British journal of pharmacology 2015 volume 172 part 6

BJP British Journal of Pharmacology DOI:10.1111/bph.13005 www.brjpharmacol.org Themed Section: Pharmacology of the Gasotransmitters Correspondence EDITORIAL Pharmacology of the ‘gasotransmitters’ NO, CO and H2S: translational opportunities Péter Ferdinandy, Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary E-mail: peter.ferdinandy@ pharmahungary.com Andreas Papapetropoulos1,2, Roberta Foresti3,4 and Péter Ferdinandy5,6 Faculty of Pharmacy, University of Athens, Athens, Greece, 2‘George P Livanos and Marianthi Simou Laboratories’, Evangelismos Hospital, 1st Department of Critical Care and Pulmonary Services, University of Athens, Greece, 3Université Paris-Est, UMR_S955, UPEC, F-94000, Créteil, France, 4Inserm U955, Equipe 12, F-94000, Créteil, France, 5Pharmahungary Group, Szeged, Hungary and 6Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary LINKED ARTICLES This article is part of a themed section on Pharmacology of the Gasotransmitters To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6 The current themed issue collates a number of reviews and original papers on the pharmacology of NO, CO and H2S These three molecules have been grouped together to form a family of signaling mediators that has become known as ‘gasotransmitters’ Authors of the articles in this issue are members of ENOG- the European Network On Gasotransmitters (COST Action BM1005, www.gasotransmitters.eu) ENOG currently numbers more than 200 researchers from 24 European Countries and is funded through the European Science Foundation Work from ENOG researchers and colleagues around the world have contributed to the understanding of the role of these molecules in physiology and disease initiation and progression In addition, substantial progress has been made in recent years in the pharmacology of CO and H2S with the development of several CO- and H2S-donors The NO field is more than decades old, but readers can find in this issue reviews on novel aspects of NO/cGMP signaling and on the therapeutic usefulness of components of this pathway in cardiovascular diseases (Papapetropoulos et al., 2015) with or without co-morbidities, such as metabolic diseases (Pechánová et al., 2015) Sexual dysfunction (Yetik-Anacak et al., 2015) and male infertility (Buzadzic et al., 2015) are additional fields where modulation of NO signaling bears therapeutic potential S-nitrosation, a NO-induced post© 2015 The British Pharmacological Society translation modification of proteins is discussed by Santos et al (2015) in the context of neuronal plasticity The H2S field has recently experienced a booming interest as evidenced by the exponentially increasing number of published articles in the field Papers on the role of H2S in ischaemic diseases (Bos et al., 2015), as well as blood pressure regulation and hypertension (Snijder et al., 2015; Brancaleone et al., 2015) can be found in this issue Interactions of H2S with myeloperoxidase are reported in an original paper by Pálinkás et al (2015); the inhibitory effect of H2S on myeloperoxidase is expected to contribute to the actions of H2S in the context of inflammation CO is a unique gasotransmitter, as its specific molecular targets are still not known and it is a more stable molecule as compared to NO or H2S However, the strong affinity of CO for metal centers can guide us in the search for the putative cellular targets E.g mitochondria rich in haeme-iron proteins are potential candidates for molecular targets for CO This concept is discussed in the review of Queiroga et al (2015) in the context of the role of endogenous and exogenous CO in pathologies of the central nervous system In addition, ion channels have been recognized as possible effectors of CO signaling and it appears that modulation of the activity of channel proteins is part of the mechanism contributing to the physiological and therapeutic actions of CO (Peers et al., British Journal of Pharmacology (2015) 172 1395–1396 1395 BJP A Papapetropoulos et al 2015) Comprehensive and conceptually challenging reviews in this issue also summarize the anti-inflammatory and tissue protective activity of CO in specific conditions, such as acute gastrointestinal inflammation (Babu et al., 2015) and preeclampsia (Ahmed and Ramma, 2015), where both H2S and CO exert anti-angiogenic properties The interaction of NO, H2S, and CO at the cellular level can be observed in several pathologies, such as ischaemic heart disease and hypertension, allowing several pharmacological approaches for modulation of these gasotransmitters in order to protect the ischaemic heart with or without co-morbidities (Andreadou et al., 2015) and to regulate blood pressure (Wesseling et al., 2015) Cardiovascular co-morbidities may alter cardioprotective signaling including gasotransmitters, therefore, co-morbidities have to be taken into account when developing cardioprotective therapies as reviewed recently elsewhere (Ferdinandy et al., 2014) The current issue also contains practical guides for scientists just entering into the interesting field of gasotransmitter research, including technical guidelines to measure NO in biological samples (Csonka et al., 2015), basic guidelines for H2S pharmacology (Papapetropoulos et al., 2015), and the chemical characteristics and biological behaviors of CO-releasing molecules (Schatzschneider, 2015) The editors of this themed issue hope that the papers gathered here will be useful for established researchers already involved in gasotransmitter research, as well as for young scientists just planning to enter the field, and for teachers and students interested in the physiology, pathology, and pharmacology of NO, H2S and CO Acknowledgements Authors acknowledge the support of the COST Action BM 1005 PF is a Szentágothai Fellow of the Hungarian National Program of Excellence (TAMOP 4.2.4.A/2-11-1-2012-0001) Bos EM, van Goor H, Joles JA, Whiteman M, Leuvenink HGD (2015) Hydrogen sulfide: physiological properties and therapeutic potential in ischaemia Br J Pharmacol 172: 1479–1493 Brancaleone V, Vellecco V, Matassa DS, d’Emmanuele di Villa Bianca R, Sorrentino R, Ianaro A et al (2015) Crucial role of androgen receptor in vascular H2S biosynthesis induced by testosterone Br J Pharmacol 172: 1505–1515 Buzadzic B, Vucetic M, Jankovic A, Stancic A, Korac A, Korac B et al (2015) New insights into male (in)fertility: the importance of NO Br J Pharmacol 172: 1455–1467 Csonka C, Páli T, Bencsik P, Görbe A, Ferdinandy P, Csont T (2015) Measurement of NO in biological samples Br J Pharmacol 172: 1620–1632 Ferdinandy P, Hausenloy DJ, Heusch G, Baxter GF, Schulz R (2014) Interaction of Risk Factors, Comorbidities, and Comedications with Ischemia/Reperfusion Injury and Cardioprotection by Preconditioning, Postconditioning, and Remote Conditioning Pharmacol Rev 66: 1142–1174 Pálinkás Z, Furtmüller PG, Nagy A, Jakopitsch C, Pirker KF, Magierowski M et al (2015) Interactions of hydrogen sulfide with myeloperoxidase Br J Pharmacol 172: 1516–1532 Papapetropoulos A, Hobbs AJ, Topouzis S (2015) Extending the translational potential of targeting NO/cGMP-regulated pathways in the CVS Br J Pharmacol 172: 1397–1414 Papapetropoulos A, Whiteman M, Cirino G (2015) Pharmacological tools for hydrogen sulphide research: a brief, introductory guide for beginners Br J Pharmacol 172: 1633–1637 Pechánová O, Varga ZV, Cebová M, Giricz Z, Pacher P, Ferdinandy P (2015) Cardiac NO signalling in the metabolic syndrome Br J Pharmacol 172: 1415–1433 Peers C, Boyle JP, Scragg JL, Dallas ML, Al-Owais MM, Hettiarachichi NT et al (2015) Diverse mechanisms underlying the regulation of ion channels by carbon monoxide Br J Pharmacol 172: 1546–1556 Queiroga CS, Vercelli A, Vieira HL (2015) Carbon monoxide and the CNS: challenges and achievements Br J Pharmacol 172: 1533–1545 Santos AI, Martínez-Ruiz A, Araújo IM (2015) S-nitrosation and neuronal plasticity Br J Pharmacol 172: 1468–1478 References Ahmed A, Ramma W (2015) Unraveling the theories of preeclampsia: Are the protective pathways the new paradigm? Br J Pharmacol 172: 1574–1586 Andreadou I, Iliodromitis EK, Rassaf T, Schulz R, Papapetropoulos A, Ferdinandy P (2015) The role of gasotransmitters NO, H2S and CO in myocardial ischaemia/reperfusion injury and cardioprotection by preconditioning, postconditioning and remote conditioning Br J Pharmacol 172: 1587–1606 Babu D, Motterlini R, Lefebvre RA (2015) CO and CO-releasing molecules (CO-RMs) in acute gastrointestinal inflammation Br J Pharmacol 172: 1557–1573 1396 British Journal of Pharmacology (2015) 172 1395–1396 Schatzschneider U (2015) Novel lead structures and activation mechanisms for CO-releasing molecules (CORMs) Br J Pharmacol 172: 1638–1650 Snijder PM, Frenay AS, de Boer RA, Pasch A, Hillebrands J, Leuvenink HGD et al (2015) Exogenous administration of thiosulfate, a donor of hydrogen sulfide, attenuates angiotensin II-induced hypertensive heart disease in rats Br J Pharmacol 172: 1494–1504 Wesseling S, Fledderus JO, Verhaar MC, Joles JA (2015) Beneficial effects of diminished production of hydrogen sulfide or carbon monoxide on hypertension and renal injury induced by NO withdrawal Br J Pharmacol 172: 1607–1619 Yetik-Anacak G, Sorrentino R, Linder AE, Murat N (2015) Gas what: NO is not the only answer to sexual function Br J Pharmacol 172: 1434–1454 BJP British Journal of Pharmacology DOI:10.1111/bph.12980 www.brjpharmacol.org Themed Section: Pharmacology of the Gasotransmitters Correspondence REVIEW Extending the translational potential of targeting NO/cGMP-regulated pathways in the CVS Stavros Topouzis, Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras 26504, Greece E-mail: stto@upatras.gr Received 31 January 2014 Revised September 2014 Accepted October 2014 Andreas Papapetropoulos1, Adrian J Hobbs2 and Stavros Topouzis3 School of Health Sciences, Department of Pharmacy, University of Athens, Athens, Greece, William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK, and 3Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece The discovery of NO as both an endogenous signalling molecule and as a mediator of the cardiovascular effects of organic nitrates was acknowledged in 1998 by the Nobel Prize in Physiology/Medicine The characterization of its downstream signalling, mediated through stimulation of soluble GC (sGC) and cGMP generation, initiated significant translational interest, but until recently this was almost exclusively embodied by the use of PDE5 inhibitors in erectile dysfunction Since then, research progress in two areas has contributed to an impressive expansion of the therapeutic targeting of the NO-sGC-cGMP axis: first, an increased understanding of the molecular events operating within this complex pathway and second, a better insight into its dys-regulation and uncoupling in human disease Already-approved PDE5 inhibitors and novel, first-in-class molecules, which up-regulate the activity of sGC independently of NO and/or of the enzyme’s haem prosthetic group, are undergoing clinical evaluation to treat pulmonary hypertension and myocardial failure These molecules, as well as combinations or second-generation compounds, are also being assessed in additional experimental disease models and in patients in a wide spectrum of novel indications, such as endotoxic shock, diabetic cardiomyopathy and Becker’s muscular dystrophy There is well-founded optimism that the modulation of the NO-sGC-cGMP pathway will sustain the development of an increasing number of successful clinical candidates for years to come LINKED ARTICLES This article is part of a themed section on Pharmacology of the Gasotransmitters To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6 Abbreviations sGC, soluble GC © 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 1397–1414 1397 BJP A Papapetropoulos et al Tables of Links TARGETS LIGANDS GPCRsa Enzymesd Angiotensin II L-arginine β2-adrenoceptor Arginase Aspirin LPS COX Ataciguat (HMR1766) Methacholine Endothelin receptors b Ligand-gated ion channels Endothelial NOS (NOS3) BAY41-2272 NADPH NMDA receptor Inducible NOS (NOS2) BH4 Naproxen Nuclear hormone receptorsc Neuronal NOS (NOS1) cGMP Nitric oxide (NO) Glucocorticoid receptor PDE family Cinaciguat (BAY58-2667) Prednisolone PPAR-α PDE2 Flunisolide Prostacyclin PPAR-γ PDE5 Glyceryl trinitrate Riociguat (BAY63-251) Soluble GC (sGC) GTP Sildenafil Hsp90 Tadalafil Isoprenaline TNF-α Isosorbide mononitrate YC-1 These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,dAlexander et al., 2013a,b,c,d) Introduction The recent progress in the generation of additional, therapeutic molecules that target the NO transduction pathway is in large part due to a