-treated cohort. Circulation 126: 25562564, 2012. 37 – R. Motterlini and R. Foresti Page 38 of 57 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. 38 59. Inoguchi T, Sasaki S, Kobayashi K, Takayanagi R, and Yamada T. Relationship between Gilbert syndrome and prevalence of vascular complications in patients with diabetes. JAMA 298: 1398-1400, 2007. 60. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, and Yamamoto M. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 275: 1602316029, 2000. 61. Johnson TR, Mann BE, Teasdale IP, Adams H, Foresti R, Green CJ, and Motterlini R. Metal carbonyls as pharmaceuticals? [Ru(CO)3Cl(glycinate)], a CO-releasing molecule with an extensive aqueous solution chemistry. Dalton Trans 15: 1500-1508, 2007. 62. Jozkowicz A, Huk I, Nigisch A, Weigel G, Dietrich W, Motterlini R, and Dulak J. Heme oxygenase-1 and angiogenic activity of endothelial cells: stimulation by carbon monoxide, inhibition by tin protoporphyrin IX. Antioxid Redox Signal 5: 155-162, 2003. 63. Kaidery NA, Banerjee R, Yang L, Smirnova NA, Hushpulian DM, Liby KT, Williams CR, Yamamoto M, Kensler TW, Ratan RR, Sporn MB, Beal MF, Gazaryan IG, and Thomas B. Targeting Nrf2-mediated gene transcription by extremely potent synthetic triterpenoids attenuate dopaminergic neurotoxicity in the MPTP mouse model of Parkinson's disease. Antioxid Redox Signal 18: 139157, 2013. 64. Kim J, Zarjou A, Traylor AM, Bolisetty S, Jaimes EA, Hull TD, George JF, Mikhail FM, and Agarwal A. In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice. Kidney Int 82: 278-291, 2012. 65. Kinderlerer AR, Pombo G, I, Hamdulay SS, Ali F, Steinberg R, Silva G, Ali N, Wang B, Haskard DO, Soares MP, and Mason JC. Heme oxygenase-1 expression enhances vascular endothelial resistance to complement-mediated injury through induction of decay-accelerating factor: a role for increased bilirubin and ferritin. Blood 113: 1598-1607, 2009. 38 – R. Motterlini and R. Foresti Page 39 of 57 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. 39 66. Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, and Witztum JL. Antioxidant vitamin supplements and cardiovascular disease. Circulation 110: 637-641, 2004. 67. Lancel S, Hassoun SM, Favory R, Decoster B, Motterlini R, and Neviere R. Carbon monoxide rescues mice from lethal sepsis by supporting mitochondrial energetic metabolism and activating mitochondrial biogenesis. J Pharmacol Exp Ther 1329: 641-648, 2009. 68. Lancel S, Montaigne D, Marechal X, Marciniak C, Hassoun SM, Decoster B, Ballot C, Blazejewski C, Corseaux D, Lescure B, Motterlini R, and Neviere R. Carbon monoxide improves cardiac function and mitochondrial population quality in a mouse model of metabolic syndrome. PLoS One 7: e41836, 2012. 69. Lavitrano M, Smolenski RT, Musumeci A, Maccherini M, Slominska E, Di Florio E, Bracco A, Mancini A, Stassi G, Patti M, Giovannoni R, Froio A, Simeone F, Forni M, Bacci ML, D'Alise G, Cozzi E, Otterbein LE, Yacoub MH, Bach FH, and Calise F. Carbon monoxide improves cardiac energetics and safeguards the heart during reperfusion after cardiopulmonary bypass in pigs. FASEB J 18: 1093-1095, 2004. 70. Lee TS, Chang CC, Zhu Y, and Shyy JY. Simvastatin induces heme oxygenase1: a novel mechanism of vessel protection. Circulation 110: 1296-1302, 2004. 71. Li C, Hossieny P, Wu BJ, Qawasmeh A, Beck K, and Stocker R. Pharmacologic induction of heme oxygenase-1. Antioxid Redox Signal 9: 2227-2239, 2007. 72. Lin Q, Weis S, Yang G, Weng YH, Helston R, Rish K, Smith A, Bordner J, Polte T, Gaunitz F, and Dennery PA. Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress. J Biol Chem 282: 20621-20633, 2007. 73. Lo Iacono L, Boczkowski J, Zini R, Salouage I, Berdeaux A, Motterlini R, and Morin D. A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species. Free Rad Biol Med 50: 1556-1564, 2011. 39 – R. Motterlini and R. Foresti Page 40 of 57 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. 40 74. Maicas N, Ferrandiz ML, Devesa I, Motterlini R, Koenders MI, van den Berg WB, and Alcaraz MJ. The CO-releasing molecule CORM-3 protects against articular degradation in the K/BxN serum transfer arthritis model. Eur J Pharmacol 634: 184-191, 2010. 75. Maines MD. The heme oxygenase system: past, present, and future. Antioxid Redox Signal 6: 797-801, 2004. 76. Maines MD, and Kappas A. Cobalt induction of hepatic heme oxygenase; with evidence that cytochrome P450 is not essential for this enzyme activity. Proc Natl Acad Sci USA 71: 4293-4297, 1974. 77. Maines MD, and Kappas A. Regulation of heme pathway enzymes and cellular glutathione content by metals that do not chelate with tetrapyrroles: blockade of metal effects by thiols. Proc Natl Acad Sci U S A 74: 1875-1878, 1977. 78. Maines MD, Trakshel GM, and Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase: only one molecular species of the enzyme is inducible. J Biol Chem 261: 411-419, 1986. 79. Martin D, Rojo AI, Salinas M, Diaz R, Gallardo G, Alam J, De Galarreta CM, and Cuadrado A. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem 279: 89198929, 2004. 80. Mazzola S, Forni M, Albertini M, Bacci ML, Zannoni A, Gentilini F, Lavitrano M, Bach FH, Otterbein LE, and Clement MG. Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs. FASEB J 19: 2045-2047, 2005. 81. Michel BW, Lippert AR, and Chang CJ. A Reaction-Based Fluorescent Probe for Selective Imaging of Carbon Monoxide in Living Cells Using a PalladiumMediated Carbonylation. J Am Chem Soc 134: 15668-15671, 2012. 82. Mizuguchi S, Stephen J, Dencev-Bihari R, Markovic N, Suehiro S, Capretta F, Potter RF, and Cepinskas G. CO-releasing molecule (CORM-3)-derived CO 40 – R. Motterlini and R. Foresti Page 41 of 57 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. 41 modulates neutrophil (PMN) migration across vascular endothelium by reducing the levels of cell surface-bound elastase. Am J Physiol Heart Circ Physiol 297: H920-H929, 2009. 83. Morimitsu Y, Nakagawa Y, Hayashi K, Fujii H, Kumagai T, Nakamura Y, Osawa T, Horio F, Itoh K, Iida K, Yamamoto M, and Uchida K. A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway. J Biol Chem 277: 3456-3463, 2002. 84. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, and Green CJ. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res 90: e17-e24, 2002. 85. Motterlini R, Foresti R, Bassi R, and Green CJ. Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress. Free Rad Biol Med 28: 1303-1312, 2000. 86. Motterlini R, Gonzales A, Foresti R, Clark JE, Green CJ, and Winslow RM. Heme oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertensive responses in vivo. Circ Res 83: 568-577, 1998. 87. Motterlini R, Green CJ, and Foresti R. Regulation of heme oxygenase-1 by redox signals involving nitric oxide. Antiox Redox Signal 4: 615-624, 2002. 88. Motterlini R, Haas B, and Foresti R. Emerging concepts on the antiinflammatory actions of carbon monoxide-releasing molecules (CO-RMs). Med Gas Res 2: 28, 2012. 