Discovery and development of phosphodiesterase 5 inhibitors

The human genome contains at least 21 genes involved in determining the intracellular levels of cAMP and cGMP by the expression of phosphodiesterase proteins or PDE's. These PDE's are grouped into at least 11 functional subfamilies, named PDE1-PDE11. PDEs are enzymes that hydrolyze cyclic adenosine 3,5-monophosphate (cAMP) and cyclic guanosine 3,5-monophospahate (cGMP), which are intracellular second messengers, into AMP and GMP. These second messengers control many physiological processes. The cAMP is formed from ATP by the enzyme adenylyl cyclase and cGMP is formed from GTP by the enzyme guanylyl cyclase which are either membrane bound or soluble in the cytosol. When soluble it functions as a receptor for nitric oxide (NO) (see figure 1). Formation of cGMP initiates several reactions in the body including influence on cGMP ion channels, cGMP binding proteins and protein kinase G (PKG). The effect on PKG reduces levels of calcium leading to relaxation of smooth muscles (see figure 2). The PDE5 enzyme is specific for cGMP which means it only hydrolyzes cGMP but not cAMP. The selectivity is mediated through an intricate network of hydrogen bonding which is favorable for cGMP but unfavorable for cAMP in PDE5. == Distribution of PDE5 in the body ==

In humans the distribution of PDE5A1 and PDE5A2 isoforms is the same and can be found in the brain, lung tissue, heart, liver, kidneys, bladder, prostate, urethra, penis, uterus and skeletal muscles. PDE5A2 is more common than PDE5A1. PDE5A3 is not as widespread as the other two isoforms, and is only found in smooth muscle tissues; it is found in the heart, bladder, prostate, urethra, penis and uterus. Exact distribution of PDE5A4 isoform was not found in the literature. PDE5 enzyme in humans has also been reported in platelets, gastrointestinal epithelial cells, Purkinje cells of cerebellum, corpus cavernosum, placenta and colon, clitoral corpus cavernosum as well as vaginal smooth muscle and epithelium. == PDE Structure and SAR ==

PDE enzymes are composed of 3 functional domains: an N-terminal cyclin fold domain, a linker helical domain and a C-terminal helical bundle domain (see figure 3). The active site is a deep pocket at the junction of the 3 subdomains and is lined with highly conserved residues between isotypes of PDE. The pocket is approximately 15 Å deep and the opening is approximately 20 by 10 Å. The volume of the active site has been calculated to be between 875 and 927 Å3. == Role in diseases ==

Erectile dysfunction Drugs that inhibit PDE5, sildenafil, tadalafil and vardenafil, have been used as treatment for erectile dysfunction. These inhibitors increase the cGMP, smooth muscle relaxation and consequently cause penis erection Pulmonary arterial hypertension Upregulation of PDE5 gene expression has been observed in animal models of pulmonary hypertension, and is thought to contribute to vasoconstriction in the lung. Long-term treatment with a PDE5 inhibitor has been shown to enhance natriuretic peptide-cGMP pathway, downregulate Ca2+ signaling pathway and alter vascular tone in pulmonary arteries in rat models. Benign prostatic hyperplasia As of 2011, the long-acting agent tadalafil is licensed for the treatment of urinary symptoms resulting from benign prostatic hyperplasia. ==Future indications for PDE5 inhibitors==

Cardiovascular diseases PDE5 inhibitors have broad-ranging effects on the cardiovascular system beyond their acute haemodynamic influence. For example, PDE5 inhibitors have been shown to improve several parameters of endothelial function. It is unclear to what extent this observation reflects the protective effects of PDE5 inhibitors against cardiovascular and renal disease. Raynaud's phenomenon Sildenafil has been shown to be at least as effective as calcium channel blockers in treating severe Raynaud's phenomenon (RP) associated with systemic sclerosis and digital ulceration. When given sildenafil for 4 weeks subjects had reduced mean frequency and duration of Raynaud attacks and a significantly lowered mean Raynaud's condition score. The capillary blood flow velocity also increased in each individual patient and the mean capillary flow velocity of all patients increased significantly. These results came without significant reductions of the systemic blood pressure. However, the therapeutic effects of PDE5 inhibitors in primary (idiopathic) RP are less well defined. Stroke Sildenafil has been shown to significantly improve neurovascular coupling without affecting overall cerebral blood flow by increasing brain levels of cGMP, evoking neurogenesis and reducing neurological deficits in rats 2 or 24 hours after stroke. These experimental data suggest that PDE5 inhibitors may have a role in promoting recovery from stroke. However, studies in humans remain inconclusive. Premature ejaculation Adding PDE5 inhibitors to SSRI drugs (e.g. paroxetine) for the treatment of premature ejaculation could result in better ejaculatory control according to recent studies. Possible mechanism is based on nitric oxide (NO)/cGMP transduction system as a central and peripheral mediator of inhibitory non-adrenergic, non-cholinergic nitrergic neurotransmission in the urogenital system. Female sexual arousal disorder PDE5 is expressed in clitoral corpus cavernosum and in vaginal smooth muscle and epithelium. Therefore, it is possible that PDE5 inhibitors could affect female sexual arousal disorder but further research is needed. Increased levels of cGMP have been shown to occur in human-cultured vaginal smooth muscle cells treated with a PDE5 inhibitor suggesting involvement of the NO/cGMP axis in the female sexual response. Sexual Exhaustion Disorder The similarity of many PDE5 inhibitors to the structure of many of the analogs of caffeine that are also adenosine antagonists suggests that in the future, it may be possible to design an PDE5 inhibitor that, like caffeine, is also an adenosine antagonist. ==Discovery==