more detailed understanding of the biochemical and mechanistic complexities of the downstream pathways this molecule triggers That is, the soluble GC (sGC)–cGMP axis cGMP is a ubiquitous intracellular signalling molecule that affects a wide spectrum of cellular, and thus physiological, processes from cell growth and apoptosis to ion channel gating Especially in the CVS in which it has been best studied, cGMP regulates many vital homeostatic mechanisms, including endothelial cell permeability, vascular smooth muscle contractility and cardiomyocyte hypertrophy (Francis et al., 2010) Of the two distinct GC systems that generate cGMP, this review exclusively focuses on the contribution of the NO-responsive arm to the detriment of the cGMP pool generated by natriuretic peptide hormones acting on membrane-bound, particulate forms of GC Whereas there is considerable functional convergence of the two systems downstream, there is overwhelming evidence of spatial compartmentalization that results from the specific cellular co-localization of both the cGMP-generating systems as well as the cGMP-degrading PDEs, exemplified by the ability of PDE2 to selectively interfere with the natriuretic-stimulated cGMP pool, whereas PDE5 targets mainly the cytosolic cGMP pool, in cardiomyocytes (Castro et al., 2006; Piggott et al., 2006; Nausch et al., 2008; Tsai and Kass, 2009; Zhang and Kass, 2011) This review will highlight the molecules and mechanisms within this pathway whose further study has recently generated successful entries in the medical arsenal, including use in some novel medical indications, thus showing great future 1398 British Journal of Pharmacology (2015) 172 1397–1414 promise in contributing to the treatment and elimination of human disease, especially disorders of the CVS Basic biology of the NO-sGC-cGMP pathway Enzymatic generation of NO Three isoforms of NOS exist, each one with a different pattern of expression (Alderton et al., 2001): neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2) and endothelial NOS (eNOS or NOS-3) nNOS and eNOS are expressed constitutively whereas iNOS is not found in healthy cells but protein expression is induced following tissue injury or infection (Nathan, 1997) NOSs are capable of associating with the cell membrane, with cytosolic proteins or with the cytoskeleton, thus exhibiting dynamic subcellular localization (Oess et al., 2006) NOSs facilitate the five-electron oxidation of the terminal guanidino moiety of the semi-essential amino acid L-arginine, utilizing NADPH and BH4 as electron sources, to generate NO and L-citrulline in the presence of molecular oxygen (Alderton et al., 2001) The regulation of NO bioavailability is complex and controlled by numerous mechanisms impacting directly NO levels, including NOS expression, substrate provision and chemical inactivation For example, production of reactive oxygen species can inactivate NO (Münzel et al., 2005), and endogenous asymmetric methylarginines appear to act as NOS inhibitors (Leiper and Nandi, 2011; Caplin and Leiper, 2012) Arginase activity decreases the availability of the NOS substrate, L-arginine (Morris, 2009), uncouples NOS (resulting in generation of cytotoxic superoxide) and is thought to underlie nitrate tolerance (Khong et al., 2012) Modulation of Translational promise for NO pathway eNOS–caveolin interactions (Garcia-Cardena et al., 1996) acts as an on/off switch for enzyme turnover and, more recently, interactions of NO with somatic haemoglobin (Straub et al., 2012) can reduce NO bioavailability Furthermore, pharmacological enhancement of NO signalling can also be achieved indirectly For example, stimulation of the β3-adrenoceptor in the heart has been shown to be coupled to the NO–cGMP pathway, to increase NO bioactivity and to prevent experimental maladaptive myocardial remodelling caused by isoprenaline or angiotensin II, an effect that deserves to be explored further clinically (Belge et al., 2014) Several molecules targeting the above mechanisms have been developed and evaluated preclinically (e.g a NOS–caveolin disruptive peptide; Bucci et al., 2000); fewer have advanced in clinical trials The latter include the arginase inhibitor N-hydroxynor-arginine, investigated in a phase I trial in coronary disease (Shemyakin et al., 2012; NCT02009527) However, no clinical approval of molecules targeting these mechanisms has yet validated these approaches cGMP biosynthesis in response to NO The major biosensor of the generated NO is the enzyme sGC, which is found as an obligate heterodimer of α (α1 and α2) BJP and β1 subunits; the α1β1 dimer seems to be the prevalent active form in most tissues with the exception of the nervous systems where equal amounts of α1/β1 and α2/β1 are detected Each sGC subunit consists of (i) an N-terminal regulatory, haem-NO/oxygen (H-NOX) domain; (ii) a central Per-ArntSim domain; (iii) a coiled-coil domain; and (iv) a C-terminal catalytic domain (Derbyshire and Marletta, 2012) There is one haem prosthetic group per heterodimer (Figure 1) that serves as the NO sensor and that is stimulated by nM concentrations of NO leading to an increase in enzymatic activity up to 400-fold (Kamisaki et al., 1986; Tsai and Kass, 2009) The α and β subunits have been proposed to be organized in a parallel fashion and the low basal activity of sGC is thought to result from the inhibitory action exerted by the binding of the catalytic domain to the regulatory domain; this inhibition is relieved upon NO binding The presence of a reduced (Fe2+, ferrous) haem group is critical in NO sensing by sGC For example, environmental cues, that increased the presence of reactive species such as superoxide (.O2−) and peroxynitrite (ONOO−) are translated into changes in the redox status of the haem group and therefore in the ability of sGC to respond to low concentrations of NO (Weber et al., 2001; Stasch and Hobbs, 2009; Figure 1) The implications of this in disease are Figure Schematic representation of the major targetable components of the NO pathway Disease-modifying NO can be generated from three main, well-studied sources: (i) cellular conversion from L-arginine; (ii) bacterial-based, enterosalivary bioconversion of food nitrates; and (iii) nitrate drugs such as glyceryl trinitrate, either spontaneously or through cellular conversion The bioavailability of NO is regulated by its generation by the synthetic NOS enzymes and by the tissue complexation and conversion of NO, for example, to nitrosyl-free radicals Initially, NO bioactivity is in major part determined by its best-described cellular ‘biosensor’: sGC coupled to reduced haem sGC ‘stimulators’ such as riociguat, which was recently approved for treatment of two forms of PH, can by themselves activate sGC or synergize with NO Chemical modification of sGC or oxidation of the haem prosthetic group and dissociation from sGC can occur in pathophysiological situations such as PH and heart ischaemia Apo-sGC has an impaired ability to respond to NO, thus ‘uncoupling’ the NO pathway This form of sGC can be ‘resuscitated’ by sGC ‘activators’ such as cinaciguat and ataciguat PDEs are themselves regulated by and participate in the catabolism of cGMP PDE5 inhibitors such as sildenafil and tadalafil are approved for erectile dysfunction and treatment of PH NO pathway modifying drugs are increasingly evaluated in clinical trials in indications as varied as heart failure, traumatic cerebral oedema and forms of skeletal muscle dystrophies British Journal of Pharmacology (2015) 172 1397–1414 1399 BJP A Papapetropoulos et al crucial: it is thought that oxidative stress, a typical trigger for cardiovascular disease, can produce an NO-unresponsive (Fe3+, Ferric) sGC that is rapidly ubiquitinylated and degraded (Evgenov et al., 2006; Stasch and Hobbs, 2009) Furthermore, this sGC ‘uncoupling’ may result from S-nitrozation of vicinal thiols in the β1 subunit in addition to oxidation of the haem group (Stasch et al., 2006; Sayed et al., 2008) Such impairment of sGC activity in cardiovascular disease, coupled to concomitant decreases in NO bioavailability, has been the bedrock on which novel NO and/or haem-independent sGC stimulators and activators have been developed and which will be examined below (Evgenov et al., 2006; Follmann et al., 2013; Gheorghiade et al., 2013) In addition to its upstream, direct effects on NO availability and sGC function, cellular oxidative stress may also interfere with the NO/cGMP pathway by inducing posttranslational activation of the downstream cGMP effector PKG-Iα and thus affect adversely the progress of disease, something that has been experimentally shown to occur in sepsis (Rudyk et al., 2013) Due to this complex, and in some cases antithetical, regulation of NO bioactivity, in such pathological settings a dual-pronged therapeutic approach, that combines upstream restoration of physiological cGMP generation and pharmacological intervention (e.g antioxidants) could be optimal to preserve the physiological function of downstream effectors It is important to note that the downstream biochemical pathway of NO is far from limited to cGMP-mediated effects: cGMP-independent changes are undeniably part of the NO signalling repertoire, including NO-triggered protein S-nitrozation (Lima et al., 2010) and effects on mitochondrial respiration and oxygen utilization (Erusalimsky and Moncada, 2007) One should keep in mind, however, that genetic inactivation of sGCβ1 (Friebe et al., 2007) and cGMPdependent kinase I (PKG1) abolishes the hallmark physiological effect of NO, that is, vasorelaxation (Pfeifer et al., 1998), emphasizing the crucial involvement of cGMP in the effects of NO It is also clear that the ‘canonical’ (cGMP dependent) NO pathway has provided the major impetus for translational progress and thus constitutes the central focus of the review Direct downstream signalling of cGMP Two main enzyme families are directly regulated and respond to cGMP, to impact the pathophysiology of the CVS: cGMPdependent PKs (PKGs) and PDEs (Figure 1) In addition, ion channel function is also directly or indirectly (e.g via PKGdependent pathways) regulated by cGMP levels, although this phenomenon is largely restricted to sensory transduction (Biel and Michalakis, 2007; Francis et al., 2010) To date, successful translational efforts have, however, focused primarily on the two upstream enzymatic targets: sGC and PDE The ability of sGC to associate with the plasma membrane (Linder et al., 2005) and the possible compartmentalization of cGMP degrading PDEs (Castro et al., 2006; Nausch et al., 2008; Zhang and Kass, 2011) may further complicate the downstream functions of spatially regulated cGMP levels and the therapeutic targeting of enzymes that regulate its levels in distinct diseases The dominant PKG in the CVS is PKG type 1, which consists of two isoforms: α and β (Hofmann et al., 2006; Burley et al., 2007) The binding of cGMP to a regulatory 1400 British Journal of Pharmacology (2015) 172 1397–1414 region of the kinase results in a conformational change that ‘unrepresses’ the catalytic activity of the kinase and permits phosphorylation on Ser/Thr residues of client proteins Pharmacological targeting of PKG is attractive but has not been successful up to now, because selective PKG activators and inhibitors are lacking In addition, PKG inhibition may result in smooth muscle dysfunction, based on experimental evidence provided by mice with genetic deletion of cGMP kinase I (Pfeifer et al., 1998) Conversely, use of PKG activators to mimic the effects of sGC and pGC turnover is theoretically desirable in cardiovascular disease, but chronic use may be ultimately undesirable, given that gain-of-function genetic mutations in PKG found in humans are causally associated with aortic aneurysms and dissections (Guo et al., 2013) The second cGMP-responsive system that has been wellstudied comprises the PDE family of cyclic nucleotidehydrolyzing enzymes, which have arguably been the most successful ‘cGMP-based’ therapeutic targets Of the 11 PDE families (PDE1-11, each consisting of one to four isozymes and their multiple isoforms), PDEs-2, -3, -5, -6 and -11 are regulated by cGMP, of which PDE2, and are expressed in the constituent cells of the CVS, with PDE11 being found in the heart PDEs exist as dimers, each monomer comprises a characteristic for the isotype N-terminal regulatory domain and a relatively high homology C-terminal catalytic domain that can undergo post-translational prenylation or phosphorylation (Conti and Beavo, 2007; Keravis and Lugnier, 2012) Whereas PDE2 and PDE5 are activated by cGMP binding to their GAF regulatory domain, PDE3 is inhibited by competitive binding of cGMP