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Treatment Of Bipolar Disorder Biology Essay

In brain cells of patients suffering from bipolar disorder there is a chemical imbalance due to increase in concentration of inositol. Lithium is an effective mood stabilizer for bipolar disorder patients and its therapeutic effect may involve inhibition of inositol monophosphatase activity in the PI signaling pathway. Lithium also affects other enzymes and aspects in the PI signaling pathway so it is not exactly clear whether modulation of PI responses can be attributed solely to the inhibition of IMPase. Lithium therapy has a lot of side effects and a narrow therapeutic window, so there is need to design an organic IMPase inhibitor as all the current IMPase inhibitors are to polar to be used as drug molecules. Drugs that mimic lithium with its respect to therapeutic mechanism of action might represent a novel approach to the treatment of bipolar disorder. Lithium is still being used despite its many side effects and narrow therapeutic window.

Introduction

Bipolar disorder also known as manic depression is a common severe, chronic and life threatening illness where patients alternate between episodes of depression and mania. Bipolar disorder affects around 340million people worldwide 1. It is a severe problem because it has no cure and the average duration of episodes of mania is around 2-3months2.With bipolar disorder not everyone’s symptoms are the same and there no simple physiological test to confirm it. The signs and symptoms of bipolar disorder are usually alterations in mood from episodes of mania to episodes of depression.

Mania has the following clinical signs and symptoms, extreme happiness, excitement, over activity, physically restless, increased appetite and self esteem, flight of ideas, pressure of speech, recklessness, psychosis, and reduced need for sleep and loss of normal social inhibitions2. Symptoms that may be experienced as part of the depressive episode includes severe low mood, loss of pleasure and interest in daily life, increased tiredness, sleep disturbance, diminished libido, low self confidence, and weight change, feelings of worthlessness, hopelessness and inappropriate guilt and the inability to concentrate or make basic decisions 2.

According to the World health organisation (WHO) mood disorders will be the major cause of global health burden in 2020 and approximately 15% of sufferers particularly those with a young age of illness onset will commit suicide2. Research has shown that bipolar disorder is associated with higher socio-economic status2.

The likely causes of bipolar disorder are complex and multifactorial. Chemical imbalance in the brain, Genetic, and environmental factors can act together to cause in bipolar disorder2. Family studies have shown that bipolar disorder run in families. Twin studies have shown that the monozygotic twins which share 100% of their genes have an approximately 65% concordance for depression which is significantly greater than the dizygotic concordance (approximately 20%) 2. This is a strong evidence for importance of genetic factors assuming that the monozygotic and dizygotic twin pairs share their environment to the same degree. Adoption studies have also shown that shared genes and not shared life experiences accounts for the fact that mood disorders run in families2. This studies shows that shared genes more than shared family environments accounts for the fact that mood disorders run in families.

The fact that concordance for monozygotic twins is not 100% also shows genes are not the only causes of mood disorder and that there must be some other factors operating to cause bipolar disorder. A number of genes act together to increase susceptibility to bipolar disorder, and the identification of these genes will play an important role in understanding the gene-environment interplay and allow for effective, better targeted treatments. Although no genes have been identified that are associated with increasing the risk for developing bipolar disease, the gene that codes for the enzyme Inositol monophosphosphatase 2 (IMPA2) is located in 18p11.2.This is thought to be a bipolar-disorder susceptible locus and polymorphism of single nucleotide in the promoter and coding region of IMPA2 has been strongly linked to bipolar-disorder.

Personality and cognitive style can put people at risk or protect them from mood disorder, for example an extravert personality can protect you against the onset of depression while mania has been associated with sensation seeking and a high need of achievement with bipolar disorder2. However it is not certain that aspects of personality and cognitive style associated with bipolar disorder are the root cause of bipolar disorder or just a consequence. Environmental stressors may be predisposing or precipitating factors to bipolar disorder such as adverse life events, trauma during childhood, divorce or job loss2.