PDE5 is an enzyme that was first purified in 1980 from a rats lung. PDE5 converts intracellular cGMP to the nucleotide GMP. Many tissues contain PDE5, such as lungs, kidneys, brain, platelets, liver, prostate, urethra, bladder and smooth muscles. Because of the localization of PDE5 in the smooth muscle tissue, inhibitors were developed for the treatment of erectile dysfunction along with pulmonary hypertension. Sildenafil did not prove effective for coronary heart disease but an interesting side effect was discovered, a penile erection. That side effect soon became the main field of investigation. The inhibitor is highly selective for the PDE5 family. Because of severe adverse effects and patients dissatisfaction with current therapy choices other inhibitors have recently been approved for clinical use. These inhibitors are udenfil, avanafil lodenafil and mirodenafil. ==Development==

Biological activity Penile erection Penile erection is a hemodynamic event in the smooth muscle of corpus cavernous. PDE5 is the main cGMP hydrolysing enzyme found in penile corpus cavernous. Erection is triggered by release of the neurotransmitter nitric oxide (NO) from non-adrenergic and non-cholinergic neurons from nerve ending in the penis as well as from endothelial cells. NO activates soluble guanylyl cyclase in smooth muscle cells in the penis which results in increased production of 3'-5'-cyclic guanosine monophosphate from guanosine-5'-triphosphate (GTP). Cyclic GMP binds to the cGMP-dependent protein kinase (PKG1) which phosphorylates several proteins that results in decreased intracellular calcium. Lower intracellular calcium leads to smooth muscle relaxation and ultimately penile erection. This pathway is demonstrated in figure 1. Erectile dysfunction PDE5 degrades cGMP and therefore inhibits erection. As demonstrated in figure 1, inhibition of PDE5 reduces degradation of cGMP and leads to penile erection. Because of this action PDE5 inhibitors have been developed for the treatment of penile erectile dysfunction. The phosphodiesterase 5 enzyme The PDE5 enzyme has a molecular mass of 200 kDa and its active state is a homodimer. Conformational change occurs when cGMP binds to the allosteric site that exposes serine and permits phosphorylation. The results for the phosphorylation of serine leads to increased cGMP hydrolysis at the catalytic domain. The affinity of the catalytic domain for cGMP increases and further increases the PDE5 catalytic domain activity. Phosphorylation of a single serine by PKG1 and the allosteric cGMP binding site activates the PDE5 catalytic activity and the result is a negative feedback regulation of cGMP/NO/PKG1 signalling. cGMP therefore interacts with both allosteric and catalytic domain of the PDE5 enzyme and PDE5 inhibitors compete with cGMP for binding at the catalytic domain resulting in higher cGMP levels. The active site of PDE5 is located at a helical bundle domain at the center of C domain (catalytic domain). The substrate pocket is composed of four subsites: M site (metal-binding site), Q pocket (core pocket), H pocket (hydrophobic pocket) and L region (lid region) as demonstrated in figure 3.{{cite journal | author = Sung, B. J. |author2=Hwang, K. Y. |author3=Jeon, Y. H. |author4=Lee, J. I. |author5=Heo, Y. S. |author6=Kim, J. H. | year = 2003 | title = Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules | journal = Nature | volume = 425 | issue = 6953 | pages = 98–102 | pmid = 12955149 |doi=10.1038/nature01914 ==Side effects==

PDE5 inhibitors are generally well tolerated, with side effects including transient headaches, flushing, dyspepsia, congestion and dizziness. However, no highly selective PDE5 inhibitors are currently in development. Patients who take nitrates, alpha blockers or sGC stimulators within 24 hours of PDE5 inhibitor administration (or 48 hours for tadalafil) may experience symptomatic hypotension, so concurrent use is contraindicated. PDE5 inhibitors are also contraindicated in patients with hereditary eye conditions such as retinitis pigmentosa due to the small increased risk of nonarteritic ischaemic optic neuropathy in patients taking the medication. Hearing impairment is one risk factor for those who are using PDE5 inhibitors and it has been reported for all available drugs on the market. This problem may be due to high level effect cGMP on cochlear hair cells. It has been reported that PDE5 inhibitors (sildenafil & vardenafil) cause transient visual disturbances likely due to PDE6 inhibition. Several reports are about approaches to improve PDE5 inhibitors, where as chemical groups have been switched out to increase potency and selectivity, which should potentially lead to drugs with fewer side effects. ==Structure–activity relationship (SAR)==

Sildenafil, the first PDE5 inhibitor, was discovered through rational drug design programme. The compound was potent and selective over PDE5 but was lacking preferable pharmacological properties. Structure-activity relationship (SAR) is demonstrated in figure 5, figure 6 and figure 7. Figure 5 demonstrates the three main groups of sildenafil, R1, R2 and R3. R1 is the pyrazolopyrimidinone ring, R2 the ethoxyphenyl ring and R3 is the methylpiperazine ring. R1 group is responsible for the binding of the drug to its active binding site of PDE5. Solubility was further increased by placing a methyl group at R positions as demonstrated in figure 7. Other phosphodiesterase-5 inhibitors were developed from the structure in figure 7. ==Other research==

Although PDE5 inhibitors main use has been for erectile dysfunction there has been a great interest in PDE5 inhibitors as a promising new therapeutic agents for treatment of other diseases, such as Alzheimer's disease. Elevation of cGMP levels through inhibition of PDE5 provides a way of improving memory and learning. PDE5 has also been considered as a potential therapeutic agent for parasitic disease such as African sleeping sickness. Strategic changes were made to the structure of sildenafil so the molecule could project into a parasite-specific pocket (the p-pocket). Similar approach has been used to design therapeutic agents Plasmodium falciparum. == PDE5-inhibitors in clinical trials ==