to its catalytic site Of these three PDEs, PDE2 and PDE3 can hydrolyse both cGMP and cAMP, while PDE5 is selective for cGMP (Bender and Beavo, 2006; Conti and Beavo, 2007) PDE5, which is highly expressed in the corpus cavernosum and in the lung, is the target of smallmolecule inhibitors that have been approved to treat erectile dysfunction and pulmonary arterial hypertension [PAH; World Health Organization (WHO) group I] (Rosen and Kostis, 2003; Croom et al., 2008) Additional preclinical data support a role for PDEs 1, 2, and 10 in pulmonary hypertension, with proof-of-concept studies in cells and tissues from patients with the disease, implying that pharmacological blockade of other PDE isoforms might be beneficial (Phillips et al., 2005; Schermuly et al., 2007; Tian et al., 2011; Bubb et al., 2014) Further consideration of the therapeutic potential of PDE inhibitors, particularly PDE5, is discussed next New lead molecules targeting the NO-sGC-cGMP pathway Innovation in targeting the NO-sGC-cGMP pathway derives from either (i) development of new molecular entities; or (ii) extended clinical applications of already-approved therapeutic molecules Research that has been conducted in the past 10–15 years has produced novel lead therapeutic molecules that have entered clinical evaluation and, on occasion, are now approved medicines Two main categories of novel chemical entities in the early or late clinical arena that target the NO-sGC-cGMP axis Translational promise for NO pathway are briefly explored below First, there are established drugs that have been coupled to an NO-donating group to alleviate undesirable side effects of the ‘parent’ molecule However, far more innovative is the second category, which includes sGC ‘stimulators’ and ‘activators’ and therefore this review will draw attention to their preclinical pharmacology and mode of action NO-donating anti-inflammatory drugs The most clinically advanced, major drug group that has been used as NO-donating, ‘carrier’ scaffold has been the steroidal and non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin These hybrid molecules are being tested in a wide array of indications, from colon cancer prophylaxis to reduction of vascular complications due to hypercholesterolaemia, not all of which can be thoroughly covered by this review The molecules that are perhaps closest to approval are NSAID conjugates whose therapeutic benefit relies (i) on the presumed gastroprotection that released NO would provide to the NSAID moiety, given the increased possibility of ulcer development (del Soldato et al., 1999; Wolfe et al., 1999; Bandarage and Janero, 2001); and (ii) on the counterbalancing of the modest, but significant, effect on blood pressure that certain NSAIDs can cause in some patient populations and that can limit the health benefit of the anti-inflammatory drug (White et al., 2011) NSAIDs are among the most prescribed drugs in the world; however, it is now well established that their use carries the risk of upper gastrointestinal damage, including life-threatening bleeding complications, as side effects of their mode of action The risk varies with the NSAID used and is especially frequent in certain populations prone to bleeding (Chan et al., 2007) There are approved pharmacological strategies to prophylactically reduce the risk of gastrointestinal events due to NSAID intake, including, for example, co-administration of proton pump inhibitors (Chan et al., 2007; Graham and Chan, 2008) There is now ample experimental evidence from preclinical models that NO-releasing forms of approved steroidal and NSAIDs, including COX inhibitors such as aspirin and glucocorticoids such as prednisolone and flunisolide, exhibit similar or increased efficacy and a more favourable side effect profile than the parent molecules in several preclinical disease settings (Fiorucci et al., 2002; Paul-Clark et al., 2003; Turesin et al., 2003; Wallace et al., 2004) Such antiinflammatory drug NO conjugates have been experimentally shown to modulate ovarian (Bratasz et al., 2008) skin (Chaudhary et al., 2013) or intestinal (Williams et al., 2004) solid tumour growth, exert anti-inflammatory activity with reduced symptoms of gastric damage properties (Wallace et al., 2004; Fiorucci et al., 2007) and protect against or accelerate improvement of experimental colitis (Fiorucci et al., 2002; Zwolinska-Wcislo et al., 2011) The increased antiinflammatory efficacy of at least one of them, the prednisolone derivative NCX-1015, may in part be attributed to glucocorticoid receptor nitration resulting in more robust signalling (Paul-Clark et al., 2003) A number of NO-conjugated COX inhibitors have also been evaluated in clinical trials For example, NCX4016 (an aspirin-NO conjugate) has completed clinical testing in preventing colorectal cancer in patients at high risk for develop- BJP ing this disease (ClinicalTrials.gov identifier: NCT00331786) and in improving walking distance in patients with peripheral arterial occlusive disease (NCT01256775); however, no published report of trial outcomes is available at the writing of this review Another 13 week clinical trial involves a naproxen–NO conjugate (naproxcinod) that is intended to treat ‘hypertensive’ patients (mean arterial pressure >125 mmHg) with osteoarthritis In these individuals, naproxen induces a small rise (3–8 mmHg) in systolic BP, which increases significantly the risk of cardiac complications in this population Naproxcinod exhibits a much lower tendency to increase systolic BP than naproxen, sparing the need for anti-hypertensive drugs taken concomitantly by this population (White et al., 2011) However, the FDA has withheld approval until longer term effects of the drug are presented In sum, none of these molecules has yet progressed to largescale clinical evaluation, while, for the moment, the clinical use of NO-donating NSAIDs awaits convincing clinical data that for approval (Fiorucci and Distrutti, 2011) sCG stimulators Pharmacology and mode of action Given that reduced NO production is a defining feature of many cardiovascular diseases, including PH, the use of PDE inhibitors is likely to be limited as the efficacy of such molecules is dependent on endogenous cGMP generation Thus, compounds that activate sGC directly, or that synergize with NO in activating the enzyme, appear a perfect fit as drug candidates in such indications The initial discovery, by Taiwanese researchers in the mid-1990s, of the first ‘sGC stimulator’, YC-1 (Wu et al., 1995), was paralleled by a wide search performed by a variety of pharmaceutical companies for molecules that could act in dual fashion: they synergize with NO in stimulating sGC and directly stimulate the enzyme in the absence of NO Both activities are, however, dependent on the presence of a reduced, sGC-bound haem moiety (Hoenicka et al., 1999) The mechanistic basis of sGC stimulation by these molecules has been extensively studied, but not conclusively elucidated (Follmann et al., 2013), mainly because there are no X-ray data of the full-length crystallized enzyme Raman spectroscopic studies with sGC stimulators and structural modelling studies (based on the somewhat tenuous similarity to the AC catalytic domain) suggest that molecules such as YC-1 and BAY 41-2272 (i) induce a (indirect) change in the prosthetic haem group geometry that has bound NO, making the enzyme more active and stabilizing the nitrosyl–haem complex; and (ii) photoaffinity labelling of BAY-41-2272 and YC-1 analogues results in labelling of the α-subunit, following binding of the compound to a domain distinct from the catalytic site However, it is not absolutely clear that the binding itself occurs on the α-subunit It is possible that the site of binding is in the interface between the sGC subunits and thus elicits an allosteric interaction that results in a more active conformational shift of the enzyme and in the labelling of the α-subunit (reviewed in Derbyshire and Marletta, 2012; Follmann et al., 2013) Alternatively, sGC stimulators have been suggested to relieve an autoinhibitory interaction between the H-NOX domain in the N-terminus, which harbours the haem moiety and the C-terminus catalytic domain (Winger and Marletta, 2005) In a recently published study, Purohit et al (2014) demonstrated that YC-1 binding to the β1 British Journal of Pharmacology (2015) 172 1397–1414 1401 BJP A Papapetropoulos et al sGC subunit overcomes the allosteric inhibition by the α1 subunit In all, the exact binding site of the sGC stimulators has not been assigned with certainty yet, and more structural studies have to be performed to finally understand how sGC stimulators bind to the protein Of the many molecules of the sGC stimulator class that have been developed, riociguat (BAY 63-2521) is the one that finished first in the translational race that led to its approval in the past year in the United States, Canada and in the European Union for the treatment of two forms of PH (Conole and Scott, 2013) Many sGC stimulator molecules, including YC-1, were abandoned because of lack of selectivity (YC-1 also inhibited PDEs) and poor pharmacokinetic characteristics (Stasch and Hobbs, 2009) One instructive reason for riociguat’s success may be that very early, before full preclinical evaluation, all fellow candidate molecules were evaluated and discarded if they possessed a poor pharmacokinetic profile (Follmann et al., 2013), allowing research to concentrate on candidates that were potent, selective and possessed a favourable bioavailability/pharmacokinetic profile At the outset, riociguat showed good bioavailability and lack of interaction with the CYP metabolizing system, thus presenting the considerable advantage of future co-administration with other drugs (Follmann et al., 2013) In vitro characterization of the drug showed strong synergy in combination with NO, ability to induce sGC activity in the absence of NO and dependence on a reduced haem prosthetic group The preclinical evaluation of riociguat in key experimental animal models in vivo displayed, crucially, a longpreserved (several weeks) hypotensive effect in rats made tolerant to organic nitrates, effective inhibition or reversal of pulmonary vasoconstriction and remodelling (muscularization of small pulmonary arteries, hypertrophy of the right ventricle) in the monocrotaline model of PH (Schermuly et al., 2008; Stasch et al., 2011; Lang et al., 2012), and reduction of heart and kidney fibrosis in the Dahl hypertensive rat, resulting in increased survival rates over time (Geschka et al., 2011) Clinical success of the sGC stimulator, riociguat There are two clinical areas where considerable progress has been made in the last few years with the sGC stimulators: PH and heart failure, with pulmonary hypertension being the most successfully targeted clinical indication, based on riociguat’s approval PH is a progressive, debilitating, multifactorial disease and exacts a high socio-economic toll Most of the approved current treatments target one subgroup: PAH, a lifethreatening form of the disease that is characterized by increased pulmonary vascular resistance, excessive remodelling of small vessels and of the pulmonary artery that lead, over time, to right heart failure and death (Baliga et al., 2011; Galiè et al., 2011; Schermuly et al., 2011) Available treatments for PAH include endothelin receptor antagonists, PDE inhibitors, prostacyclin analogues and Ca2+ channel blockers (Baliga et al., 2011; Galiè et al., 2011) The necessity of additional supportive drug therapy to treat concurrent pathophysiologies, which includes oral anticoagulants, digoxin for arrhythmias and diuretics to regulate fluid accumulation and blood pressure (reviewed by Galiè et al., 2011) increases the risk of undesirable drug–drug interactions, especially with the 1402 British Journal of Pharmacology (2015) 172 1397–1414 anticoagulants Approval of any new pharmacological options that are well-tolerated and display minimal drug– drug interactions would be a welcome addition to this therapeutic arsenal Among other PH forms, persistent PH of the neonate can be effectively treated with administration of inhaled NO (Roberts et al., 1992; Vosatka et al., 1994), but NO donors are not clinically useful for chronic treatment of PH because of partial patient response, development of severe tolerance over time, short-lived duration of the pulmonary vasodilation and the danger of methaemoglobinaemia with high NO doses (Ichinose et al., 2004; Galiè et al., 2011) The exact molecular ‘defect’ in the NO-sGC-cGMP axis that may contribute to the development of the various forms of pulmonary hypertension in adults remains debatable and experimental