Bipolar disorder is assessed by doctors, they interview and examine the patient, the interview includes observing the patients mental state and history. Doctors also take a physical examination to rule out any secondary symptoms to a physical illness. Sometimes a history from an informant, along with assessment their risk to themselves or to others is also important when assessing bipolar disorder. In severe cases of mania when patients are found to be at high risk to themselves, to others or are suicidal they are hospitalized compulsorily under a section of the Mental Health Act2.

This literature review will focus more on the biochemical abnormalities in the brain cells of patients suffering from bipolar disorder and how the treatment (lithium therapy) affects this process. The current treatment is lithium therapy to treat both manic and depressive episodes, lithium has been used as a mood stabiliser for 50years, but how it works is still not clear. It is thought that lithium works on the inositol-phosphate-dependent second-messenger pathways.

Lithium therapy

The anti-manic effect of lithium was first discovered accidentally by an Australian psychologist John Cade in 1949 3. Cade was able to administer lithium to manic patients, with positive clinical results and in 1970 lithium became established as an effective treatment for bipolar disorder. Inhibition of Inositol Monophosphatase a key enzyme pathway produces the effects on the PI signalling that mimic those effects produced by lithium 4. Pharmacological intervention aimed at inhibiting Inositol monophosphatase, by lithium therapy, leads to a depletion of free available Inositol4.

Problems associated with lithium~ complications of lithium treatment. The Pharmacological aspects of lithium: Dosage, absorption, distribution, excretion and the effects of lithium.

Lithium is given orally as the carbonate salt (lithium carbonate) or as a liquid (lithium citrate). It is rapidly absorbed from the gut within 8hours of administration5. Li+ does not bind to serum proteins and it is evenly distributed in total body water both intra and extracelularly5. It cannot be pumped out of cells as efficiently as sodium but it can be actively transported across cell membranes. Li+ uptake into tissues is not uniform; it is rapidly taken up by the kidney and more slowly into the liver, muscle and bone. The distribution of lithium in the brain is even slower and delayed by 24hours compared with plasma. Lithium is not evenly distributed also between the serum and the CSF, the concentration of lithium in the cerebrospinal fluid and serum is a 1:3 to 1:4 ratios5. This is due to its transport out of the cerebrospinal fluid by the brain capillary endothelium.

Excretion of lithium is via the kidney; about half of the oral dose is excreted within 12hours. The remainder is taken up by cells and excreted over 1-2 weeks. This means that with regular dosage lithium accumulates slowly over 2weeks before a steady state is reached. The narrow therapeutic window (0.5-1.5mM) and long duration of action means that the dose and plasma concentration must be monitored. The main toxic effects that occur during treatment include nausea, vomiting, tremor of the hands, polydipsia and polyuria. Signs of lithium toxicity include drowsiness and confusion, plasma concentrations above 2-3mM, life-threatening seizures and coma may occur and death if the with plasma concentration reaches 3-5Mm5.

It is difficult to get the level of lithium in the body right, at concentrations to low it doesn’t work, to high and it becomes toxic, so regular blood tests are usually done to monitor plasma concentrations . The concentration of lithium in the blood is also very sensitive to how much or how little water there is in the body, when dehydrated the level of lithium in the blood rises and are more likely to suffer from the side effects and it takes months or longer for lithium to work properly. As a result of the side effects profile of lithium and the narrow therapeutic window an appreciable number of bipolar patients cannot tolerate lithium therapy. When lithium therapy is discontinued there is greatly increased risk of manic relapse and so indefinite continuation is often required and recommended2. With all these limitations of lithium therapy it has become an important task to find more effective drugs with a safety clinical profile than lithium in treating bipolar disorder . To do this the biochemical effects in the brain and the therapeutic action of lithium in the brain have to be understood properly. Research has shown that inositol monophosphatase is the target of lithium therapy in the treatment of bipolar disorder6. This enzyme also hydrolyses myo- inositol monophosphatase into inositol and phosphate group7. It also plays an important role in the regeneration of free inositol monophosphate.8

Section 1

This section focuses on the Phosphatidylinositol (PI) signalling pathway in bipolar disorder and how lithium affects this pathway by blocking the key enzyme inositol monophosphatase in the PI signalling pathway and the central effects of lithium.

