Activators of Soluble Guanylate Cyclase for the Treatment of Male Erectile Dysfunction

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Activators of soluble guanylate cyclase for the treatment of male erectile dysfunction

JD Brioni1*, M Nakane1, GC Hsieh1, RB Moreland1, T Kolasa1 and JP Sullivan1

1Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois, USA

Soluble guanylate cyclase (sGC) is an important enzyme in corpus cavernosum smooth muscle cells as it is one of the regulators of the synthesis of cGMP. The efficacy of sildenafil (ViagraTM)in the treatment of male erectile dysfunction indicates the importance of the cGMP system in the erectile response as the increased levels of cGMP induce relaxation of the corpus cavernosum. sGC is physiologically activated by nitric oxide (NO) during sexual stimulation, and its activity can be pharmacologically enhanced by several NO-donors. Agents like YC-1 can also activate sGC after binding to a novel allosteric site in the enzyme, a site different from the NO binding site. YC-1 can relax rabbit cavernosal tissue and it facilitates penile erection in vivo. This review summarizes the enzymology, biochemistry and pharmacology of this novel allosteric site and its relevance for the regulation of penile function. This type of sGC activators represent a new class of compounds with a different pharmacological profile in comparison to the classical NO-donors and they could be beneficial for the treatment of male erectile dysfunction. International Journal of Impotence Research (2002) 14, 8–14. DOI: 10.1038=sj=ijir=3900801

Keywords: guanylate cyclase; YC-1; penile erection; erectile dysfunction; corpus cavernosum; sildenaﬁl; cGMP

Introduction that was initiated by nitric oxide (NO). These processes are mediated in part by nerves expressing nitric oxide synthase I (neural-NOS), as sexual The treatment of male erectile dysfunction (MED) stimulation results in the peripheral release of NO. has evolved during the last decade from surgical This neurally-derived NO mediates the initial options or intracavernosal – intraurethral injections dilation of the helicine resistance arterioles as well to the recent development of oral therapies such as as the trabecular smooth muscle, resulting in an TM sildenafil (Viagra ). The efficacy of IC351 and increase of arterial blood flow which further acti- vardenafil in clinical trials has strengthened the vates the endothelial NOS (type II) releasing more notion that blockade of phosphodiesterase 5 (PDE5) NO down into the smooth muscle layers. NO in the corpus cavernosum is a successful approach. activates soluble guanylate cyclase (sGC) in the The efficacy of apomorphine in a sublingual for- corpus cavernosum smooth muscle. sGC is respon- mulation in phase III trials and its recent approval sible for the enzymatic conversion of GTP to cyclic by the CPMP in Europe also indicate that MED can GMP (cGMP), and the increase of cGMP mediates be treated by agents that act at the neuronal level via relaxation of cavernosal smooth muscle leading to central mechanisms. Several other targets may also penile erection. be useful for the treatment of MED, and the The discovery that NO-sGC-cGMP system is one biochemical and neurological bases to facilitate the of the major effectors in penile smooth muscle 1 erectile response has recently been reviewed. relaxation and erectile function has led to the Penile erection is the end result of corpus development of two classes of agents: (a) agents that cavernosum trabecular smooth muscle relaxation inhibit cGMP degradation like the PDE inhibitors; (b) agents that elevate cGMP levels through poten- tiation of cGMP synthesis. Sildenafil increases *Correspondence: JD Brioni, PhD, Project Leader, cGMP levels by inhibiting PDE5, the enzyme that Neuroscience Research, Global Pharmaceutical Research & degrades cGMP. NO and NO-donors are well-known Development, Abbott Laboratories, Abbott Park, IL 60064, sGC activators and