and clinical data seem often contradictory (Giaid and Saleh, 1995; le Cras et al., 1996; Xu et al., 2004) Pharmacological potentiation of the NO pathway (Rossaint et al., 1993; Klinger, 2007; Vermeersch et al., 2007; Geschka et al., 2011) has been the basis for the development of smallmolecule inhibitors of PDE5A such as sildenafil and tadalafil, which were introduced in this clinical area in the past decade (Galiè et al., 2009; 2011; Stasch and Hobbs, 2009) The issue, particularly in PAH, is reduced NO bioavailability: PAH is considered an NO-deficient state (Stasch and Evgenov, 2013) Because sGC expression is maintained or even up-regulated in PH, targeting it with a sGC stimulator (which can synergize with NO) seems a particularly beneficial approach Clinical trials with the sGC stimulator, riociguat, in two forms of PH were successfully concluded in 2013: the treatment met primary end points in patients diagnosed with PAH and with chronic thromboembolic pulmonary hypertension (CTEPH or WHO group IV) In the phase III trial (PATENT ClinicalTrials.gov) in PAH patients who received riociguat alone or in combination with approved endothelin receptor antagonists or prostanoids for 12 weeks, the walk distance (6-MWD) increased by 36 m compared with placebo In addition, there was significant improvement in pulmonary vascular resistance, cardiac output, N-terminal pro-B-type natriuretic peptide (NT-proBNP) plasma levels, time to clinical worsening, WHO functional class, Borg dyspnoea score and quality-of-life assessment In addition, the benefit was also manifest at 24 weeks (Ghofrani et al., 2013b) The second, 16 week phase III trial (CHEST-1) included patients diagnosed with CTEPH who were either inoperable or showed persistent or recurrent PH despite having undergone pulmonary endarterectomy, a standard surgical option for this group for which no pharmacological options exist Riociguat increased the 6-MWD by 46 m compared with placebo and produced significant improvement in pulmonary vascular resistance, cardiac output, N-terminal pro-B-type natriuretic peptide level and WHO functional class (Ghofrani et al., 2013a) In both trials, the safety profile of the sGC stimulator was reassuring, a major plus that warrants further evaluation of the molecule in additional indications In addition to the above indication, riociguat is also being tested clinically, and has shown beneficial effects, in proof of concept, pilot or phase II studies in patients with PH secondary to interstitial lung disease and chronic obstructive pulmonary disease (Bonderman et al., 2013; Hoeper et al., 2013; Stasch and Evgenov, 2013) The first report of a phase IIb trial Translational promise for NO pathway in patients with pulmonary hypertension caused by systolic left ventricular dysfunction, an indication with no approved medication, shows that treatment with riociguat did not meet the primary end point, which was the decrease in mean pulmonary artery pressure at 16 weeks (Bonderman et al., 2013); however, it improved the secondary outcomes cardiac index and systemic and pulmonary resistance Despite an attempt to decipher possible effects in patient populations after stratification, the study was not powered or designed to answer some critical questions, for example, whether riociguat elicited pulmonary vasodilation (inferred by the calculated drop in pulmonary vascular resistance) or whether variation of the drug dose and duration of treatment in specific patient subpopulations would successfully reach the primary end point The mitigated results may leave the door open for a more prolonged trial, where long-term ventricular function is monitored and where, given riociguat’s safety profile, higher doses are tested Riociguat is also in early clinical stage evaluation for improvement of flow to the digits in Raynaud’s syndrome patients (NCT01926847) sGC activators Preclinical pharmacology of sGC activators Additional screening of a compound library following the discovery of sGC stimulators at Bayer and further examination of hits revealed that a second series of dicarboxylic acids could up-regulate sGC activity in an NO-independent and haem-independent manner, thus inaugurating a quite different molecular class, termed sGC activators More companies also arrived at similar-acting molecules (Schindler et al., 2006; Costell et al., 2012; Follmann et al., 2013) Most of the second-generation molecules contain only one monocarboxylic acid moiety An example of an activator that lacks carboxylic acid moieties also exists (HMR176) The mechanistic basis for the mode of action of sGC activators is arguably better understood than that of sGC stimulators Data from functional, mutational and spectroscopic studies indicate that sGC activators bind in the haem cavity within the H-NOX domain of the β1 subunit, competing with the native ligand (Pellicena et al., 2004; Martin et al., 2010; Follmann et al., 2013) The His105 in the β1 H-NOX domain, which serves as a fifth coordination for the haem iron and is crucial for sGC activation, is displaced from the ‘inactive’ form, causing the rotation of the helix that harbours His105 to a degree that depends on the sGC activator used (Follmann et al., 2013) In this way, this class of compounds activate sGC in the absence of a haem moiety (Pellicena et al., 2004; Follmann et al., 2013) Of the sGC activators, the molecular mechanism of action of BAY 58-2667 (cinaciguat) has been characterized in most detail (Martin et al., 2010) The carboxylic groups of BAY 58-2667 displace the haem propionic acids and interact with Tyr135 and Arg139 of the β1 subunit and sGC activation results from a signal transmission triad composed of His105, Tyr135 and Arg139 (Schmidt et al., 2004) Cinaciguat, and possibly other sGC activators, can prevent the degradation of sGC subunits that occurs following haem oxidation, apo-sGC formation and subunit ubiquitination in disease conditions The ability of cinaciguat to closely mimic haem binding rescues sGC from proteasomal degradation by stabilizing the apo-sGC structure and thus possesses a dual mechanism of action (maintenance of sGC BJP levels and sGC activation) in diseases associated with increased oxidative stress (Evgenov et al., 2006; Martin et al., 2010; Follmann et al., 2013) A more conclusive assessment of the sGC haem redox state in whole cells and in tissues would help improve decision making on which diseases might benefit from the administration of sGC activators (Ahrens et al., 2011) There are two, recently described, methods that may allow this in different contexts in the future, provided that they are validated and confirmed by other laboratories Fluorescence dequenching can be measured after the attachment of the biarsenical fluorophore FlAsH to the haem moiety (Hoffmann et al., 2011) via energy transfer from this fluorophore to the haem However, this technique for now is limited to live cells in vitro and has yet to be extended to in vivo applications In addition, a biochemical determination can be performed by assessing the degree of sGC-Hsp90 complexation: the binding of Hsp90 is limited to the haem-lacking enzyme and Hsp90 is dissociated once sGC has incorporated a haem prosthetic group (Ghosh and Stuehr, 2012) Similar methods, once established, could be very useful in better directing the therapeutic applicability of sGC activators This class of NO- and haem-independent sGC activators, therefore, raised the possibility of therapeutic use in situations where sGC is present in its haem-free form Increased levels of apo-sGC (leading to its ubiquitination and proteasomal degradation) occur during oxidative stress, exemplified by either full-blown, acute inflammatory responses or chronic, low-level inflammation (Stasch et al., 2002; 2006) In these situations, the effect of PDE inhibitors or sGC stimulators is inherently limited (Evgenov et al., 2006) due to a lack of intact NO–sGC signalling Thus, sGC activators have been extensively characterized in preclinical models of disease to determine if they offer a greater therapeutic potential For example, drugs modifying the haem-oxidized or haem-free enzyme would target diseased tissue This proved to be the case with encouraging results observed in models of myocardial infarction, hypertension or congestive heart failure (reviewed by Follmann et al., 2013) Cinaciguat, in a fast ventricular pacing model of congestive heart failure in dogs (Boerrigter et al., 2007), reduced mean arterial, right atrial, pulmonary artery and pulmonary capillary wedge pressure; increased cardiac output and renal blood flow; and preserved glomerular filtration rate and sodium and water excretion, making it a prime therapeutic candidate for cardiovascular indications where sGC is impaired because of oxidative stress In addition, cinaciguat was shown to antagonize crucial profibrotic mechanisms in vitro (Dunkern et al., 2007), thought to operate in many pathological remodelling processes in chronic cardiovascular diseases GSK2181236A a sGC activator developed by GlaxoSmithKline, was tested in spontaneously hypertensive stroke-prone rats on a high salt/fat diet, demonstrating organ-protective effects and reducing left ventricular hypertrophy (Costell et al., 2012) Yet another sGC activator, HMR 1766 (ataciguat), was shown to improve ex vivo vascular function and reduce platelet activation (Schäfer et al., 2010) Ataciguat also prevents and reverses pulmonary vascular remodelling and right ventricular hypertrophy in a mouse model of PH (Weissmann et al., 2009) Collectively, these results warranted clinical evaluation in similar indications British Journal of Pharmacology (2015) 172 1397–1414 1403 BJP A Papapetropoulos et al Clinical testing of sGC activators HMR1766 (ataciguat) has been evaluated in two indications and trials have been completed: in the first, the primary end point was the reduction of pain in patients with neuropathic pain (NCT00799656) and in the second, the primary end point was improvement of intermittent claudication in patients with Fontaine stage II peripheral arterial disease (NCT00443287) The conclusions from these trials are still being awaited Cinaciguat has been tested in patients with acute decompensated heart failure, an indication where it seemed to be perfectly poised to succeed because of the strong evidence of NO pathway impairment in this disease and because of the experimentally based ability of the drug to limit fibrosis (reviewed by Tamargo and López-Sendón, 2011; Gheorghiade et al., 2013) Cinaciguat was delivered by i.v administration at dose rates of 50–150 μg·h−1 and patients were monitored for up to 48 h The trial, however, was terminated prematurely because of an increased occurrence of hypotension with all three doses (Gheorghiade et al., 2012; Erdmann et al., 2013a), which is an unfavourable occurrence in this patient population; in addition, there was no discernible effect of this treatment on either dyspnoea or on cardiac index and the small patient numbers did not allow stratification (Gheorghiade et al., 2012) Although some of these clinical results have been disappointing, human genome-wide association studies have identified mutations in the genes encoding α1 (GUCY1A3) and β1 (GUCY1B3) subunits of sGC, and in the sGC-stabilizing protein CCTη, which increase the risk of hypertension, thrombosis and myocardial infarction (Ehret et al., 2011; Erdmann et al., 2013b) Thus, there is strong evidence for a direct involvement of sGC impairment in thromboembolic human disease and in the regulation of blood pressure Individuals carrying such mutations may be prime candidates for treatment with sGC stimulators or activators, as they are likely to be disease modifying However, the ethnic divergence in phenotype which is associated with GUCY SNPs suggests that patient stratification to sGC modulating drugs may be necessary NOS cofactor supplementation One particular approach aiming to augment NO production is supplementation of the NOS cofactor tetrahydrobiopterin (BH4) Its bioavailability is reduced in a variety of cardiovascular pathologies, such as in atherosclerosis, at least in part as a result of overproduction of oxygen radicals, and correlates with NOS uncoupling (Förstermann and Li, 2011; Li and Förstermann, 2013) Pharmacological augmentation of BH4, therefore, aims to re-establish a healthy cofactor stoichiometry (Alkaitis and Crabtree, 2012; Starr et al., 2013) and direct eNOS catalytic activity towards producing NO rather than O2− To achieve just that, several clinical trials have been conducted or are in progress in disease conditions that include systolic or systemic hypertension and peripheral artery disease; however, for