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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 International Journal of Impotence Research (2002) 14, 8–14 ß 2002 Nature Publishing Group All rights reserved 0955-9930/02 $25.00 www.nature.com/ijir 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).
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