1.1 Phosphatidylinositol (PI) signalling and bipolar disorder

In response to an external stimuli, Phosphatidylinositol-4, 5- bisphosphate is hydrolysed to the secondary messengers inositol-1, 4, 5-trisphosphate and 1, 2- diacylgycerol. Once Inositol-1, 4, 5-trisphosphate has been released it is dephosphorylated by enzymes to ultimately give myo-inositol. IMPase catalyses the final step in this sequence of dephosphorylation (figure1). There is an abnormal increase in the myo-inositol levels in brains of bipolar patients9 and this increases of the resynthesis of the secondary messenger precursors. The over activity of this cellular response mechanism is thought to cause the violent mood swings associated bipolar patients10.

Inositol

Inositol (3,4)P2

Inositol (1) P inositol(3) P Inositol(4)P

(1)P

Phosphatidyl-inositol

CMP-Phosphatidate

Diacylgycerol

Phosphatidyl-inositol (4, 5) P2

Inositol (1, 4,5)P3

Inositol (1,3,4,5)P4

Inositol (1,4 )P2

Inositol (1,3)P2

Inositol monophosphatase

Inositol (1,3,4) P3

Figure1: The Phosphatidylinositol pathway showing the inositol cycle

1.2 The central effect of lithium evidence for other enzymes involved in bipolar disorder.

Protein kinase C (PKC) might play an important role in some of the cognitive features of mania11. PKC activation results in manic like behaviour while inhibition of PKC by lithium results in behaviour that might be antimanic in knock-out mice12. This is because Lithium results in increased concentration of diacylgycerol and this reduces the activity of protein kinase C13. Lithium inhibits hormone induced cAMP production and blocks cellular responses, such as the response of thyroid to thyroid stimulation hormone and the renal tubular cells to antidiuretic hormone although it does not have a pronounced effect in the brain13. Lithium inhibits glycogen synthase kinase 3, which phosphorylates the key enzymes involved in the pathway leading to amyloid formation and apoptosis. GSK3 inhibition resulted in behavioural changes (in knock –out mice) that are relevant to both mania and depression12,14-16. From the behavioural data regarding GSK3 in knockout mice it suggested that lithium’s therapeutic effect comes from its activity as a GSK3 inhibitor 12,14-16. Some researchers also believe that lithium works by stabilizing the serotonin receptors, thereby preventing wide shifts in neural sensitivity2.

1.4 Evidence of the role of inositol Monophosphatase in bipolar treatment (The inositol depletion hypothesis)

There is an assumption inositol Monophosphatase is involved in bipolar disorder, from the hypothesis that lithium treats bipolar disorder by reducing the responses of the PI signalling system and PI neurotransmitters that are hyperactive during depression or mania. But there is no direct evidence to show that a dysfunctional PI signalling is associated with the pathophysiology of bipolar disorder. Lithium reduces the level of inositol in the brain by its inhibition of the enzyme inositol monophosphatase17,6. Inositol required for the resynthesis of PI is derived from the breakdown of inositol monophosphate by inositol monophosphatase18. The enzyme inositol monophosphatase was measured in human red blood cells of psychiatrically healthy control subjects, Lithium-free bipolar patients, and Lithium-treated bipolar patients. Inositol monophosphatase was found to be reduced by almost 80% in Li-treated bipolar patients, this findings support the concept that chronic Lithium at therapeutic concentrations inhibits inositol monophosphatase4.

