Targeting the Cyclic AMP Signal Pathway

1.               Table 1 Receptor binding measured as % displacement of fluorescent natural ligand

ED50 for compound X = 1.939e-007

ED50 for compound Y =7.085e-008

 Table 2  The increase in heart rate in rats due to adrenaline as % of the increase observed in control rats

ED50 for compound X= 1.639e-006

ED50 for compound Y= 1.176e-007

2.  Compound X and Y are beta blockers which show an antagonist activity when it binds to the beta adrenergic receptors. The ligand binding occurs in the pocket formed by the 7 transmembrane helices. The amine group from the compounds forms an iconic bond with a COO^– in TM domain 3. The beta OH groups forms a hydrogen bond with the NH from domain 6. The benzene ring forms a hydrophobic bond with phenylalanine in TM domain 6. The OH groups on the compound from hydrogen bonds with the OH groups of two serine’s in TM domain 5. Due to this binding, it causes a conformational change in the receptor that is transmitted through an essential cysteine residue at base of domain 6. The 3^rd cytoplasmic loop is opened and it forms a binding site for the G protein. This is the site at which the G-protein Gs binds and becomes activated.

Beta adrenergic receptors are found in the heart and when they are stimulated, they increase the heart rate. This causes the heart to contract. Compound X and Y are beta blockers because they have a similar structure to adrenaline.  Most beta blockers inhibit the same structure. When beta receptors are coupled to Gs-proteins, it activates adenylyl cyclase to form Camp from ATP. An increase in cAMP activates a cAMP dependent protein kinase that phosphorylates L-type calcium channels. This causes an increase calcium so there is more calcium entering the body. An increase in calcium entry leads to an increase of calcium  which causes the heart to contract.

 Compound X or Y can bind onto the receptors and prevent this from happening. From table 1, it shows that at higher concentration, compound X and Y binds with the receptor more efficiently at 99% than the ones at a lower concentration. Table 2 also supports this as it shows that a higher concentration, it shows a decrease in the heart rate. By having a decrease in heart rate, it will reduce the number of heart failures. Compound Y has a better effect when it binds to the beta 1 adrenergic receptors compared to compound X.  Isoprenaline is medication used for the treatment of heart block and it’s an analogue of adrenaline. From table 3,  it can be concluded that the PKA activity of isoprenaline on it own is lower compared to forskolin and dbcAMP. Forskolin is an activator of cAMP and dbcAMP is a cell soluble cAMP analogue. When isoprenaline is combined with Compound X and Y, the PKA activity is much lower. Isoprenaline on its own has an  activity of 1.25 where as when its combined, it has an activity of 0.23 and 0.22. This significantly reduced the PKA activity.  With forskolin and dbcAMP, when the compounds were combined, the PKA activity did not decrease effectively compared to isoprenaline. PKA activity can be used a measure of cAMP because second messengers bind to intracellular proteins and change their biological activity. These are often protein kinases that can in turn phosphorylate and change the activity of target protein. The activity of PKA is dependent on the on the levels of cAMP. Therefore, the decrease in activity of PKA will suggest that there is low level of cAMP in the body.

This can further be supported by figure 2, the CREB phosphorylation.

3.  As classified before, Compound X and Y are beta blockers. Most beta blockers contain an amino group because the amine is the main site of drug metabolism. In vivo, the metabolism of drugs depends on MAO and COMT. However in vitro, there are other factors such as oxidation and racemization. Due to this reason, the potency of the compounds will be different. As mentioned before, there are different factors which can affect the potency of in vitro such as sunlight which can be oxidised. In vivo, they are metabolised by MAO and COMT. The role of MAO is to convert catecholamines into aldehydes. Following this, aldehyde dehydrogenase converts the aldehyde into a carboxylic acid which is excreted in the urine. If we compare the structures of Compound X and Y, it can be seen that Compound X contains two OH groups on the structure which affects the COMT metabolism, however in compound Y, there is only one OH group which suggests that it is less influenced by COMT metabolism which makes compound Y more potent. Increased bulkiness of the alkyl substituents on N atom produces Beta specificity and resistance to MAO. Compound Y has bulkier substituents on the N atom than Compound X which makes Compound Y more potent. Compound X is more polar due the extra OH group and to be an effective drug, the drug should be non-polar so that it can cross the cell membrane. This makes Compound Y more effective as it is non polar.

the amino group is important

because the amine is a main site of drug metabolism and so

drug inactivation. Also, this functional group causes the

compounds which are predominantly ionized at physio-

logic pH and protonated amine group is required for b

receptor binding.

the amino group is important

because the amine is a main site of drug metabolism and so

drug inactivation. Also, this functional group causes the

compounds which are predominantly ionized at physio-

logic pH and protonated amine group is required for b

receptor binding.