the examples of drugs that work USA. E-mail: [email protected] as NO-donors include nitroglycerine, minoxidil, Received 27 June 2001; revised 10 September 2001; sodium nitroprusside, and S-nitrosylated deriva- accepted 6 October 2001 tives of other drugs. So far, among the various sGC Activators of soluble guanylate cyclase JD Brioni et al 9 catalyze the conversion of GTP to cGMP. They are expressed in particulate and soluble forms, and while they share similar structural characteristics, they differ in their mechanisms of physiological regulations. Most importantly, sGC contains a heme group and binds NO that activates the enzyme, while particulate GC is stimulated by natriuretic peptides. sGC has been purified to apparent homogeneity and it exists as a heterodimer (Figure 2). The larger molecular weight subunit is designated a (82 kDa) and the smaller subunit, b (70 kDa). The first sGC Figure 1 Chemical structure of YC-1, Isoliquiritigenin and ODQ. 2 YC-1 and Isoliquiritigenin are activators of soluble guanylate cDNA isolated was for the b subunit from the rat. cyclase. However, no enzyme activity was observed when the b subunit cDNA was transfected into L cells, an established cell line derived from mouse connective tissue.3 A novel sGC subunit was isolated from rat kidney using the catalytic domain sequence of sGC by PCR 4 and subsequently designated the b2 subunit. This subunit is preferentially expressed in rat kidney and liver. Recently, co-expression of the rat b2 subunit revealed that a1b2 heterodimers are inactive and that 5 this subunit may be an inhibitor of the a1b1 subunit. Harteneck et al isolated a cDNA coding for a second novel subunit of sGC from human fetal brain and 6 designated as a2 subunit. Co-expression of the a2 Figure 2 Schematic representation of soluble guanylate cyclase subunit with the b1 subunit showed active sGC showing the catalytic site and the two allosteric sites. One activity indistinguishable from a1b1 heterodimer, allosteric site is defined by the NO binding site (the heme) and the demonstrating the interchangeability of the subunit second allosteric site is represented by the binding of YC-1. sGC isoforms. exists as a heterodimer of a (82 kDa) and b (70 kDa) subunits. Giuili et al have also isolated cDNAs correspond- ing to both a and b subunits of sGC from human activators, only NO-donors have been considered as 7 brain, and designated them a3 and b3. However, drug candidates for MED. Zabel et al re-sequenced these cDNA and found that A novel type of sGC activators is represented by 8 they were in fact authentic human a1 and b1. To YC-1 (Figure 1). This agent is not an NO-donor, but date, only a1, a2, b1 and b2 have been cloned and causes activation of sGC especially in the presence sequenced. of NO. The binding site for NO was the only Although two isoforms for each subunit have site reported to modulate sGC activity (Figure 2). been cloned, a1 and b1 are the main isoforms However, the finding that YC-1 activates sGC expressed in most of the mammalian tissues includ- by binding to an allosteric site on the enzyme ing brain, lung and liver.3 Behrends et al character- opened the possibility to discover a new class of ized the sGC in human corpus cavernosum by compounds with a different pharmacological profile revserse transcription-polymerase chain reaction in comparison to the NO-donors. (RT-PCR), and found that a1 and b1 are also the In this review we summarize the biochemical and main isoforms expressed in human corpus caverno- pharmacological data that indicate that activation of sum.9 Studies conducted in our laboratory also sGC via an allosteric site in the enzyme can lead to confirmed the expression of a1 and b1 subunits as facilitation of penile responses in animals. The the main isoforms in human corpus cavernosum.10 regulation of sGC activity by this type of sGC activators may represent a novel approach to regulate penile erection. Enzymology of soluble guanylate cyclase