the moment, results from these trials either not reveal statistically significant changes or are still not reported (Alkaitis and Crabtree, 2012; Cunnington et al., 2012) Characteristically, supplementation of BH4 in patients with coronary artery disease, although it produced increased levels of BH4 in saphenous vein (but not in internal mammary artery), resulted in the presence of the 1404 British Journal of Pharmacology (2015) 172 1397–1414 oxidation product BH2, which lacks NOS cofactor properties, and failed to either reduce superoxide levels or improve vascular function (Cunnington et al., 2012) These results demonstrate that, while supplementation of NOS cofactor(s) is based on a sound therapeutic rationale, the establishment of a favourable target BH4 : BH2 ratio is hard to achieve Therefore, a fundamentally different approach targeting BH4 may be more useful, such as indirectly increasing its recycling and preservation Indeed, in atherosclerotic patients, supplementation with 5-methyl-tetrahydrofolate (which prevents peroxynitrite-driven oxidation of BH4) has been shown to reduce peroxynitrite-mediated BH4 oxidation, to ameliorate the BH4/total biopterin ratio and to increase NOS coupling, thus preserving in vivo and ex vivo vascular endothelial function (Antoniades et al., 2006) Repositioning of existing medicines and combination approaches New molecular entities and modes of action have unquestionably boosted excitement in the NO field, and have advanced understanding of the physiology and pathology of sGC–cGMP signalling However, significant translational progress has also been made with older, approved drugs Quite a few of these have been, or are currently being, evaluated in indications that are either poorly served by available medications, or where an improvement of the currently obtainable therapeutic effect is desired One such example is the small (six patient), pilot clinical trial with a combination of the tried-and-tested organic nitrate, isosorbide mononitrate (ISMN), and the PDE5 inhibitor, sildenafil, in achieving better regulation of the blood pressure in patients afflicted with ‘resistant’ hypertension (Oliver et al., 2010) Monotherapy with either drug alone effectively reduced brachial systolic and diastolic blood pressure, and central systolic and diastolic arterial pressure Combination of sildenafil and ISMN elicited significantly stronger reduction of brachial systolic blood pressure and central arterial systolic pressure, compared with either drug alone Reduction of central arterial pressure with the combination reached a maximum of 26/18 mmHg (systolic blood pressure/ diastolic blood pressure) compared with placebo (Oliver et al., 2010), thus opening the way for a study involving more patients and evaluation of longer administration of this combination in this challenging patient population Sildenafil also showed improvement in non-ischaemic, non-failing diabetic cardiomyopathy (i.e at a relatively early stage) in a small, month trial in 59 diabetic patients (NCT00692237), improving left ventricle contraction and preventing cardiac remodelling through, presumably, direct intramyocardial effects, independent of endothelial vasodilatation (Giannetta et al., 2012) Longer term results are expected in the next 48 months More impressively, in a year prospective trial in 45 patients with stable, systolic heart failure, sildenafil, at months and year, improved left ventricle ejection fraction and elicited reverse remodelling of left atrial volume index and left ventricle mass index These structural and functional ameliorations by sildenafil were coupled with improved exer- BJP British Journal of Pharmacology DOI:10.1111/bph.12688 www.brjpharmacol.org Themed Section: Pharmacology of the Gasotransmitters Correspondence REVIEW Novel lead structures and activation mechanisms for CO-releasing molecules (CORMs) Professor Dr Ulrich Schatzschneider, Institut für Anorganische Chemie, Julius-MaximiliansUniversität Würzburg, Am Hubland, D-97074 Würzburg, Germany E-mail: ulrich.schatzschneider @uni-wuerzburg.de Received December 2013 Revised 28 February 2014 Accepted March 2014 U Schatzschneider Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany Carbon monoxide (CO) is an endogenous small signalling molecule in the human body, produced by the action of haem oxygenase on haem Since it is very difficult to apply safely as a gas, solid storage and delivery forms for CO are now explored Most of these CO-releasing molecules (CORMs) are based on the inactivation of the CO by coordinating it to a transition metal centre in a prodrug approach After a brief look at the potential cellular target structures of CO, an overview of the design principles and activation mechanisms for CO release from a metal coordination sphere is given Endogenous and exogenous triggers discussed include ligand exchange reactions with medium, enzymatically-induced CO release and photoactivated liberation of CO Furthermore, the attachment of CORMs to hard and soft nanomaterials to confer additional target specificity to such systems is critically assessed A survey of analytical methods for the study of the stoichiometry and kinetics of CO release, as well as the tracking of CO in living systems by using fluorescent probes, concludes this review CORMs are very valuable tools for studying CO bioactivity and might lead to new drug candidates; however, in the design of future generations of CORMs, particular attention has to be paid to their drug-likeness and the tuning of the peripheral ‘drug sphere’ for specific biomedical applications Further progress in this field will thus critically depend on a close interaction between synthetic chemists and researchers exploring the physiological effects and therapeutic applications of CO LINKED ARTICLES This article is part of a themed section on Pharmacology of the Gasotransmitters To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6 Abbreviations BODIPY, boron dipyrromethene difluoride; CORM, CO-releasing molecule; EPR, electron paramagnetic resonance; ET-CORM, enzyme-triggered CORM; HO, haem oxygenase; Mb, myoglobin; MbCO, carboxymyoglobin; MO, molecular orbital; MOF, metal-organic framework; PhotoCORM, photoactivatable CORM; sGC, soluble guanylate cyclase; YFP, yellow fluorescent protein Carbon monoxide: a small signalling molecule with specific target structures Carbon monoxide (CO) is now well established as the third small signalling molecule in higher organisms, including 1638 British Journal of Pharmacology (2015) 172 1638–1650 humans (Wu and Wang, 2005; Kim et al., 2006; Piantadosi, 2008) It is endogenously generated by the action of haem oxygenase (HO) on haem (see Alexander et al., 2013c), which also leads to the formation of iron(II) and biliverdin The latter is further converted to bilirubin by biliverdin reductase The enzymatic mechanism has been elucidated at the molecular level by a combination of spectroscopic and © 2014 The British Pharmacological Society CO-releasing molecules (CORMs) theoretical approaches (Chen et al., 2008; Lai et al., 2010; Matsui et al., 2010) as well as X-ray crystal structure analysis (Friedman et al., 2003; Rahman et al., 2008; 2009) The two isoforms of HO, constitutively expressed as HO-2 and inducible HO-1, are involved in a large number of important physiological processes, in particular vasodilatation and responses to stress conditions (Morse and Choi, 2002; Ryter et al., 2002; 2006; Abraham and Kappas, 2008; Piantadosi, 2008; Rochette et al., 2013) CO signalling is intimately intertwined with that of the other two small signal-transducing molecules, NO and H2S (Ignarro, 1999; Mustafa et al., 2009; Kajimura et al., 2010; 2012; Szabo, 2010; Li et al., 2011) However, there is a fundamental difference in the reactivity of CO compared to NO and H2S (Fukuto et al., 2012), since at ambient pressure and temperature and in the absence of special catalysts, CO will only bind to transition metal centres, whereas the other two species react rapidly with both metal centres and many of the organic constituents of biological systems This relative inertness and selective reactivity of CO has special implications for both the cellular target structures of CO and the development of delivery systems for CO in potential therapeutic applications (Motterlini and Otterbein, 2010; Wegiel et al., 2013) Thus, the present review will first summarize the most important properties of CO and its binding to metal centres for the non-chemist Then, a short but critical view will be given on the currently discussed cellular target structures of CO The main focus, however, is on artificial systems for the delivery of CO to cells and tissues and the activation mechanisms that trigger the release of CO from such carriers The most important lead structures of CO-releasing molecules (CORMs) will be critically discussed, with particular emphasis on the latest development in this field during the past few years Another important issue is the in vitro and ultimately in vivo detection and tracking of CO and CORMs, which will be discussed before this review concludes with an outlook on the challenges of turning CORMs into viable drug candidates Carbon monoxide: properties and binding to metal centres CO is a colourless and odourless flammable gas, which, in spite of its signalling function, is highly toxic to organisms at elevated concentrations In that context, it should be noted that it is actually not so much an interference with oxygen transport in the blood due to tight binding of CO to the haem iron centres in haemoglobin, but rather the increase in tissue CO concentrations occurring upon inhalation, which disturb normal mitochondrial function, that is responsible for CO toxicity As elegantly demonstrated by Goldbaum and co-workers, mammals actually have a rather large ‘built-in’ margin of safety in the oxygen-carrying capacity of the blood, and haemoglobin levels available for O2 transport can be reduced to about 20–30% of the normal without detrimental effects, as long as high lung CO levels not shift the equilibrium towards increased tissue accumulation (Goldbaum et al., 1975; Foresti and Motterlini, 2010) With a melting point of −205.1°C and a boiling temperature of −191.5°C, CO exists as a gas at ambient conditions, which is only sparingly soluble in water (0.35 L of CO per L of BJP H2O or 14 mM) The short carbon-oxygen bond distance (1.1282 Å in the gas phase) and high dissociation energy (1070 kJ·mol−1) are in line with the C≡O triple bond character This is particularly evident from the molecular orbital (MO) diagram shown in Figure 1A The overlap of the 2s atomic orbitals of carbon and oxygen leads to two σ and σ* MOs, whereas the 2p atomic orbitals form three bonding and three anti-bonding MOs, a pair of σ/σ* character and two each of π and π* character Four of the bonding but only one of the anti-bonding MOs are doubly occupied, resulting in this exceptionally strong bonding However, of particular importance for the reactivity and binding of CO to metals, is the presence of relatively low-lying empty MOs of π* character As shown in Figure 1B, the energetically highest filled orbital of CO, which is of σ* character, can overlap with symmetry-adapted empty d orbitals on a transition metal centre along the M-C vector to form a bonding σ-donor interaction At the same time, there is also a π-donor interaction between the next highest occupied π orbital on the CO with properly oriented empty metal d orbitals In contrast, occupied metal d orbitals can also overlap with the empty low-lying MOs on the CO, forming an additional π-acceptor interaction, the so-called backbonding, which is among the characteristic features of transition metal carbonyl complexes and is responsible for the particular stability of this type of compound (Elschenbroich, 2006) Therefore, carbonyl complexes preferentially form with transition metal centres in relatively low oxidation states, since these provide filled d orbitals of proper energy to facilitate the backbonding Furthermore, the strength of the M-(CO) bond can be modulated by the presence of electron-donating or withdrawing coligands on the metal-carbonyl fragment A decrease in the metal d electron density will result in subsequent weakening of the backbonding and thus facilitate CO release from the metal coordination sphere This can be achieved, for example, by chemical or electrochemical oxidation of a low-valent metal centre Alternatively, electronic excitations due to absorption of light of a proper wavelength might also reduce the metal charge density in the excited state, particularly when metal-to-ligand charge transfer transitions are involved These