4. Drug Targets in the cyclic AMP Signal Pathway that are not GPCRs

In the human body, there are messengers. First messengers are known as extracellular signals, but they cannot enter cells directly so they are converted into intracellular signals which are known as secondary messengers. Intracellular second messengers include cAMP, cGMP, nucleotides, lipids and small molecules.(Tilley, 2011) Biochemical reactions in the body occur due to the recognition process between intracellular second messengers and extracellular receptors. Adenylate cyclase converts ATP into cAMP which stimulates cAMP-dependent protein kinase (PKA). Some proteins can be phosphorylated by PKA. (Tilley, 2011)The phosphorylation of the cAMP response element binding protein (CREB) is important in the regulation of gene transcription. (Tilley, 2011)G protein coupled receptors are the largest family of membrane receptors which are targeted by drugs. Over 700 drug targets GPCRS and when the GPCR binds to a specific ligand, it becomes activated. There is a conformational change in the receptor. This causes the enzyme adenylyl cyclase to activate which catalyses the conversion of ATP into cAMP. However, there are new potential drug targets in the cAMP signal pathway that are not GPCRS. The potential drug targets are adenylyl cyclase, PDE and PKA.

As stated previously, the role of the enzyme adenylyl cyclase is to convert ATP into cAMP. Many drugs target cAMp signalling pathway through GPCR, however adenylyl cyclase has not been considered as drug target due to their potential side effects. Over the years, they have been studies on the physiological functions of the different mammalian AC isoforms. (Pierre et al., 2009)There are 9 AC isoforms which are expressed in different tissues. ACs 1 to 8 are activated by forskolin which is a diterpene. Forskolin is a drug which targets adenylyl cyclase and increases the cAMP concentration. The activation of adenylate cyclase occurs via the direct action of the diterpene on the catalytic subunit of adenylate cyclase enzyme without interacting with cell surface receptors. (Sapio et al., 2016)Targeting the cAMP levels has shown anticancer effects such as the induction of mesenchymal to epithelial transition and the inhibition of proliferation. (Sapio et al., 2016) Furthermore, it also can lower blood pressure, promote vasodilation and bronchodilation. There are studies which have shown forskolin inhibit the growth of the human gastric cancer cell lines by decreasing the activity and expression of protein kinase C. (Sapio et al., 2016)It also prevented the growth and induced apoptosis of myeloid and lymphoid cells. (Pierre et al., 2009) It has been reported that forskolin can be used a drug to target patients with Alzheimer’s disease. (YAN et al., 2016)However, forskolin has not been proven to be an effective anticancer agent in humans. More clinical studies are required to support this potential drug as an anticancer agent.

There are therapeutic potential of isoform selective compounds in different clinical settings such as neuropathic pain and heart failure. (Pierre et al., 2009) Some of the AC isoforms have already reached clinical usage. An example of this will be Colforsin Daropate hydrochloride (NKH447) which has been approved in Japan for the treatment of advanced congestive heart failure. (Pierre et al., 2009)  There are also other compounds which are in its preclinical stage. Congestive heart failure is common in the US as it affects more than five million people. There are 54 different types of drugs which has increase survival however the prognosis of patient remains poor. There has been vivo studies where the overexpression of AC6 in mice improved cardiac function. In this investigation, cardio myopathic mouse  was used and when the AC6 expression was activated, there was an improvement in systolic and diastolic functions.AC5 and AC6 are expressed in the mammalian hearts. These two isoforms regulate the heart rate an contractility. However, these two isoforms have different roles.AC5 knockout models showed a protective phenotype against chronic failure and inflammatory pain. Inhibitors of AC5 has been suggested as a treatment for chronic failure but there are no AC5 inhibitors which are used clinically.

AC isoforms can also be used to improve asthma. Asthma is an inflammatory disease  and the common treatment for asthma is the beta 2-adrenergic receptor. AC9 expressions are found in multiple human lung cell types. A common polymorphism in AC9, Met for Ile at position 772, results in loss of enzyme activity. (Pierre et al., 2009)In cells where that express AC9-Met772, treatment with glucocorticoid indicated a significant increase of the albuterol-stimulated AC response. This led to a conclusion that AC-Met772 could be used to improve albuterol bronchodilator response after glucocorticoid treatment in asthmatics. (Pierre et al., 2009)There was a 4 year study where 436 asthmatic children took part in. The children either received a placebo or the inhaled corticosteroid budesonide. AC9-Met772 carriers showed an improvement. These findings hint the potential use of AC activators as bronchodilators.

cAMP is broken down by an enzyme called cAMP dependent phosphodiesterase (PDE). There are different types of isoforms and PDE3 enzyme targets this conversion. (Boswell-Smith, Spina and Page, 2009)Inhibition of this enzyme will prevent cAMP from breaking down, therefore the concentration of cAMP can be increased. PDE3 has a high affinity for cAMP but at the same it can hydrolyse cGMP. It acts as a competitive inhibitor for cAMP because it hydrolyses cAMP at a higher rate. (Boswell-Smith, Spina and Page, 2009) Due to this, it was identified as a potential therapeutic target in cardiovascular disease and asthma. It also relaxed vascular and airway smooth muscle and the inhibition of increased platelet. There were development of PDE3 inhibitors such as milrinone to treat patients with heart failure. However, this increased the risk of mortality. Cilostazol is another PDE3 inhibitor which blocks platelet aggregation. This relaxes vascular smooth muscle and in the heart, it cause positive inotropic and chronotropic effects.