Cloning and expression of soluble guanylate sGC is a heterodimeric protein consisting of a and 11 cyclase subunits b subunits. The a and b subunits contain a C- terminal homologous domain to adenylate cyclase that constitutes the catalytic center.12 The N-term- The guanylate cyclases [GTP pyrophosphatase-lyase inal domains of both subunits are essential for the (cyclizing); EC 4.6.1.2] are a family of enzymes that stimulation of the enzyme by NO, although heme

International Journal of Impotence Research Activators of soluble guanylate cyclase JD Brioni et al 10 binding occurs in the b subunit. sGC contains one submaximally activating NO. In the presence of YC- mole prosthetic heme per heterodimer attached to 1, the NO concentration – response curve is shifted histidine 105 of the b subunit, which is required for to the left, indicating that YC-1 sensitized sGC the activation of the enzyme by NO.13 towards NO. Several agents that modulate the activity of sGC have been identified (Figure 1). ODQ, a quinoxaline derivative, is a potent inhibitor of sGC in brain slices Biochemistry of the NO-cGMP system: (IC50 ¼ 20 nM), providing an important tool to allosteric sites in sGC identify cGMP-dependent signaling. ODQ binds to sGC in an NO-competitive manner and inhibits NO- stimulated activity, leaving basal activity un- The structure of sGC yields clues as to its mechan- changed. However, the inhibitory effect of ODQ is ism of action. The two subunits described above due to oxidation of the heme ion, indicating that share extensive homology in the C-terminal region ODQ may affect various heme-containing enzyme to the active site of adenylate cyclase.12 Membrane activities.14 The ODQ analog, NS 2028, was de- bound adenylate cyclase is activated by either the scribed as another potent inhibitor of sGC binding of specific G-proteins or by the terpenoid 15 16 17 (IC50 ¼ 30 nM). Methylene blue and LY-83583 forskolin. sGC is activated by NO via a heme were reported as inhibitors of sGC, but they are less prosthetic group bound in the fifth position to potent than ODQ and NS 2028. Furthermore, they histidine 105 of the b1 subunit (Figure 3). This inhibit not only sGC activity but they affect the process is thought to occur by the rapid binding biological activity of NO synthase and glucose (t1 ¼ 0.21 ms) of NO to the sixth position in the 2 transporters.18,19 ferrous heme yielding a six liganded complex.25 Isoliquiritigenin, isolated from Dalbergia odori- This intermediate then undergoes a relatively slow fera T, was originally identified as a vasorelaxant transition (t1 ¼ 630 ms) breaking the protein histi- 2 and later was found to activate sGC.20 It induces dine bond, oxidizing the iron to the ferric state and relaxation of rat aortic rings and increases intracel- leading to activated sGC. In this activated state, both 2þ lular Ca in heart muscle, but the effects of the Km for GTP decreases and the Vmax for cGMP isoliquiritigenin may be due to inhibition of PDE formation increases.26 Since the X-ray crystallo- activity rather than activation of sGC.21,22 graphic structure of sGC has yet to be reported, it YC-1, on the other hand, was initially identified is unclear exactly what transpires to activate sGC. as an inhibitor of platelet aggregation.23 Soon after Sedimentation or CD-ORD spectral analyses do not the discovery of the pharmacological effect of YC-1 detect gross changes in conformational structure. on platelets, YC-1 was found to be a modulator of With analogy to forskolin-activated adenylate sGC activity.24 YC-1 stimulates basal sGC and it is cyclase, there could be a subtle conformational not blocked by NO scavengers indicating that the change, favoring a more efficient GTP cyclizing effect of YC-1 is NO-independent. Interestingly, activity at the active site. sGC is unusual as a YC-1 highly potentiates the stimulatory effect of hemoprotein in that the protein-heme bond breaks

Figure 3 Diagrammatic representation of NO activation of soluble guanylate cyclase. Pentagons represent histidine 105 on the sGC b1 subunit. GSH: glutathione; GSSG: oxidized glutathione; GSNO: S-nitroso-glutathione. Spectral maximums (lmax) are indicated for the heme absorbance. Rate constants have been extrapolated for 37 C (Denninger and Marletta,12 and Zhao et al25).