binding characteristics have important implications for the triggering of CO release from stable prodrug compounds, but also on the potential cellular target structures of CO Another special feature of CO as a ligand is that it can coordinate terminally to a metal centre but also act as a μ2- and even μ3-ligand, bridging two or three metal centres respectively Furthermore, in a suitable environment, it can even change its coordination mode from terminal to bridging and vice versa (Figure 1C) Cellular target structures of carbon monoxide Keeping in mind the special stability of the M-(CO) bond due to the interplay of bonding and backbonding interactions, it is very hard to imagine a recognition and binding of CO without the involvement of a transition metal centre, at least under physiological conditions Therefore, it is not surprising that, in particular, haem proteins have been implicated as the British Journal of Pharmacology (2015) 172 1638–1650 1639 BJP U Schatzschneider A B C Figure (A) Molecular orbital (MO) diagram of CO; (B) bonding and backbonding interactions in transition metal-CO complexes; and (C) CO coordination modes (Elschenbroich, 2006) primary cellular targets for CO binding Soluble guanylate cyclase (sGC) has been the focus of many earlier works in this field It is a haem protein in which the haem co-factor is covalently anchored to the protein via an axial histidine ligation to the iron to give rise to a five-coordinate metal centre, with the position trans to the histidine able to bind either NO or CO, but not O2 (Poulos, 2006; Derbyshire and Marletta, 2012) This leads to an increase in cGMP production from GTP by about two orders of magnitude, at least in the case of NO The cGMP then triggers further down-stream signalling events, including phosphodiesterase and protein kinase activation as well as modulation of gated ion channels The study of sGC structure and function has, however, for a long time been hampered by problems with the overexpression of the full-length protein, and thus, usually only shortened constructs are investigated Furthermore, sGC activation by CO alone only leads to a two- to fourfold increase in cGMP production Additional synthetic sGC activators such as YC-1 were shown to potentiate these effects (Ibrahim et al., 2010), but the search for natural analogues thereof has so far remained elusive Based on the very low sGC activation by CO alone, it is questionable whether this is indeed the main target of normal CO signalling in cells Other haem proteins, where this activity seems to be better defined, have been identified more recently Especially mentioned here is cystathionine-β-synthase, one of the enzymes involved in H2S biosynthesis from sulphur-containing amino acids, and thus one of the sites of cross-talk between the different smallmolecule signalling systems (Puranik et al., 2006; Weeks et al., 2009; Li et al., 2011) Other targets include nuclear hormone 1640 British Journal of Pharmacology (2015) 172 1638–1650 receptors (Gupta and Ragsdale, 2011), and particularly in the case of very simple organisms, different gas sensor proteins (Uchida and Kitagawa, 2005; Podust et al., 2008) Particularly intriguing, however, is the recent discovery that the activity of different ion channels, such as KCa1.1 (also know as BKCa), ENaC, K2P2.1 (also known as TREK-1), Kv (see Alexander et al., 2013b), P2X2 and P2X4 (Alexander et al., 2013a), can be modulated by the application of CO (Wilkinson and Kemp, 2011) Taking the KCa1.1 channels as an example, it was demonstrated that they can be directly stimulated by CO and thus act as a gas sensor (Hou et al., 2009) However, the initial hypothesis that haem bound to the channel is the CO binding site is not in line with more recent results and the location of the haem binding site and the CO interaction seem to be spatially separated Through site-directed mutagenesis, a number of histidine and asparagine residues in the RCK1 domain were shown to be essential for the CO sensitivity, particularly His365, His394 and Asp367 (Hou et al., 2008) However, the molecular mechanism of the interaction of CO with this part of the ion channel is still elusive In the absence of an X-ray crystal structure of the channel or at least of the RCK1 domain, homology arguments have been drawn upon to suggest that this sensory domain does not contain a haem co-factor or any other metal Since membrane proteins are notoriously difficult to handle and purify, it is possible that a relatively labile metal site has been lost during the channel isolation and preparation Clearly, more careful biophysical characterization of the native ion channels is urgently required, including metal analysis of the different preparations with inductively coupled plasma mass spectrometry or CO-releasing molecules (CORMs) atomic absorption spectroscopy, and electron paramagnetic resonance (EPR) as well as Mössbauer spectroscopy to try to detect a metal centre, with the latter method particularly well-suited for iron-containing biomolecules An intriguing prospect is based on the observation that haem oxygenase (HMOX2) seems to associate in the cellular membrane with KCa1.1 channels Thus, it could be envisaged that both the ferrous iron as well as the CO generated by HMOX2’s action on haem actually migrate within such a multi-enzyme cluster, without ever escaping to the cytosol, to form a very sensitive metal site in situ by iron coordination to the abovementioned amino acid-derived ligands, which then binds the CO, but is easily lost upon handling during the purification procedure A particularly well-studied example of such internal gas channels is found in dual-functional CO dehydrogenase/acetyl-coenzyme A synthase as part of the Wood-Ljungdahl pathway of anaerobic CO2 and CO fixation in certain microorganisms (Ragsdale et al., 2012) Furthermore, since NO has been shown to interact with iron-sulphur clusters, leading to the formation of dinitrosyl iron complexes (Tinberg et al., 2010), one could speculate that CO might also target this very important class of metal centres, having functions in electron transfer and catalysis in a very wide range of proteins Delivery systems for carbon monoxide gas One option to modulate the concentration of CO in an organism for biomedical applications is of course the pharmacological stimulation of haem oxygenase-1 activity (Li et al., 2007; Abraham and Kappas, 2008) However, many of the current approaches are more focused on an exogenous delivery of CO The simplest way to achieve this is inhalation of a gas mixture containing CO Due to the very high general toxicity of CO, this has to be carried out in a carefully controlled way, to prevent adverse effects on the patient and the medical personnel In fact, there are devices now available that will adjust the quantity of CO gas delivered, depending on the breathing rate as well as other parameters, and immediately shut off the supply under abnormal conditions (Motterlini and Otterbein, 2010) Although this mitigates the safety issues associated with this mode of delivery, a fundamental drawback of the application of CO by inhalation remains This is related to the fact that once the CO is taken up via the respiratory system, its further distribution is determined by the partition ratio between the different body fluids and tissues Since this is a fixed value, it is very difficult to specifically address particular disease sites in the organism using this form of application, particularly when they are not part of the vascular system CORMs: general design principles and activation mechanisms for CO release Due to the above-mentioned inherent limitations in the use of CO gas, about 10 years ago, Motterlini and co-workers BJP Figure The ligand environment around a metal-carbonyl complex can be divided in a ‘CORM sphere’ and a ‘drug sphere’ While the former determines the stoichiometry and kinetics of the CO release from the metal centre by proper choice of metal, metal oxidation state and coligands, the latter, as mainly defined by the periphery of the coligands L, can be modulated to enable tissue-specific targeting (Romao et al., 2012) introduced the concept of using a chemically bound form of CO as a prodrug for physiological CO release, for which the term CO-releasing molecules (CORMs) was coined (Motterlini et al., 2002) Although a number of organic compounds was initially explored for their potential as CORMs, including haloalkanes, aldehydes, oxalates and silacarboxylic acids (Mann, 2010; Romao et al., 2012), it turned out that their release rate and toxicological profile were not favourable to justify further development Consequently, due to the strong but modifiable binding of CO to transition metal centres, most of the research on CORMs has focused on metal carbonyl complexes A notable exception, however, is a family of main group boranocarbonates introduced by Alberto’s group (Motterlini et al., 2004; Pitchumony et al., 2008; 2010) Furthermore, very recently, two organic systems based on unsaturated cyclic 1,2-diketones and xanthene-9carboxylic acid were introduced as the first really viable purely organic CORMs (Antony et al., 2013; Peng et al., 2013) Nevertheless, it is still the transition metal-based CORMs that offer the greatest flexibility in terms of molecular design and thus they will be the focus of the discussion herein This is due to the fact that by proper selection of the metal centre, the number and spatial arrangement of CO ligands around it, and the nature of the coligands completing the preferred coordination sphere of the metal, it is possible to tune the CO-release properties in a wide range (Figure 2) Consequently, this ‘inner’ part of such a molecule has been termed the CORM sphere or coordination sphere by Romao et al (2012) Key parameters are the number of CO molecules that can be released from the metal coordination sphere, the kinetics of the CO release process and the trigger mechanism required to initiate the liberation of the CO Currently, there is no consensus on whether a high or a low number of CO ligands per metal complex unit would be more desirable and whether the CO release should be slow or fast Monocarbonyl compounds of course have the simplest release kinetics, while in the case of more than one labile CO present per molecular unit, these might be liberated consecutively at a different speed, thus making kinetic analysis and identification of potential intermediates much more difficult Furthermore, it is not clear whether a low but steady release of CO from CORMs would British Journal of Pharmacology (2015) 172 1638–1650 1641 BJP U Schatzschneider Figure Common trigger mechanisms to initiate CO release from a metal coordination sphere: (A) ligand-exchange triggered, (B) enzyme-triggered and (C) photoinduced release be most beneficial, or rather a quick burst of CO liberated Very likely, there is no single answer to these questions and, instead, different sets of CORMs should be designed with these properties specifically optimized for a particular medical application in mind In addition to the CORM sphere, which can be tuned to control the CO release, it is also possible to modify the (co)ligand periphery pointing away from the metal centre This is particularly important and the most significant advantage of CORMs over simple CO gas, since it allows the modulation of their partition ratio between the different body fluids and tissues and might also enable an active or passive targeting of specific cell subpopulations Thus, the ‘outer part’ of CORMs has been termed the drug sphere (Figure 2), since it will dominate the pharmacological profile of these compounds (Romao et al., 2012) Unfortunately, much research has so far mainly focused on the CO-release properties and the proper tuning of the drug sphere has been more or less neglected, although this is absolutely vital if CORMs are indeed to be used beyond simple tools for fundamental biological studies on small-molecule signalling and turned into real drugs However, the optimization of the drug sphere will require a clear focus on selecting and treating a particular medical condition, as nicely illustrated by liver targeting of CORMs in recent work from the Romao group (Marques et al., 2012) In the context of targeting specific cells and tissues, another currently unresolved question is whether it will actually be necessary for the CORM to exert its biological action to enter the cells by passive or active uptake, or whether it will be sufficient to liberate the CO close enough to the target site, which it will then reach by diffusion through cellular mem1642 British Journal of Pharmacology (2015) 172 1638–1650 branes