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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.
"Bipolar disorder is a common severe, chronic and life threatening illness where patients alternate between episodes of depression and mania"
Why bipolar disorder is a problem and why the current drugs don't work. Bipolar disorder affects around 340million people worldwide. It is a severe problem because it has no cure and the average duration of episodes of mania is around 2-3months. The current treatment is lithium therapy. As a result of the side effects profile of lithium, an appreciable number of bipolar patients cannot tolerate lithium therapy.
Signs and symptoms ~ not everyone's symptoms are the same and no simple physiological test to confirm
Diagnosis~ how it is assessed~ interview and examine the patient, take history from an informant, assess their risk to themselves or to others
The likely causes are (multifactorial) complex~ genetic factors (biological) environmental (physical and psychosocial) and hormonal abnormalities (chemical imbalance in the brain)
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-bisphosphate. 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 signaling mediators. 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 1: The hydrolysis of Myo-inositol monophosphate catalysed by inositol monophosphatase
Scheme 1: The hydrolysis of myo-inositol monophosphate by IMPase
The gene encoding for Inositol monophosphosphatase 2 (IMPA2) is located in 18p11.2; this is a bipolar-disorder susceptible locus. Single-nucleotide polymorphism in the promoter and coding region of IMPA2 has been strongly linked to bipolar-disorder.
(PI) cell signalling and bipolar disorder
In response to an external stimuli, Phosphatidylinositol-4, 5- bisphosphate is hydrolysed to 1,2- diacylglycerol and
Indirect evidence for altered signaling in bipolar disorder patients comes from
Evidence of the role of inositol Monophosphatase in bipolar disorder and in antibipolar 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 signaling 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 monophosphatase . Inositol required for the resynthesis of PI is derived from the breakdown of inositol monophosphate by inositol monophosphatase. 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 and 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 monophosphatase .
1.4 The crystal structure of human Myo-Inositol Monophosphatase (substrate binding)
Inositol monophosphatase is a dimeric protein containing 277 amino acids in each monomer unit (figure 2). 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 Î±Î²Î±Î²Î± arrangement.
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.
Figure2: 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.
Gd3+ and sulphate are competitive inhibitors for Mg2+ and phosphate. Source :http://www.rcsb.org/pdb/images/2hhm_bio_r_500.jpg?bioNum=1
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 group as shown in figure 3. 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 3: The coordination sphere of Mg2+ 1 in the optimised structure of Mg2+ enzyme substrate complex
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, 93 and 220 (figure 4).
Figure 4: The coordination sphere of Mg2+ 2 in the Optimised structure of Mg2+ enzyme substrate complex.
The key role of Mg2+ 2 ion is to act as a Lewis acid and deprotonate the water molecule (nucleophile)
The key amino acids involved in substrate and metal binding
Nucleophilic water activation
Table 1 (source: Bipolar medications mechanisms of action)
Section 2: Chemistry of lithium (lithium and IMPase metal binding) ~ lithium and enzymes/ proteins~ key active site interactions~ Mechanisms.
2.1 How does lithium work ~ what lithium affects in the brain
lithium and the Phosphatidylinositol (PI) signalling pathway
IMPase the main target for lithium
The anti-manic effect of lithium was first discovered accidentally by an Australian psychologist John Cade in 1949. Inhibition of Inositol Monophosphatase a key enzyme ( critical for the regeneration of secondary messenger) in the PI signalling pathway produce the effects on the PI signalling that mimic those effects produced by lithium. Pharmacological intervention aimed at inhibiting inositol monophosphatase, by lithium therapy, leads to a depletion of free available inositol.
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, 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
Kinetic studies on IMPase
At low Mg2+ concentrations each monomer is required to bind with one Mg2+ ion for full activity, and the binding of the second Mg2+ after substrate binding is facilitated by the binding of the first ion. The enzyme is inhibited at high concentrations of Mg2+ .Lithium non-competitively inhibits IMPase with respect to its substrate at low concentration or high concentrations.
Structural requirements for substrate activity
The inositol ring binds to the active site cleft through its hydroxy groups, 2-OH, and 4-OH are involved in binding to the enzymes. 6-OH is involved in catalysis (removal of the 6hydroxy group stops catalytic activity, it coordinates to the water molecule (functions as the nucleophile) to complete the shell of Mg2+ 2 to give the octahedral geometry. 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-213.
The active site nucleophile is a water molecule co-ordinated to the second Mg2+ ion ~ Mg2+ activates the nucleophile. The nucleophile (Mg2+ 2 bound 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. This agreed with the x-ray crystal (inactive) structure of the di-Mn2+ phosphate product complex.
In-line nucleopilic attack (by Merck, Sharp and Dohme)
According to the authors ~Mg2+ 1 bound water molecule is in H-bonding with the 3-OH group of Thr-95 and is activated by Glu-70. (Implies a decrease in nucleophility)
2.3 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 administration. Li+ does not bind to serum proteins and it is evenly distributed in total body water both intra and extracelularly. 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 ratios. 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 3-5mM.
2.4 Alternatives inhibitory of inositol Monophosphatase
Substrate based inhibitors ~
L-690,330 competitive inhibitor of IMPase synthesised on the basis of the substrate inositol monophosphatase, very potent, at very high concentrations was able to produce an accumulation of inositol monophosphate, but L-690, 330 could not cross the cell membrane in tissue culture so the prodrug L-690,488 was produced by esterification of the bisphosphonate group, to reduce the high charge.
Gani group phosphate inhibitors ~ 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 showed no substrate activity.
Phosphate inhibitors synthesised by the Merck group~ Series of inhibitors made by the deletion of each hydroxyl group in the natural substrate, some turned out to be highly potent inhibitors of IMPase.
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.
Section 3; Central effect of lithium evidence for other enzymes involved in bipolar disorder
3.1 The alternative targets of lithium~.
3.2 lithium's effect on cyclic AMP
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 brain.
3.3 Lithium's effect on protein kinase C (PKC)
Protein kinase C might play an important role in some of the cognitive features of mania. PKC activation results in manic like behaviour while inhibition of PKC results in behaviour that might be antimanic in knock-out mice. Lithium results in increased concentration of diacylgycerol and this reduces the activity of protein kinase C and
3.4 Lithium's effect on glycogen synthase kinase 3 (GK3)
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 depression. From the behavioural data regarding GSK3 in knockout mice it suggested that lithium's therapeutic effect comes from its activity as a GSK3 inhibitor.
Section 4; Alternative drugs in the treatment of bipolar disorder and, Future models of new drugs
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.
4.2 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.
4.3 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
4.4 Future models development of new compounds with the clinical profile of lithium with less side effects and a narrow therapeutic window. IMPase inhibitors (future target) mimic the effects of lithium on the PI cycle
The substrate based inhibitors of IMPase were not suitable for use in invivo
Design of bioavailable drug molecules for IMPase, ( structure based drug design)
More investigation into the IMPase mechanism ( probing the role of the catalytic water molecule)
Design of a pharmacophore ~ FOR HOSPHATE BINDING INHIBITORS