1.4 The role of inositol Monophosphatase

IMPase is a key enzyme in a brain secondary messenger system, it provides free myo-inositol for the biosynthesis of the secondary messenger precursor Phosphatidylinositol-4, 5-bisphosphate8. Inositol monophosphatase also hydrolyses inositol monophosphate, inositol 3-phosphate and inositol 4-phosphate to inorganic phosphate and inositol, which is the precursor for all inositol-based signalling mediators7. The over activity of this cellular response mechanism is linked to the violent mood swings associated bipolar patients. The Suppression of IMPase activity by an inhibitor such as lithium reduces the levels of myo-inositol in the brain and therefore slows down the resynthesis of second messenger precursors.

Figure 2: The hydrolysis of Myo-inositol monophosphate catalysed by inositol monophosphatase

Scheme 1: The hydrolysis of myo-inositol monophosphate by IMPase

Kinetic studies on Inositol monophosphatase

Experimental studies have shown that two divalent Mg2+ ions are required for IMPase activity20. At low concentrations each monomer is required to bind with one Mg2+ (1) ion for full activity and the binding of the second Mg2+ (2) after substrate binding is facilitated by the binding of the first ion20. The enzyme is inhibited at high concentrations of Mg2+ and Li+. Lithium is an uncompetitive inhibitor of IMPase with respect to its substrate at low concentration and at high concentrations it non-competitively inhibits IMPase20. Uncompetitive inhibition by Li+ and Mg2+ are similar this implies that they both bind in the same site. From kinetic studies it has shown that this site is the site for the catalytic Mg2+ (2) ion. Therefore Lithium ion binds in place of Mg2+ (2) and prevents the inorganic phosphate from unbinding21 (as seen in scheme 2). Other divalent cations and trivalent cations such as Ca2+, Zn2+, Mn2+, Be2+ and Gd3+ can inhibit IMPase22. These cations are competitive inhibitors of IMPase and compete with Mg2+ for the binding site thereby preventing the Mg2+ dependent hydrolysis of the substrate23. Lithium is used therapeutically instead of these other cations because it is highly potent and its small size allows it to be able to cross the blood brain barrier.

Structural requirements for substrate activity

A lot of Studies have been done to understand the substrate binding interactions by deleting the OH groups around the inositol ring to be able to identify the binding and catalytic OH groups. This showed that inositol ring binds to the active site cleft through its hydroxy groups, 2-OH, and 4-OH which are involved in binding to the enzymes24.The 6-OH group has also been identified to be involved in catalysis, it coordinates to the water molecule which functions as the nucleophile to complete the shell of Mg2+ 2 to give the octahedral geometry24. Removal of the 6hydroxy group stops catalytic activity. Neither the 3-OH nor 5-OH has an effect on catalysis or binding .2-OH forms H-bonding with the side chain of Asp-93, and to Ala-196. 4-OH forms H-bonding with the carboxylate group of Glu-21324.

Mg2+ ion activates the inositol ester oxygen and makes it more vulnerable to nucleophilic attack by water molecule activated by Glu-70 and Thr95 as well as Mg2+ in site 1. The active site nucleophile is a water molecule co-ordinated to the first Mg2+ ion, Mg2+ activates the nucleophile. The water molecule is deprotonated in the presence of Mg2+ ion and attacks as a hydroxide group. The role of the catalytic water molecule coordinated to Mg2+ is still unclear and this water molecule has been shown to be essential for the catalysis of the reaction. It is thought that the catalytic water molecule can donate a proton to the free oxygen of the phosphate group, thereby polarising the P-O bond and making it the phosphorous atom more electrophilic and more susceptible to nucleophilic attack.31. The catalytic water molecule is also thought to donate a proton to the inositolate leaving group oxygen, making it a good leaving group and stabilising the formation of the product while stopping the backward reaction.( assisting in the formation and release of the inositol product)

The nucleophile Mg2+ 1 bound to water molecule attacks the phosphorous from the opposite side of the O-atom which is hydrogen bonded to the NH of Gly-94 and Thr-95. In the product complex formed the side chain of Thr-95 and Mg2+ 1 interact with the pseudo rotated phosphate O-atom (retention of stereochemistry). This agreed with the x-ray crystal (inactive) structure of the di-Mn2+ phosphate product complex.