Since the diffusion behaviour and maximum free path length of CO in complex biological systems is not known at present, it is difficult to estimate how close will be close enough However, some mathematical modelling on the ‘sphere of action’ of H2S has already been carried out and it would be highly desirable if such methodology is also applied to the study of CO distribution (Cuevasanta et al., 2011) Since CORMs serve as prodrugs for CO delivery, a very important question is also the trigger mechanism by which the CO liberation is actually induced, since this will be important to control site-specific delivery In most of the metalcarbonyl complexes initially investigated for their suitability as CORMs, the release of CO from the metal coordination sphere is evoked by ligand exchange reactions with the medium (Figure 3) For example, while CORM-3, [RuCl(glycinato)(CO)3], has a half-life of 98 h in distilled water at 37°C, this is reduced by three orders of magnitude to just 3.6 in human plasma, probably due to an interaction with the thiol from glutathione (Johnson et al., 2007) Since upon i.v delivery, CORMs will immediately be exposed to high concentrations of bio(macro)molecules as potential ligands, the tissue distribution and targeting capability of such ligand-exchange triggered ‘conventional’ CORMs will largely depend on the fine balance between their half-life in complex medium and the time required to reach and accumulate at a specific target site in the body One way to modulate this is by incorporation of the CORM inro a local hydrophobic environment and such an approach has indeed been explored by incorporation of CORM-3 into a triblock co-polymer composed of a COreleasing domain flanked by hydrophilic and hydrophobic CO-releasing molecules (CORMs) sections, which self-assemble to a CO-releasing micelle with release kinetics slowed down compared with the free parent compound (Hasegawa et al., 2010) In addition, researchers have started to explore alternative trigger mechanisms, in which a metal-carbonyl complex serves as a prodrug entirely stable towards ligand exchange even with highly abundant biological nucleophiles, and the CO release is only induced by a proper internal or external stimulus (Figure 3) The most attractive external stimulus is probably light, since highly focused and pulsed light sources will allow for a very precise spatial and temporal control of the biological activity of such photoactivatable CO-releasing molecules (PhotoCORMs) (Schatzschneider, 2011; Rimmer et al., 2012) In addition to topical applications, for example, in the treatment of skin diseases, it can generally also be envisioned to target sites deeper inside solid tissue due to modern developments in waveguide technology, but the direct correlation between excitation wavelength and tissue penetration depth remains a major problem (Lane, 2003; Szacilowski et al., 2005; Agostinis et al., 2011) Red light or even infrared photoactivation is the ultimate goal, both to ensure that structures deeper inside the body can be reached and to minimize photodamage to healthy tissue in the beam pathway A different way of elegantly combining external stimulation with ligand-exchange triggered CO-release from metal carbonyl complexes was recently reported by Kunz et al (2013) In this work, a catecholate-modified CORM-3 was anchored to maghemite (Fe2O3) nanoparticles, which, upon exposure to an alternating magnetic field, locally heat up; this then accelerates the ligand exchange of CO with medium, leading to a twofold increase in CO release in some of the models studied More recently, work has also started to utilize differences in cellular microenvironments for a localized control of the CO release While parameters like cellular pH and redox environment might also be exploited in this context, an interesting alternative approach was taken by the group of Schmalz, which is based on potential differences in cellular enzyme expression rates These researchers prepared metal carbonyl complexes in which an organometallic ligand is ‘trapped’ in one of two possible tautomeric forms by ester formation or O-phosphorylation Exposure of these CO prodrugs to esterases or phosphatases leads to a hapticity change of the metalcarbonyl unit, resulting in increased sensitivity to dioxygen and subsequent CO release (Romanski et al., 2011; 2012a,b; Botov et al., 2013) Although these enzyme-triggered CO- BJP releasing molecules (ET-CORMs) are still at an exploratory stage, this approach also holds great promise for tissuespecific, internally-triggered, controlled CO bioactivity CORMs: important lead structures and novel developments A significant number of different CORMs based on transition metal as well as main group elements have been reported during the past decade and a ‘periodic table of CORMs’ is shown in Figure Since this extensive body of work has already been reviewed, for example, by Mann and co-workers (Mann, 2010; Romao et al., 2012; Zobi, 2013), no attempt will be made here to provide a comprehensive coverage of all CORMs published to date Instead, the discussion will focus on some of the most important and most widely used CORMs with a particular focus on their benefits and limitations, and then particularly new developments and further prospects for the field Initially, iron pentacarbonyl (Fe(CO)5) and dimanganese decacarbonyl (Mn2(CO)10) were explored However, these were found to be only poorly bioavailable due to their nonpolar nature and also require photoactivation (Motterlini et al., 2002) Thus, most studies on the cellular delivery and biological activity of CO today utilizes either CORM-2 ([RuCl(μ-Cl)(CO)3]2) or CORM-3 ([RuCl(glycinato)(CO)3]) Although many interesting results have been obtained employing these two CORMs (Figure 5), they are far from ideal for this purpose In particular, CORM-3 shows a very complicated and solvent-dependent speciation in solution, which is also further influenced by the pH of the medium A large number of different isomers and adducts can form, which are difficult to track down and might have variable CO release kinetics This is particularly evident in the case of CORM-3, which shows a wide variation in half-life depending on the medium (Johnson et al., 2007) Furthermore, the Ru(CO)n fragment was shown to bind to surface-accessible amino acid side chains in proteins such as lysozyme (Santos-Silva et al., 2011; Santos et al., 2012) Since ruthenium complexes are widely explored in the context of anticancer chemotherapy (Alessio et al., 2004; Melchart and Sadler, 2006; Bratsos et al., 2007; Levina et al., 2009; Süss-Fink, 2010), it is very important to keep in mind that the metal–coligand fragment remaining after liberation of CO from the coordination sphere might well possess biological Figure Periodic table of CO-releasing molecules (CORMs) Elements for which CORMs have been reported to date are highlighted in grey British Journal of Pharmacology (2015) 172 1638–1650 1643 BJP U Schatzschneider Figure Selected examples of CO-releasing molecules, by activation mechanism: (A) ligand-exchange triggered CORMs, (B) enzyme-triggered CORMs (ET-CORM), and (C) photoactivated CORMs (PhotoCORMs) activity on its own Unfortunately, these reaction products, termed inactivated CORMs (iCORMs), are often not structurally characterized and, in many cases, not assayed for bioactivity separately Thus, unless very careful controls are included in a study, using both CO gas as a positive and well-characterized iCORMs as a negative control, it remains doubtful whether the biological effects observed are actually indeed due to the CO only, or rather result from the metal– 1644 British Journal of Pharmacology (2015) 172 1638–1650 coligand fragment remaining, or at least a combination of both Furthermore, since these CORMs have not been designed for a specific biomedical application, their drug sphere is probably not ideal in terms of cellular targeting, uptake and intracellular distribution A notably different approach has been taken by Romao et al., who very carefully modified and explored a large family of molybdenum(0) tricarbonyl complexes of the general CO-releasing molecules (CORMs) formula [Mo(CO)3(L)3], in which L is a neutral monodentate ligand, for the treatment of acute livery injury resulting from poisoning with acetaminophen (Marques et al., 2012) It turned out that isonitriles were the ligands of choice, and the correct selection of peripheral functional groups indeed led to the desired tissue selectivity Furthermore, for a related molybdenum(0) compound, Na[Mo(histidinate)(CO)3], it was shown that this is finally metabolized to a polyoxomolybdate cluster [PMo12O40]3−, as demonstrated by X-ray crystallography of this species bound to lysozyme (Seixas et al., 2013) Thus, instead of somewhat arbitrarily preparing and screening further metal carbonyl complexes, there is a very clear need for compounds that are designed for a very particular biomedical application and thoroughly examined not only for their CO release behaviour, but also the follow-up products Since the compounds discussed previously all start to undergo CO release as soon as they are dissolved in the medium, it is largely their half-lives in the circulation after i.v injection that determine the target structures that can be addressed in the body Recent work is therefore aimed at CORM prodrugs that are stable in serum and only get triggered in the targeted tissue by a very specific stimulus A very interesting example of this class of compounds is the enzyme-triggered CORMs (ET-CORMs) from Schmalz’s group (Romanski et al., 2011; 2012a,b; Botov et al., 2013) This concept is based on the trapping of an α,β-unsaturated ketone, such as cyclohexenone, in the enolate form by ester formation or O-phosphorylation The resulting diene, with the acyloxy or phosphoryloxy group in either the or position, then acts as a η4-ligand to the Fe(CO)3 fragment (Figure 5), which is introduced by reaction with diiron nonacarbonyl Cleavage of the C–O or P–O bond by suitable esterases or phosphatases regenerates the dienol, which undergoes a hapticity change from η4 to η2 These intermediates are much more prone to oxidative decomposition, giving rise to ferric iron and the α,β-unsaturated ketone, as well as the release of all three equivalents of carbon monoxide Differences in tissue expression rates of esterases or phosphatases between organs as well as healthy and diseased sites might allow for a locally restricted CO release and thus bioactivity Although the potency of the iron(III) as well as the α,β-unsaturated ketone needs to be further explored in separate controls, this system also gives rise to well-defined follow-up products, which can be independently prepared and assessed An alternative, external trigger is based on the use of light to induce CO release from a transition metal carrier system (Schatzschneider, 2010; 2011; Rimmer et al., 2012) In addition to the problems with independent evaluation of the iCORM products, which is inherent to all CORMs (vide supra), these PhotoCORMs face an additional difficulty due to the inverse correlation between the tissue penetration depth of light and the incident wavelength Thus, the further the PhotoCORM absorption can be shifted to the red or even infrared part of the electromagnetic spectrum, the more likely the deeper structures can be addressed inside the tissue (Lane, 2003; Szacilowski et al., 2005) In addition to tuning the PhotoCORM absorption itself, alternative strategies might be based on its conjugation to established photosensitizers, the exploration of two-photon excitation and the use of up-converting nanoparticles A sig- BJP nificant number of such compounds have been explored in the last few years, in particular based on iron and manganese compounds (Niesel et al., 2008; Gonzalez et al., 2011; 2012a,b; Ward et al., 2012) A particularly interesting PhotoCORM has been reported by Pierri and Ford The rhenium(I) compound [Re(bpy)(CO)3(PR3)]+, with R = CH2OH, upon photolysis at 405 nm, undergoes specific liberation of only one of the three carbonyl ligands, the one trans to the phosphane group (Pierri et al., 2012) The most interesting feature of this system is due to the fact that both the starting material as well as the resulting aqua compound are luminescent and can be tracked inside cells by confocal fluorescence microscopy with excitation at 405 nm and detection at 465– 495 nm versus 660 nm, respectively, for the PhotoCORM and the follow-up product (Figure 5) Also, very recently some of the first non-metal-based CORMs with photoactivation have been reported (Antony et al., 2013; Peng et al., 2013) An alternative external