International Journal of Impotence Research Activators of soluble guanylate cyclase JD Brioni et al 11 as part of activation, a process not seen in hemoglo- therapeutically. Ideally one would wish to develop bin, P450s, oxidases or the cytochromes.12 Once an sGC activator that binds to the allosteric non- activated, thiols such as glutathione can reduce the heme site that in itself has little effect on increasing ferric-NO heme complex to ferrous heme-histidine the basal activity, but that in the presence of NO, 105 bond once again.27 The sGC inhibitor ODQ this activator would greatly amplify the effects of works by oxidizing the ferrous heme in the first NO. complex to a five liganded ferric intermediate and The endogenous ligand that binds to the YC-1 site this prevents both binding and activation by NO. as well as the potential role of this site on the self- Allosteric sites are defined as topographically regulation of sGC activity is unknown at the present distinct sites from the catalytic domain on an time, but it may represent an attractive area of enzyme or protein.28 An allosteric effector is defined scientific inquiry in the future. These multiple sites as a substance that changes the kinetic properties of for allosteric regulation may provide sGC with a an enzyme but structurally it does not resemble the sensitive and versatile way to respond to intracel- substrate, and allosteric effects are thought to occur lular changes in the cell leading to relaxation or through conformational changes in the enzyme contractile events. upon binding of the molecules.29 For sGC, allosteric modulators would be any molecules that do not resemble GTP. Examples would include activators In vitro and in vivo pharmacology like NO, NO-donors, YC-1 and inhibitors such as ODQ. In biological systems, the most common allosteric effect is feedback or product-induced sGC is expressed in most cells of the cardiovascular inhibition characterized in many biosynthetic path- system and in many other cell types.33 The levels of ways (for example, gluconeogenesis, tricarboxcylic sGC mRNA and protein often change in response to acid cycle, amino acid synthetic pathway). How- different physiological conditions and development ever, allosteric effects can include activation rather stages. By formation of cGMP as a second messen- than inhibition. In these cases, changes in the ger, sGC plays an important role in different kinetic behavior of the enzyme include decreases physiological processes and is considered the key in the Km (increased affinity for substrate requiring enzyme mediating vascular relaxation induced by less for activity) and=or increases in Vmax, the NO and NO-releasing agents. The ability of NO to maximum velocity of the enzyme. The latter can relax smooth muscle has been described in multiple be thought of as making the enzyme more efficient models and muscle types, including vascular so that more product per unit time is produced. In smooth muscle,34 tracheal smooth muscle,35 corpus the case of sGC, both NO, NO-donors (sodium cavernosum smooth muscle,36,37 and myome- nitroprusside) and YC-1 act as allosteric activators trium.38 Carbon monoxide (CO), another physiolo- by increasing both Km and Vmax (see later). gical activator of sGC, is also capable of binding to While NO mediates its physiological actions via the heme group of sGC and converts GTP to cGMP binding to an allosteric site in sGC (the heme), YC-1 after activation. However, NO is more potent binds to a second allosteric site in the enzyme.30 activator of sGC than CO, and the purified enzyme Although YC-1 has been described in the literature is activated 100 to 200-fold by NO, but only about 4- as an NO-independent activator, this issue warrants fold by CO.39 further clarification. YC-1 binds to a ‘heme-inde- In recent years, the introduction of the potent and pendent site’ but its effects on enzyme activity can specific sGC inhibitor ODQ40 has helped to identify be defined as ‘NO-independent’ (effect on basal more precisely the sGC-mediated effects in various activity in the absence of NO) and ‘NO-dependent’ tissues. Using rat artery rings, Homer et al, demon- (effect on enzyme activity in the presence of NO). strated that ODQ (3 mM) completely abolished the YC-1 binds to sGC at a different site from the relaxant responses to glyceryl trinitrate, isosorbide heme and this is evidenced by studies in which YC- dinitrate and sodium nitroprusside.41 This inhibitor 1 can act as an allosteric activator in the absence of has been extensively used in vitro to examine the NO and lead to even more activation in the presence specificity of sGC activation and it was shown that of NO, decreasing the Km for GTP and increasing the the sensitivity of ODQ-mediated inhibition of 26 Vmax of cGMP formation. This site has recently vascular relaxation appeared to be different among been mapped using a photoaffinity labeling techni- NO donors.42 The vasorelaxation can be induced que to a subunit cysteines 238 and 243 of human through exclusive activation of the heme site of sGC sGC.31 This report is not in agreement with earlier for the NO donors such as sodium nitroprusside. On work that suggested that cysteine 541 of the b the other hand, some NO donors such as S- subunit was important for the YC-1 interaction and nitrosothiols may also activate cellular vasodilating that YC-1 binds to the heme-binding domain.32 This mechanisms through interaction with sulfhydryl may reflect the nature of the conformational change site(s) of sGC and such biochemical mechanism(s) as well as the initial binding of YC-1. For sGC to may not be available to other NO donors that only possess two different allosteric sites is fortuitous bind to conventional NO binding site.42