SECTION 2

This section focuses on the crystal structure of myo-inositol monophosphatase, and how it interacts with its substrate inositol monophosphate and lithium.

2.1 The crystal structure of human Myo-Inositol Monophosphatase (substrate binding)

Inositol monophosphatase is a dimeric protein containing 277 amino acids in each monomer unit20 (figure 3). The enzyme exists as a dimmer of identical sub-units each folded into a five layered sandwich of three pairs of alpha helices and two beta sheets in a αβαβα arrangement19. The X-ray crystal structure was generated from diffraction data collected for a gadolinium sulphate form of the human brain enzyme. Gd3+ and sulphate are competitive inhibitors of Mg2+ and phosphate respectively; therefore they bind in the same site as Mg2+ 1 and the phosphate in the enzyme product complex20. This crystal structure with gadolinium sulphate bound (Gd3+), highlights Mg2+ (1) metal binding site as Glu-70, Asp-90, Ile -92, Thr-95, the sulphate binds to metal ion.

This structure suggested that there was one metal binding site per subunit this was because the crystal structure was obtained in the presence of high concentrations lithium which was bound to the second metal binding site. Therefore there are two metal binding sites; the other metal binding site had Li+ in it and this site was masked because lithium cannot be observed in X-ray crystallographic structure.

Figure3: The crystal structure of human inositol monophosphatase determined by using x-ray crystallography. The sulphate and Gd3+ are bound at identical sites on each of the subunits and take the position of the active sites.

Source

To determine the structure of the active enzyme complex the Gd3+ ion in the crystal structure was replaced by Mg2+, and the sulphate group was replaced with the phosphate group of the substrate. It was found that the position of Mg2+ (1) in the substrate complex corresponded to the position of Gd3+ in the x-ray structure for inositol monophosphatase substrate complex. The active enzyme complex showed that Mg2+ (1) coordinates to the hydroxy group of Thr-95, the side chain carboxylate groups of Glu-70 and Asp-90, to the carbonyl O-atom of IIe-92 as well to the two non-bridging O-atoms of the phosphate group20 as shown in figure 4. The water molecule is unable to co-ordinate with the phosphate group of the substrate.Mg2+ 1gets to the active site before any of the other species, and it remains there throughout the whole catalytic process, even at high saturating concentration of the substrate.

Figure 4: The coordination sphere of Mg2+ 1 in the optimised structure of Mg2+ enzyme substrate complex

Source;

The second metal active site for Mg2+ is formed in the enzyme substrate complex, with ligands from the substrate and enzyme. Mg2+ is coordinates to the substrate through the bridging ester O-atom and one of the equivalent O-atoms of the phosphate group, it coordinates to the enzyme through the carboxylate groups of the three aspartate groups, Asp-90, Asp-93 and Asp-220 (figure 5).

Figure 5: The coordination sphere of Mg2+ 2 in the Optimised structure of Mg2+ enzyme substrate complex, showing the interactions with the key amino acid residues.

Source:

2.2: Interactions between inositol monophosphate, lithium and its substrate

The substrate inositol monophosphate binds to inositol monophosphatase with mg2+ in metal binding site 1(Mg2+ remains bound to the enzyme throughout the process). A second Mg2+ binds (after substrate binding) in metal binding site 2 and interacts with the three aspartate residues (Asp 90, 93 and 220). The binding of the second Mg2+ activates the inositol ester oxygen and makes it more susceptible to nucleophilic attack by water molecule activated by site 1 Mg2+, Glu70 and Thr95. The nucleophilic attack results in the hydrolysis of the phosphate bond, releasing inositol and leaving the inorganic phosphate with both Mg2+ ions. In normal conditions the Mg2+ in site 2 and the inorganic phosphate unbinds to regenerate the enzyme with Mg2+ bound in site 1, for the hydrolysis of the substrate in the next round. But in the presence of lithium (scheme2), when the Mg2+ in site 2 unbinds, the lithium occupies this site. This prevents the inorganic phosphate from unbinding; this leaves inositol monophosphatase in an inactive state.