stimulus to trigger CO-release locally from a prodrug system is the application of an alternating magnetic field to CORM-loaded magnetic nanoparticles, which leads to local heating and thus acceleration of the ligand exchange reaction of CO with medium, as demonstrated by Kunz et al (2013) Finally, a number of carrier systems have been used to confer some target specificity to CORMs This includes conjugation to biomolecules and macromolecules as well as soft and hard nanomaterials For example, Zobi and co-workers have attached rhenium-based CO-releasing groups to cobalamin as a potential naturally occurring carrier system (Zobi et al., 2012), whereas the Schatzschneider group as well as others use peptide conjugates for CORM conjugation (Pfeiffer et al., 2009; 2013; Matson et al., 2012) Other hard and soft nanomaterials used for the attachment of CORMs include silica nanoparticles as well as carbon nanomaterials (Dördelmann et al., 2011; 2012), dendrimers (Govender et al., 2013), and micelles (Hasegawa et al., 2010) Alternative approaches not depend on the attachment of molecular metal-carbonyl units to nanoscale carrier systems, but are instead based on the direct loading of porous nanomaterials like metal-organic frameworks (MOFs) with CO gas, which is liberated again upon controlled degradation of the material (Ma et al., 2013) Of course, good biocompatibility of both the organic and the inorganic parts of the MOFs has to be ensured for potential therapeutic applications In summary, a wide range of different trigger mechanisms to induce the CO release from the metal coordination sphere and a significant number of core structures now exist, and these have been explored for use as CORMs Future generations of CORMs to be added to this arsenal certainly have to be designed with very clear therapeutic applications in mind, in order to properly tune the release kinetics and stoichiometry to the desired range, and with a specific emphasis on the drug-like properties of any new compound Thus, much more emphasis has to be placed on the choice and variation of the ‘drug sphere’ in the future and the biomedical community has to provide information on the required amount of CO at a target site to elicit a physiological response (high or low) and the time scale over which a certain concentration level needs to be kept (fast or slow CO release) British Journal of Pharmacology (2015) 172 1638–1650 1645 BJP U Schatzschneider Detecting and tracking CO and CORMs The quantification of the number of CO ligands released from a CORM as well as the release kinetics and the nature of potential intermediates, if the CO ligands are released in a stepwise instead of a concerted process, is a very important parameter in the characterization of CORMs The simplest method for this purpose is the so-called ‘myoglobin assay’, in which the conversion of dexoymyoglobin (Mb) to carboxymyoglobin (MbCO) is followed spectrophotometrically, by the decrease in intensity of the band of Mb at 557 nm and the increase of the MbCO absorption at 540 and 577 nm The amount of CO released per molecular unit of carbonyl compound is then determined from the plateau value at the end of the measurement, which is reached once no more spectral changes can be observed at prolonged reaction time, and the kinetics can be obtained from a proper fitting of the increase in MbCO absorption with time For this type of experiment, one always has to ensure that the molar amount of Mb exceeds the maximum number of moles of CO that a CORM may potentially release, in order to provide at least one free Mb for each CO liberated and thus prevent premature signal saturation Although the myoglobin assay will probably remain the procedure of choice for initial screening for CORM activity since it is very easy to carry out, there are some severe limitations with this method First of all, especially for highly coloured metalcarbonyl compounds, which is usually the case with PhotoCORMs, there might be an overlap in the absorption bands of the myoglobin, the CORM, and potentially also the follow-up iCORM (inactivated CORM) products (Figure 6), which will require complicated spectral deconvolution techniques (Atkin et al., 2011) Furthermore, the assay has to be carried out under an atmosphere of dinitrogen or argon protective gas in order to keep the iron centre in the ferrous (+II) state and to prevent formation of oxymyoglobin from air However, some CORMs, particularly the ET-CORMs from the Schmalz group, require an oxidative process to complete the CO release from the metal coordination sphere (Romanski et al., 2011) The reduction of the myoglobin is usually carried out by the addition of a large excess of aqueous sodium dithionite, which causes additional problems due to Figure Application of a trigger to a CO-releasing molecule (CORM) prodrug leads to the release of CO from the metal coordination sphere In addition to the CO thus generated, inevitably a metal–coligand fragment, termed inactivated CORM (iCORM), is also formed, which might have biological activity on its own in addition to that of the CO Therefore, iCORMs should always be independently prepared, fully characterized and included in bioassays as a control 1646 British Journal of Pharmacology (2015) 172 1638–1650 sulphite species generated from the reductant, which might react with either the CORM or the myoglobin (McLean et al., 2012) Since commercially available dithionite is often not of the purity required for such experiments, it needs to be carefully checked and, if necessary, recrystallized before use in the myoglobin assay (McKenna et al., 1991) Another currently unresolved question is whether the release of the CO from the CORM and its binding to the myoglobin are two totally uncoupled processes, with free CO intermediately present in solution, or whether there might be a more intimate association between the CORM and the myoglobin, with a more or less direct transfer of CO between the two metal centres, at least in some special cases Therefore, a number of alternative methods have been explored to study the release kinetics of CO and to quantify the amount of CO liberated per mole of CORM They are usually based on a separation of the CORM solution from the sensor unit (Mann, 2010; Rimmer et al., 2012) and also circumvent the use of reducing or anoxic conditions The most accurate but also most expensive technique is GC, which is to be considered the gold standard in the field (Vreman et al., 2005; 2011; Bernardi et al., 2008) An even more challenging field is the in vitro/in vivo detection of either CO endogenously generated by HO activity or exogenously delivered from CORMs Only very recently, two fluorescent sensor systems have been described, which are initial proof of principle for this concept (Yuan et al., 2013) Thus, He and co-workers developed a genetically encoded fluorescent probe, which is based on the CO binding affinity of the CooA protein, which has been fused to yellow fluorescent protein (YFP) (see Figure 7A) Upon CO binding to the CooA domain, the whole construct, called ‘COSer’ (CO Sensor), undergoes a conformational change, which leads to the YFP emission lighting up (Wang et al., 2012) Significant signal response was found down to 1–2 μM CO and little interference was observed from imidazole, cyanide, NO and dioxygen However, a significant drawback of this system is the need to transfect cells to be studied with a COSercontaining expression vector, which will very likely severely limit its broader application In contrast, Chang et al followed an approach based on a synthetic small molecule, which is composed of a BODIPY (boron dipyrromethene difluoride) unit and an attached cyclometalated dimeric palladium(II) moiety, which has been termed ‘COP-1’ (CO Probe 1) (see Figure 7B) While this compound is non-luminescent, addition of CO leads to CO insertion in the carbon-palladium bond with subsequent carboxylation of the pendant substituent and the probe then lights up (Michel et al., 2012) Challenge experiments with a number of small molecules were carried out and a detection limit of about μM was estimated Although this small-molecule probe is much more suitable for general use, both in solution and in cell cultures, important questions remain Firstly, what is the fate of the palladium metal liberated during the CO binding process by the probe, and secondly, what is the membrane permeability of COP-1 and whether a uniform distribution inside cells can generally be reached In addition, the concentration ratio of COP-1 to CORM as well as the proper order and timing of the addition of both the fluorescent probe and the CORM to cell cultures seems to play a very important role in obtaining meaningful results Thus, although these two systems finally CO-releasing molecules (CORMs) BJP Figure (A) ‘CO Sensor’ (COSer) and (B) ‘CO Probe 1’ (COP-1) as fluorescent switch-on probes for CO provide some initial tools to study in vitro/in vivo CO generation and delivery, there is clearly an urgent need for further improvement in fluorescent CO detection in complex biological environments Summary, conclusion and outlook: challenges to turn CORMs into drugs CO is now well established as a small signalling molecule in higher organisms While its endogenous production by the action of HO enzymes on haem and the regulation of this process is quite well understood now at the molecular level, this is not the case with the direct down-stream effects of the CO generated Although the chemical properties of CO strongly suggest that binding to a transition metal centre will be required for its recognition, surprisingly little is known about its primary targets in the cell Although implicated for a long time, sGC is rather questionable since its activation by CO is very low compared to NO in the absence of additional synthetic effectors, for which no natural analogue has been found so far However, a fascinating prospect is the activation of ion channels by CO, although further light needs to be shed on the molecular details of this process It is quite possible that a rather labile metal site has so far been lost during isolation and purification of these delicate to handle membrane proteins and better application of current biophysical techniques such as Mössbauer and EPR spectroscopy as well as metal analysis will certainly be required to solve this riddle To modulate these signalling pathways by exogenous application of CO sources, for either fundamental biological studies or potential therapeutic applications, metal carbonyl complexes as CORMs have emerged as very valuable tools and a wide variety of such compounds with different trigger mechanisms (ligand exchange, enzymatic activation, photoactivation) is now available with modifiable release stoichiometry and kinetics Recent efforts are also under way to modify the outer periphery of such compounds, the so-called ‘drug sphere’, to modulate the target specificity of CORMs, either by careful choice of peripheral functional groups or attachment to carrier systems such as bio(macro)molecules or hard and soft nanomaterials Further progress in the field will depend on reliable methods to study the CO release from these carrier systems and in particular on the tracking of CORMs as well as CO in complex biological environments, both in vitro and in vivo Although some recent progress has been made in the latter field with the introduction of the first fluorescent probes for CO, further development is urgently needed The major challenge is now to design systems suitable for real clinical applications and take them beyond animal models of disease British Journal of Pharmacology (2015) 172 1638–1650 1647 BJP U Schatzschneider Acknowledgements Our own work in this field was greatly facilitated by COST 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consent does not extend to other kinds of copying, such as copying for general distribution for advertising or promotional purposes, for creating new collective works, or for resale.Special requests should be addressed to: permissionsuk@wiley.com ... http://dx.doi.org/10.1111/bph .2015. 172. issue -6 Abbreviations sGC, soluble GC © 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 1397–1414 1397 BJP A Papapetropoulos et al Tables of Links... Pharmacol 172: 1587– 160 6 Babu D, Motterlini R, Lefebvre RA (2015) CO and CO-releasing molecules (CO-RMs) in acute gastrointestinal inflammation Br J Pharmacol 172: 1557–1573 13 96 British Journal of Pharmacology. .. two isoforms: α and β (Hofmann et al., 20 06; Burley et al., 2007) The binding of cGMP to a regulatory 1400 British Journal of Pharmacology (2015) 172 1397–1414 region of the kinase results in