89. Motterlini R, Mann BE, and Foresti R. Therapeutic applications of carbon monoxide-releasing molecules (CO-RMs). Expert Opin Investig Drugs 14: 13051318, 2005. 90. Motterlini R, Mann BE, Johnson TR, Clark JE, Foresti R, and Green CJ. Bioactivity and pharmacological actions of carbon molecules. Curr Pharmacol Design 9: 2525-2539, 2003. 41 – R. Motterlini and R. Foresti monoxide-releasing Page 42 of 57 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. 42 91. Motterlini R, and Otterbein LE. The therapeutic potential of carbon monoxide. Nat Rev Drug Discov 9: 728-743, 2010. 92. Motterlini R, Sawle P, Bains S, Hammad J, Alberto R, Foresti R, and Green CJ. CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J 19: 284-286, 2005. 93. Motterlini R, Sawle P, Hammad J, Mann BE, Johnson TR, Green CJ, and Foresti R. Vasorelaxing effects and inhibition of nitric oxide in macrophages by new iron-containing carbon monoxide-releasing molecules (CO-RMs). Pharmacol Res 68: 108-117, 2013. 94. Nakao A, Faleo G, Shimizu H, Nakahira K, Kohmoto J, Sugimoto R, Choi AM, McCurry KR, Takahashi T, and Murase N. Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts. Kidney Int 74: 1009-1016, 2008. 95. Nakao A, Kimizuka K, Stolz DB, Neto JS, Kaizu T, Choi AM, Uchiyama T, Zuckerbraun BS, Nalesnik MA, Otterbein LE, and Murase N. Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury. Am J Pathol 163: 1587-1598, 2003. 96. Nakao A, Toyokawa H, Tsung A, Nalesnik MA, Stolz DB, Kohmoto J, Ikeda A, Tomiyama K, Harada T, Takahashi T, Yang R, Fink MP, Morita K, Choi AM, and Murase N. Ex vivo application of carbon monoxide in University of Wisconsin solution to prevent intestinal cold ischemia/reperfusion injury. Am J Transplant 6: 2243-2255, 2006. 97. Naughton P, Hoque M, Green CJ, Foresti R, and Motterlini R. Interaction of heme with nitroxyl or nitric oxide amplifies heme oxygenase-1 induction: involvement of the transcription factor Nrf2. Cell Mol Biol 48: 885-894, 2002. 98. Niesel J, Pinto A, Peindy N'dongo HW, Merz K, Ott I, Gust R, and Schatzschneider U. Photoinduced CO release, cellular uptake and cytotoxicity of a tris(pyrazolyl)methane (tpm) manganese tricarbonyl complex. Chem Commun 15: 1798-1800, 2008. 42 – R. Motterlini and R. Foresti Page 43 of 57 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. 43 99. Nobre LS, Seixas JD, Romao CC, and Saraiva LM. Antimicrobial action of carbon monoxide-releasing compounds. Antimicrob Agents Chemother 51: 4303-4307, 2007. 100. Ollinger R, Wang H, Yamashita K, Wegiel B, Thomas M, Margreiter R, and Bach FH. Therapeutic applications of bilirubin and biliverdin in transplantation. Antioxid Redox Signal 9: 2175-2185, 2007. 101. Ollinger R, Yamashita K, Bilban M, Erat A, Kogler P, Thomas M, Csizmadia E, Usheva A, Margreiter R, and Bach FH. Bilirubin and biliverdin treatment of atherosclerotic diseases. Cell Cycle 6: 39-43, 2007. 102. Otterbein LE, Mantell LL, and Choi AMK. Carbon monoxide provides protection against hyperoxic lung injury. Am J Physiol 276: L688-L694, 1999. 103. Otterbein LE, Soares MP, Yamashita K, and Bach FH. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol 24: 449-455, 2003. 104. Otterbein LE, Zuckerbraun BS, Haga M, Liu F, Song R, Usheva A, Stachulak C, Bodyak N, Smith RN, Csizmadia E, Tyagi S, Akamatsu Y, Flavell RJ, Billiar TR, Tzeng E, Bach FH, Choi AM, and Soares MP. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nature Med 9: 183-190, 2003. 105. Pamplona A, Ferreira A, Balla J, Jeney V, Balla G, Epiphanio S, Chora A, Rodrigues CD, Gregoire IP, Cunha-Rodrigues M, Portugal S, Soares MP, and Mota MM. Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria. Nat Med 13: 703-710, 2007. 106. Piantadosi CA. Biological chemistry of carbon monoxide. Antioxid Redox Signal 4: 259-270, 2002. 107. Pierri AE, Pallaoro A, Wu G, and Ford PC. A luminescent and biocompatible photoCORM. J Am Chem Soc 134: 18197-18200, 2012. 43 – R. Motterlini and R. Foresti Page 44 of 57 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. 44 108. Poss KD, and Tonegawa S. Reduced stress defense in heme oxygenase 1deficient cells. Proc Natl Acad Sci USA 94: 10925-10930, 1997. 109. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, and Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 114: 1248-1259, 2004. 110. Rice-Evans C. Flavonoid antioxidants. Curr Med Chem 8: 797-807, 2001. 111. Rimmer RD, Richter H, and Ford PC. A photochemical precursor for carbon monoxide release in aerated aqueous media. Inorg Chem 49: 1180-1185, 2010. 112. Romanski S, Kraus B, Schatzschneider U, Neudorfl JM, Amslinger S, and Schmalz HG. Acyloxybutadiene iron tricarbonyl complexes as enzyme-triggered CO-releasing molecules (ET-CORMs). Angew Chem Int Ed Engl 50: 2392-2396, 2011. 113. Ryan MJ, Jernigan NL, Drummond HA, McLemore GR, Jr., Rimoldi JM, Poreddy SR, Gadepalli RS, and Stec DE. Renal vascular responses to CORMA1 in the mouse. Pharmacol Res 54: 24-29, 2006. 114. Ryter SW, Alam J, and Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiological Reviews 86: 583-650, 2006. 115. Ryter SW, and Choi AM. Heme oxygenase-1/carbon monoxide: from metabolism to molecular therapy. Am J Respir Cell Mol Biol 41: 251-260, 2009. 116. Sammut IA, Foresti R, Clark JE, Exon DJ, Vesely MJJ, Sarathchandra P, Green CJ, and Motterlini R. Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1. Br J Pharmacol 125: 1437-1444, 1998. 117. Sandouka A, Balogun E, Foresti R, Mann BE, Johnson TR, Tayem Y, Green CJ, Fuller B, and Motterlini R. Carbon monoxide-releasing molecules (CO-RMs) modulate respiration in isolated mitochondria. Cell Mol Biol 51: 425-432, 2005. 44 – R. Motterlini and R. Foresti Page 45 of 57 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. 45 118. Sandouka A, Fuller BJ, Mann BE, Green CJ, Foresti R, and Motterlini R. Treatment with carbon monoxide-releasing molecules (CO-RMs) during cold storage improves renal function at reperfusion. Kidney Int 69: 239-247, 2006. 119. Sawle P, Hammad J, Fairlamb IJ, Moulton B, O'Brien CT, Lynam JM, DuhmeKlair AK, Foresti R, and Motterlini R. Bioactive properties of iron-containing carbon monoxide-releasing molecules (CO-RMs). J Pharmacol Exp Ther 318: 403-410, 2006. 120. Scapagnini G, Foresti R, Calabrese V, Stella AM, Green CJ, and Motterlini R. Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers. Mol Pharmacol 61: 554-561, 2002. 121. Song R, Kubo M, Morse D, Zhou Z, Zhang X, Dauber JH, Fabisiak J, Alber SM, Watkins SC, Zuckerbraun BS, Otterbein LE, Ning W, Oury TD, Lee PJ, McCurry KR, and Choi AM. Carbon monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects. Am J Pathol 163: 231-242, 2003. 122. Stein AB, Bolli R, Dawn B, Sanganalmath SK, Zhu Y, Wang OL, Guo Y, Motterlini R, and Xuan YT. Carbon monoxide induces a late preconditioningmimetic cardioprotective and antiapoptotic milieu in the myocardium. J Mol Cell Cardiol 52: 228-236, 2012. 123. Steinhubl SR. Why have antioxidants failed in clinical trials? Am J Cardiol 101: 14D-19D, 2008. 124. Stocker R. Induction of haem oxygenase as a defence against oxidative stress. Free Radic Res Commun 9: 101-112, 1990. 125. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, and Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 235: 1043-1046, 1987. 126. Suematsu M, Goda N, Sano T, Kashiwagi S, Egawa T, Shinoda Y, and Ishimura Y. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 96: 2431-2437, 1995. 45 – R. Motterlini and R. Foresti Page 46 of 57 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. 46 127. Suliman HB, Carraway MS, Ali AS, Reynolds CM, Welty-Wolf KE, and Piantadosi CA. The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy. J Clin Invest 117: 3730-3741, 2007. 128. Suliman HB, Carraway MS, Tatro LG, and Piantadosi CA. A new activating role for CO in cardiac mitochondrial biogenesis. J Cell Sci 120: 299-308, 2006. 129. Sun J, Brand M, Zenke Y, Tashiro S, Groudine M, and Igarashi K. Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network. Proc Natl Acad Sci U S A 101: 1461-1466, 2004. 130. Sun J, Hoshino H, Takaku K, Nakajima O, Muto A, Suzuki H, Tashiro S, Takahashi S, Shibahara S, Alam J, Taketo MM, Yamamoto M, and Igarashi K. Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. Embo J 21: 5216-5224, 2002. 131. Sussan TE, Rangasamy T, Blake DJ, Malhotra D, El Haddad H, Bedja D, Yates MS, Kombairaju P, Yamamoto M, Liby KT, Sporn MB, Gabrielson KL, Champion HC, Tuder RM, Kensler TW, and Biswal S. Targeting Nrf2 with the triterpenoid CDDO-imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice. Proc Natl Acad Sci U S A 106: 250-255, 2009. 132. Taille C, Foresti R, Lanone S, Zedda C, Green CJ, Aubier M, Motterlini R, and Boczkowski J. Protective role of heme oxygenases against endotoxin-induced diaphragmatic dysfunction in rats. Am J Respir Crit Care Med 163: 753-761, 2001. 133. Takamiya R, Hung CC, Hall SR, Fukunaga K, Nagaishi T, Maeno T, Owen C, Macias AA, Fredenburgh LE, Ishizaka A, Blumberg RS, Baron RM, and Perrella MA. High mobility group Box 1 contributes to lethality of endotoxemia in heme oxygenase-1 deficient mice. Am J Respir Cell Mol Biol 41: 129-135, 2008. 134. Tsoyi K, Lee TY, Lee YS, Kim HJ, Seo HG, Lee JH, and Chang KC. Hemeoxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and 46 – R. Motterlini and R. Foresti Page 47 of 57 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. 47 improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo. Mol Pharmacol 76: 173-182, 2009. 135. Urquhart P, Rosignoli G, Cooper D, Motterlini R, and Perretti M. Carbon monoxide-releasing molecules modulate leukocyte-endothelial interactions under flow. J Pharmacol Exp Ther 321: 656-662, 2007. 136. Vandegriff KD, Young MA, Lohman J, Bellelli A, Samaja M, Malavalli A, and Winslow RM. CO-MP4, a polyethylene glycol-conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats. Br J Pharmacol 154: 1649-1661, 2008. 137. Vesely MJJ, Exon DJ, Clark JE, Foresti R, Green CJ, and Motterlini R. Heme oxygenase-1 induction in skeletal muscle cells: hemin and sodium nitroprusside are regulators in vitro. Am J Physiol 275: C1087-C1094, 1998. 138. Wang G, Hamid T, Keith RJ, Zhou G, Partridge CR, Xiang X, Kingery JR, Lewis RK, Li Q, Rokosh DG, Ford R, Spinale FG, Riggs DW, Srivastava S, Bhatnagar A, Bolli R, and Prabhu SD. Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart. Circulation 121: 1912-1925, 2010. 139. Wang J, Karpus J, Zhao BS, Luo Z, Chen PR, and He C. A Selective Fluorescent Probe for Carbon Monoxide Imaging in Living Cells. Angew Chem Int Ed Engl 51: 9652-9656, 2012. 