International Journal of Impotence Research Activators of soluble guanylate cyclase JD Brioni et al 12 YC-1 is a direct activator of sGC through a this allosteric site in the enzyme can lead to penile pathway that does not involve the interaction with erection in mammals.50,51 YC-1 in combination with the heme moiety and causes a pronounced activa- SNP caused a strong decrease in mean arterial tion of the enzyme in the presence of NO.30 YC-1 pressure in hypertensive as well as normotensive induced a concentration-dependent relaxation rats after intravenous injections.52 The hypotensive (EC50 ¼ 2.9 mM) in rat aortic rings precontracted with effect may be related to the activation of sGC in phenylephrine, and the effects of YC-1 were blocked vascular tissue, and in view of the cardiovascular by ODQ, whereas YC-1 (30 mM) exhibited no effects liabilities of sildenafil in MED patients, the cardio- on the contractile force of ventricular muscle vascular effects of YC-1 deserve further investiga- strips.43 cGMP-increasing effects of YC-1 have been tion. Although YC-1 has already been proposed for reported in vascular smooth muscle cells and an the treatment of cardiovascular and thrombotic increase in responsiveness toward NO has been disorders, YC-1 as well as activators of sGC may shown to be substantially higher in intact cell than also be beneficial for the treatment of MED. in cell-free purified enzyme system.24 Stimulation of the vascular endothelial cells with YC-1 increased cGMP accumulation up to 100-fold, an effect that Male erectile dysfunction: physiology and 44 was further potentiated by NO. The maximal effect pathophysiology of YC-1 was diminished in the presence of NOS inhibitors. As YC-1 does not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP The tone of the corpus cavernosum smooth muscle accumulation is apparently caused by the synergis- is controlled at the level of the peripheral as well as tic activation of sGC by YC-1 and basal endogenous the central nervous system (CNS). Within the CNS, NO released from the endothelial cells. Using an many different neurotransmitters (such as dopamine aortic rings organ bath preparation, O’Reilly et al and oxytocin) as well as different brain regions (for also demonstrated that a non-vasorelaxant concen- example, cortical association centers, medial pre- tration of YC-1 enhanced the ability of organic optic area of the hypothalamus) are involved in nitrates to relax vascular smooth muscle and elevate penile erection. The reader is referred to more intravascular cGMP levels.45 detailed reviews on the subject.1,46 Penile erection involves relaxation of corporal Ultimately, corpus cavernosum smooth muscle smooth muscle during sexual stimulation.46 During tone regulates penile flaccidity and erection. During the last decade, the application of in vitro model flaccidity, the helicine resistance arterioles are systems of isolated corpus cavernosal tissue has constricted, principally through a-adrenergic me- significantly enhanced our understanding of the chanisms although endothelin and constrictor pros- biochemical mechanisms of potentially therapeutic tanoids may play a role. Penile erection is the end drugs for the treatment of MED.1 Primary cultures of result of smooth muscle relaxation and this relaxa- corpus cavernosal cells from human or animals have tion can be initiated by sensory stimulation that provided useful insights into factors that can activates CNS pathways. NO from NANC nerves modulate the intracellular components important mediates the dilation of the helicine arterioles as to the corporal activity. The corpus cavernosal well as the trabecular smooth muscle via activation smooth muscle in organ bath preparations has also of sGC. NO diffuses into the smooth muscle also been widely used to assess the pharmacological activating sGC and further enhancing relaxation. As effects of the contractile or relaxation agents. the corpus cavernosum sinuses relax and fill with Submicromolar concentrations of NO have been blood, intracavernosal pressure and volume in- shown to cause significant changes in smooth creases and veno-occlusion ensues. muscle tone and increases in cGMP levels in the MED is defined as the consistent inability to corpus cavernosum cells, and a positive correlation attain or maintain penile erection satisfactory for was found between the magnitudes of relaxation intercourse. It has become recognized that 75% of and cGMP formation.37 Recently, Liu et al, reported this condition has organic origins. Pathophysiology that YC-1 enhanced the relaxing effects of sodium of MED can be thought to have a structural nitroprusside in rabbit corpus cavernosum strips in component as well as a metabolic component.53 a concentration-dependent manner and demon- The penis is comprised of soft tissue and functions strated that relaxation is mediated via increasing as a blood-filled capacitor of sufficient rigidity intracellular cGMP levels.47 during erection for vaginal penetration. The corpora Pharmacological studies in vivo support these in cavernosa, that are integral to this function are vitro findings. The systemic administration of YC-1 composed of a specialized vascular bed that has a significantly prolonged tail bleeding in mice at 3 – high content of connective tissue (48 – 55%). The 30 mg=kg doses, consistent with the inhibition of corpus cavernosum smooth muscle cells also platelet aggregation exhibited by YC-1 in vitro.48,49 synthesize connective tissue that contributes to the Systemic injections of YC-1 facilitate penile erec- structural integrity of the corpora, and a functional tions in rats, indicating that the activation of sGC via corpus cavernosum smooth muscle=connective

International Journal of Impotence Research Activators of soluble guanylate cyclase JD Brioni et al 13 tissue ratio is necessary for veno-occlusion.53 Thus, References regardless of the amount of corpus cavernosum smooth muscle relaxation, veno-occlusion cannot 1 Moreland RB, Hsieh G, Nakane M, Brioni JD. The biochemical occur in some patients due to higher content of and neurologic basis for the treatment of male erectile connective tissue and an inability to occlude the dysfunction. J Pharmacol Exp Therap 2001; 296: 225 – 234. draining venules. One area of active research in 2 Nakane M et al. Molecular cloning of a cDNA coding for 70 MED is to identify vasoactive factors, cytokines, kilodalton subunit of soluble guanylate cyclase from rat lung. autacoids and=or neurotransmitters which may play Biochem Biophys Res Commun 1988; 157: 1139 – 1147. 3 Nakane M et al. 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