Scheme 2: summary of the mechanism of substrate hydrolysis and inhibition by lithium of inositol monophosphatase

E = enzyme

InsP = inositol monophosphate

Li= lithium

Pi = inorganic phosphate

Mg (1) and Mg (2) binding at metal site1 and 2 respectively

InsP Mg

E.Mg (1). E.Mg (1).InsP

Pi

E.Mg (1).InsP.Mg (2)

E.Mg (1).Pi E.Mg (1).Pi.Mg (2)

Li Mg Ins

E.Mg (1).Pi.Li

3D interactions between inorganic phosphate and Lithium to IMPASE

Lithium acts as an uncompetitive inhibitor of IMPase at therapeutic levels. It works by preventing the release of the inorganic phosphate. Li+ interacts with the O-atoms of Asp-90, Asp-93 and Asp-220 and one of the phosphate O-atoms in a tetrahedral geometry as seen in figure 6.

2.3 Mechanisms for the hydrolysis of inositol monophosphate by IMPase

Figure 7: Phosphate hydrolysis mechanisms.

Inline association mechanism

In this mechanism the nucleophile attacks the phosphorous before the inositolate leaving group departs (resulting in inversion of configuration.

Inline dissociative mechanism

In this mechanism the leaving groups departs before the nucleophile attacks.

Associative sn2 like mechanism in which the nucleophile attacks and the inosilate leaving group departs at the same time.

Non-line associative mechanism.

In this mechanism the nucleophile attacks from the same face as where the leaving group departs, this requires a pseudo-rotation step around phosphorous centre.

The Merck, Sharp and Dohme group using site-directed mutagenesis studies in conjunction with kinetic and molecular-modelling data were able to identify that the Mg2+1 coordinated to nucleophilic water molecule is hydrogen bonded to the 3-OH group of the Thr95 and is activated by Glu-70 25. The mechanism proceeds through the inline displacement with inversion of configuration at phosphate and the Mg2+ 2 was proposed to assist in phosphate coordination and charge stabilization during the transition state. It is still unclear if the inline mechanism will proceeds via a two step mechanism (a) or if the reaction undergoes an SN2 like mechanism (c) in which the leaving group attacks and the inositolate leaving group departs at the same time.

In contrast, on the basis of kinetic data for the hydrolysis of different substrates, the Gani and co-workers suggested that although Mg2+ 1 coordinates the phosphate group, it is Mg2+ 2 that would chelate and activate the water molecule for nucleophilic attack on phosphorus26, 20. In this proposal, the substrate 6-OH group hydrogen bonds to the nucleophilic water molecule so that it is positioned for a non-inline attack on the phosphate P atom with adjacent displacement of the inositol group26. This non-inline nucleophilic attack will require a pseudo-rotation step (d) of the ligands at the phosphorous centre. The process will result in retention of configuration at the phosphorous centre26. This mechanism proved to be wrong by studies on chiral phosphates which demonstrated the reaction proceeded with inversion of configuration at the phosphorous27.

 The recent determination of the stereochemical course of the reaction demonstrated that hydrolysis occurs with inversion at phosphorus27, indicating that the water nucleophile is indeed associated with Mg2+ 1. However, this interpretation excluded a role for the substrate 6-OH group which is essential for catalysis28, 29, and 30. A new unified mechanism was proposed by the Gani and co-workers group in which the Mg2+ 2-bound water molecule acts as a proton donor for the inositolate leaving group and the 6-OH substrate group hydrogen bonds to this water to make it more susceptible for further for inline attack of the 1-inositolate anion31 (b).