In 1970s, PDE4 inhibitor called rolipram was developed as potential drug to treat depression. It indicated that an increase in cAMP would enhance noradrenergic neurotransmission in the central nervous system. (Boswell-Smith, Spina and Page, 2009)However, this drug had side effects such as nausea and gastrointestinal disturbance.  PDE4 inhibitors also have the ability to induce relaxation of isolated human bronchus. (Boswell-Smith, Spina and Page, 2009)CDP840 was developed in 1997 which was the first orally active PDE4 inhibitor. It showed beneficial effects in patients with asthmas at different doses and it showed no serious adverse effect. Cilomilast is an orally active PDE4 inhibitor in late clinical development. In chronic obstructive pulmonary disease (COPD) patients, cilomilast improved forced expiratory volume. (Boswell-Smith, Spina and Page, 2009) However there are side effects and it was rejected by FDA in 2003 due to the concern over the efficacy and safety of the drug.

 Roflumilast is another PDE4 inhibitor and it has been approved by FDA in the US in 2011. Studies have indicated that patients who have asthma and COPD, the lung function was improved and that it could be a new potential drug. There are side effects such as gastrointestinal therefore it is still going under further clinical evaluations. 3-isobutyl-1-meth-ylxanthine is another common inhibitor. This has been developed based on S-adenosylmethionine. It functions as an inflammatory drug and it has indicated as an effective PDE4 inhibitor for the treatment of chronic inflammatory disease.

Pentoxifylline, a PDE inhibitor, increases the cAMP level and it acts as an immunosuppressant. It has anti-fibrotic activity and improves the dynamic of the blood flow in the human body. There has been observations found in mice, that it increases bone mass, therefore it used in the treatment of osteoporosis. It can also block macrophage activation and the production of nitric oxide. (YAN et al., 2016)PDE can be a potential drug target to design drugs as it shows beneficial effects .There are side effects and if the benefits outweigh the risk, then PDE inhibitors can be a successful drug. There are still challenges going on to find a better PDE inhibitor which has a reduced number of side effects and more selective.

Protein kinase is an enzyme which is dependent on cAMP as it only gets activated if there is cAMP. The role of PKA is to phosphorylate other proteins in the body. PKA inhibitors can be used as potential drug.  H89 is a PKA inhibitor. It is known that the activation of beta adrenergic receptors increase the concentration of cAMP thus activates PKA. This leads to the L type calcium channels opening which results in contraction of the heart. By having PKA inhibitors, this should decrease this effect. In a recent study, an isolated rat heart was used as an experimental model and it was found that H89 is a potential cardioprotective agent. (Lochner and Moolman, 2006)It improved contractile recovery and reduced infarct size. (Lochner and Moolman, 2006) There was another study which indicated that h89 blocks prostaglandin E2 (PGE2), nitric acid and inflammatory effects. However, the use of H86 is only in laboratory agent and there no clinical evidences and whether it is safe to take them.

Taking everything into consideration, there are alternatives to GPCRS in the cAMP signal pathway. GPCRs is a large protein family of receptors therefore there many drugs which target these and there are many clinical evidences to support this. As it has strong clinical data, FDA has approved many drugs in the market which targets GPCRs. The research methods used to find a drug target in the cAMP signal pathway should be improved and this could be done by high-throughput screening as it is a well-developed technology. Adenylyl cyclase has 9 isoforms that different function in each tissues has a huge potential in the drug discovery. From all the evidences provided, adenylyl cyclase can be a major drug target in the cardiac function. Most of the AC isoforms as a drug target is only based on mice. There are no clinical evidences whether it is successful on humans and the safety of the drug. Not only AC, PKA and PDE  have been successful as drug targets in some cases. It has the potential to be targeted by many drugs if there are proven clinical evidence which support it.

References

1. Tilley, D. (2011). G Protein–Dependent and G Protein–Independent Signaling Pathways and Their Impact on Cardiac Function. Circulation Research, 109(2), pp.217-230.
2. Pierre, S., Eschenhagen, T., Geisslinger, G. and Scholich, K. (2009). Capturing adenylyl cyclases as potential drug targets. Nature Reviews Drug Discovery, 8(4), pp.321-335.
3. Sapio, L., Gallo, M., Illiano, M., Chiosi, E., Naviglio, D., Spina, A. and Naviglio, S. (2016). The Natural cAMP Elevating Compound Forskolin in Cancer Therapy: Is It Time?. Journal of Cellular Physiology, 232(5), pp.922-927.
4. Boswell-Smith, V., Spina, D. and Page, C. (2009). Phosphodiesterase inhibitors. British Journal of Pharmacology, 147(S1), pp.S252-S257.
5. YAN, K., GAO, L., CUI, Y., ZHANG, Y. and ZHOU, X. (2016). The cyclic AMP signaling pathway: Exploring targets for successful drug discovery (Review). Molecular Medicine Reports, 13(5), pp.3715-3723.
6. Lochner, A. and Moolman, J. (2006). The Many Faces of H89: A Review. Cardiovascular Drug Reviews, 24(3-4), pp.261-274.