140. Wegiel B, Gallo D, Csizmadia E, Roger T, Kaczmarek E, Harris C, Zuckerbraun BS, and Otterbein LE. Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase. Proc Natl Acad Sci U S A 108: 18849-18854, 2011. 141. Wegiel B, Hanto DW, and Otterbein LE. The social network of carbon monoxide in medicine. Trends Mol Med 19: 3-11, 2013. 142. Wegiel B, and Otterbein LE. Go green: the anti-inflammatory effects of biliverdin reductase. Front Pharmacol 3: 47, 2012. 47 – R. Motterlini and R. Foresti Page 48 of 57 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. 48 143. Wilks A. Heme oxygenase: evolution, structure, and mechanism. Antioxid Redox Signal 4: 603-614, 2002. 144. Wojakowski W, Tendera M, Cybulski W, Zuba-Surma EK, Szade K, Florczyk U, Kozakowska M, Szymula A, Krzych L, Paslawska U, Paslawski R, Milewski K, Buszman PP, Nabialek E, Kuczmik W, Janiszewski A, Dziegiel P, Buszman PE, Jozkowicz A, and Dulak J. Effects of intracoronary delivery of allogenic bone marrow-derived stem cells expressing heme oxygenase-1 on myocardial reperfusion injury. Thromb Haemost 108: 464-475, 2012. 145. Yabluchanskiy A, Sawle P, Homer-Vanniasinkam S, Green CJ, Foresti R, and Motterlini R. CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke. Crit Care Med 40: 544-552, 2012. 146. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, and Koizumi S. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 103: 129-135, 1999. 147. Yore MM, Kettenbach AN, Sporn MB, Gerber SA, and Liby KT. Proteomic analysis shows synthetic oleanane triterpenoid binds to mTOR. PLoS One 6: e22862, 2011. 148. Zhang DD. Bardoxolone Brings Nrf2-Based Therapies to Light. Antioxid Redox Signal 19: 517-518, 2013. 149. Zhang WQ, Whitwood AC, Fairlamb IJ, and Lynam JM. Group 6 carbon monoxide-releasing metal complexes with biologically-compatible leaving groups. Inorg Chem 49: 8941-8952, 2010. 150. Zobi F, Blacque O, Jacobs RA, Schaub MC, and Bogdanova AY. 17 e- rhenium 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. 48 – R. Motterlini and R. Foresti Figure Legends Page 49 of 57 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. 49 – R. Motterlini and R. Foresti 49 Page 50 of 57 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. 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). 50 – R. Motterlini and R. Foresti Page 51 of 57 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. 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). 51 – R. Motterlini and R. Foresti Page 52 of 57 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. 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). 52 – R. Motterlini and R. Foresti Page 53 of 57 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. 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). 53 – R. Motterlini and R. Foresti Page 54 of 57 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. 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). 54 – R. Motterlini and R. Foresti Page 55 of 57 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. 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 α-β 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). 55 – R. Motterlini and R. Foresti Page 56 of 57 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. 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 µM 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). 56 – R. Motterlini and R. Foresti Page 57 of 57 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. 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. 57 – R. Motterlini and R. Foresti