It has also been proposed by some research groups that there is a third Mg2+ involved in catalysis25. Evidence suggesting the 3rd Mg2+ ion is involved in catalysis came from mutagenesis studies on Lys-36. The proposed role of the Mg2+ 3 ion is to assist the deprotonation of the nucleophile coordinated to Mg2+ 137, for inline nucleophilic attack by the water molecule coordinated to Mg2+.

Results from mechanistic studies have shown that the associative inline mechanism is the most favoured route for IMPase. The results agree with experimental studies which show that. It also suggests that the catalytic water molecule is required to transfer a proton to the inositolate leaving group to assist in the release of the product, because if the leaving group is not protonated the negative charge on the inositolate group would coordinate to the Mg2+ 2 ion making it more difficult to be released.

SECTION 3- Alternatives inhibitory of inositol Monophosphatase

Lithium has a narrow therapeutic window as discussed in the introduction, and above 2mM just twice the therapeutic dose severe toxicity is observed .Therefore there is need for the design of organic compounds that can mimic inositol monophosphate so as to reduce the activity of inositol monophosphatase. This section focuses on such inhibitors of inositol monophosphatase.

Substrate based inhibitors

L-690,330 is a competitive inhibitor of IMPase, it was synthesised on the basis of the substrate inositol monophosphate32. It is very potent, at very high concentrations it was able to produce an accumulation of inositol monophosphate, but L-690,330 could not cross the cell membrane in tissue culture due to its highly charged bisphosphonate group32. The prodrug L-690,488 was produced by esterification of the bisphosphonate group, to reduce the high charge. It was thought that once L-690,488 was in the cell it would be able to yield the parent drug L-690,333 33. It proved to be more potent than L-690,333, in causing accumulation of inositol monophosphate, it worked well in vitro in rat cortical slices and Chinese hamster ovary cells transfected with the human muscarinic M1 receptor and human platelets. 33

Gani group phosphate inhibitors31, 34, 35, 36

These are substrate like inhibitors possessing 6-substituents other than the 6-hydroxy group of the substrate, where able to bind to the enzyme. With the exception of 6-amino phosphate and 6-methlamino phosphate (km values of 300µM and 140µM respectively) this had hydrogen bond donor at carbon 6. This helped to establish that primary and secondary amino groups at carbon 6 where able to interact with Mg2+ ion bound to water molecule. The remaining phosphate analogues compound 1, 2 and 3 shown below showed no substrate activity.

Phosphate inhibitors synthesised by the Merck group

This series of inhibitors 6, 7, 8 and 9 were made by the deletion of each hydroxyl group in the natural substrate, some turned out to be highly potent inhibitors of IMPase. This also showed the function an importance of the 6-OH, 1-O, 2-OH and 4-OH in catalysis and binding. However the compound shown below (10) was synthesised by adding a lipophilic side chain to increase its binding ability and it is known to be the most potent IMPase inhibitor till date.

Terpenoid inhibitors ~ Two structurally similar inhibitors where Isolated from Memnonniella (Ki value of approximately 500 µM) and Stachybotris species (Ki value 450µM). They were both non-competitive inhibitors with respect to inositol 3 phosphates and Mg2+ with a different inhibition profile to lithium.

Tropolone Inhibitors ~ Puberulonic acid was a competitive inhibitor of IMPase isolated from several penicillium strains, with an IC50 value of 10µM. Although they are less polar than the substrate based inhibitors their bioavailability is too low to be used as drugs.

Phosphonate inhibitors synthesised by A.M Mcloed et al (33) in ref

Less polar than the substrate –based inhibitors but they are still too polar to be used as drugs.

Reduced charge phosphonate inhibitors

With doubly charged phosphate moiety these compounds were unable to cross the lipophilic blood-brain barrier to reach IMPase therefore they would require modifications to be able to serve as drug candidate. Aim is to reduce the charge but still retain the steric demands of the active site for binding, so the phosphate group was replaced with a phosphonate group.

ph

Section 4; Alternative drugs in the treatment of bipolar disorder and, Future models of new drugs

This section will focus on the other drugs used in the treatment of bipolar disorder, in comparison to lithium, if they are more effective and have less side effects and a safety profile in treating mania or depression associated with bipolar disorder. The future models of new drugs will also be considered

4.1 mood stabilising medications: Valproic acid (VPA and inositol depletion) and Carbamazepine.

Valproic acid and Carbamazepine are used to treat manic episodes in bipolar patients that are unresponsive to lithium. These drugs are gaining favour for the treatment of bipolar patients because of their less side effects and safety profile. These drugs are anticonvulsants and are usually used to treat epilepsy, but are now an alternative to lithium. Valproate produces altered patterns of expression in of protein kinase C, thereby altering cellular response and gene expression.

Why lithium is better in treating bipolar disorder.

Lithium is more effective at preventing episodes of mania in bipolar disorder than treating it. In Contrast to lithium Valproic acid and Carbamazepine are thought to be better in preventing the recurrence of depressive episodes in bipolar patients.

4.2 Why the new drugs fail~ not polar, cant cross the blood brain barrier,

Currently all the IMPase inhibitors which are potent enough to be used as drug molecules are to polar to cross cell membranes and be used as drugs

L-690,330 ~ could not penetrate the cell membrane because of its highly charged bisphosphonate, instead the tetrapivaloymethyl ester of prodrug of this compound L-690,488 was synthesised. L-690,488 was more permeable than L690, 333 and it served as a prodrug releasing the parent compound L-690,333 when in the cell, that will then be able to inhibit IMPase. But this also failed in vivo as a result of its lipohilicity, when injected into animals neither it nor the parent compound L-690,333 could be detected in the plasma or brain. This suggested that the compound remained at the site of injection.

Puberulonic acid ~ the anhydride was supposed to hydrolyse to give biscarboxylic acid and biscarboxylic acid was supposed to chelate Mg2+ ion in the active site of IMPase, but the biscarboxylic acid was shown to the inactive

Several of the inhibitors are potent but the subtrate-based inhibitors are all too polar to be used as drug molecules. These molecules have poor bioavailability and will not cross the cell membrane and therefore will not be successful as drug molecules. The reduced charge have increased bioavailability but the binding of the molecules is decreased by reduction in charge.

4.3 Summary and Future work

The substrate based inhibitors of IMPase were not suitable for use in in-vivo. In the future development of new compounds with the clinical profile of lithium with less side effects and a narrow therapeutic window should be focused on. IMPase inhibitors mimic the effects of lithium on the PI cycle, this can be a future target for new drugs that can

Future/ further work.

Identification of the mobile regions of a protein and how they may be exploited in drug design. To use computational and experimental studies to further categorize the mobile loops. The use of computational chemistry is a powerful tool in drug design will help guide the synthesis of target drug molecules and help with investigations into the mechanism of enzymatic reaction pathways.

Design of bioavailable drug molecules for IMPase, (structure based drug design) using the mobile regions of IMPase as a target

More investigation into the IMPase mechanism (probing the role of the catalytic water molecule)

Future work should consider the influence of the 3rd Mg2+ ion in the active site, which some x-ray structures have shown to be present. The protonation state of the nucleophile should also be studied in more detail (mechanistic studies performed with water as the nucleophile instead of hydroxide group. When the Mg2+ ion deprotonates the water nucleophile it is still unclear whether the proton is accepted by the side chain of an amino acid or by a water molecule in solution further study should be done on this.

Design of a pharmacophore ~ FOR PHOSPHATE BINDING INHIBITORS


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