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Cholinesterases •acetylcholinesterase •a serine hydrolase that belongs to the esterase family, which acts on different types of carboxylic esters. •biological role is the termination of impulse transmissions at cholinergic synapses within the nervous system by rapid hydrolysis of the neurotransmitter, acetylcholine (Schumacher et al, 1986) •butyrylcholinesterase •Acetylcholinesterases •found in all excitable tissue, whether muscle or nerve, central or peripheral, cholinergic or adrenergic, motor or sensory, in erythrocytes, and in placental tissue; but not found in plasma •high affinity for acetylcholine, •hydrolyses acetylcholine to acetic acid and choline •low affinity for noncholine esters •specific substrate is acetyl-β-methylcholine
Acetylcholine •synaptic transmitter at •central nervous system •autonomic ganglia •postganglionic parasympathetic nerve endings •postganglionic sympathetic nerve endings to eccrine sweat glands •neuromuscular junction •undergoes rapid hydrolysis •at the synapse by acetylcholinesterase •in plasma by butyrylcholinesterase Acetylcholine receptors •nomenclature based on the selective cholinoceptor stimulants •muscarinic (G protein-linked) •G-protein coupled type with 7 segments arranged in a serpentine fashion across the membrane •nicotinic (ion channel) •receptors are part of •transmembrane polypeptide Distrubution of cholinoceptors
•exists in two classes of molecular forms •simple oligomers of a 70,000 MW catalytic subunit, contains a hydrophobic surface, found in the plasma membrane •elongated form of complex molecular structures, consist of tetramers of catalytic subunits linked by disulphide bonds to either lipid-linked subunits or strands of collagen-like subunits (MW ~ 106), localised in the outer basal lamina of the synaptic cleft, primarily found in the neuromuscular junction •3 distinct domains on acetylcholinesterase •constitute binding sites for inhibitory ligands •form the basis for specific differences between acetylcholinesterase and butyrylcholinesterase •active site is composed of two subsites: •the esteratic subsite (acyl pocket) which contains the catalytic machinery of the enzyme •the anionic subsite that accommodates the positive quaternary pole of acetylcholine. •peripheral anionic site (PAS) or binding site •plays a role in the confirmation of the residues within the aromatic gorge and active site Glu His Ser substrate
•esteratic subsite •consists of the catalytic triad (Glu334-His447Ser203) which combines with the carbonyl group of the ester linkage and is responsible for the hydrolysis of the ester bond •Serine binds to one of the carbons of the acetate part of the molecule, while •Aspartic acid (Asp) has a negative charge that attracts the positive charge on the ACh nitrogen •Histidine helps to split the bond that holds the acetyl and choline parts together •anionic subsite •termed "anionic" is misleading as the site is actually uncharged and lipophilic •located at or near the rim of the aromatic gorge •binds the quaternary group of acetylcholine, •plays a role in the confirmation of the residues within the aromatic gorge and active site •plays a crucial factor in enzymatic regulation, if the site becomes bound the enzyme will become inactivated due to conformational changes •Butyrylcholinesterase •aka plasma cholinesterase, pseudocholinesterase •synthesised in the liver •found in the liver, plasma, kidney, and the intestine •low affinity for acetylcholine •specific substrate is benzoylcholine •likely physiological function is the hydrolysis of ingested plant esters •responsible for the hydrolysis of succinylcholine, mivacurium and the ester local anaesthetics •capacity to hydrolyse succinylcholine is enormous •influences the action of succinylcholine by determining the amount of drug reaching the end plate; only a small fraction of the injected dose reaches the neuromuscular junction •hypotheses of existence •prevents inhibition of acetylcholinesterase by removal of choline esters formed during metabolism •prevents inhibition of acetylcholinesterase by organophosphorus compounds during villous growth and organogenesis •hydrolysis of extraneuronal acetylcholine which has diffused into the smooth muscle regions •some individuals possess a genetically-determined abnormal variant of plasma cholinesterase which has a very low capacity to hydrolyze succinylcholine and may therefore experience prolonged blockade (4 hours compared to a few minutes) •the enzyme variants may be distinguished by determining the dibucaine number which reflects the ability of an individual to hydrolyze succinylcholine •measurement of activity •adding plasma to benzoylcholine and following the reaction by spectophotometry •use of enzyme-linked immunosorbent assays and DNA amplification and sequencing utilizing the polymerase chain reaction
•measurement of activity by adding plasma to benzoylcholine •normal range of activity is 0.8-1.2 units (mmol of benzoylcholine hydrolysed/min/ml of plasma) •0.4 units for the activity of cholinesterase in an individual who is homozygous for the atypical gene •to clarify the phenotype, the reaction is carried out in the presence of inhibitors to this reaction such as dibucaine (cinchocaine, nupercaine) which is an amide local anaesthetic, sodium fluoride and specific inhibitor known as Ro2-0683 •Acquired enzyme deficiency •the newborn •reaching adult levels by 2-6 months •pregnancy •acute or chronic liver diseases •malnutrition •collagen diseases •chronic anaemia •uraemia •myxoedema •chronic debilitating diseases •severe burns •MAO inhibitors •trimethaphan •cytotoxic drugs •azathioprine •echothiopate eye drops •hexafluorenium bromide, tacrine •quinidine •propanidid •oral contraceptive •chlorpromazine •chronic pesticide exposure and accidental poisoning •Increased activity of butyrylcholinesterase seen in •obesity •type IV hyperlipoproteinaemia •nephrosis •toxic goitre •Inherited enzyme defects •plasma cholinesterase is coded for by two allelomorphic genes on an autosomal chromosome •four variants are described, •normal gene N •dibucaine resistant gene D •fluoride resistant gene F •silent gene S •dibucaine-related variants are the most important •the most frequent atypical form, the dibucaine resistant gene, has a far lower affinity for succinylcholine at normal serum concentrations •D-gene population prevalence ~ 1:53 •incidence ~ 1:2800 •the usual laboratory estimates of plasma cholinesterase do not differentiate between the varieties
•Dibucaine number •the local anaesthetic dibucaine inhibits normal plasma cholinesterase to a far greater extent than the atypical enzyme (Kalow & Genest) •dibucaine number (DN): •the percentage inhibition of plasma cholinesterase produced by a standard titre of dibucaine (10-5 mmol/l) •under standard test conditions, dibucaine inhibits the normal enzyme by about 80% and abnormal enzyme by about 20% • Inherited enzyme defects Genotype SCh sensitivity DN FN Incidence(%)
NN ND DD NF NS DF DS FF FS SS normal mildly increased greatly increased mildly increased normal greatly increased greatly increased greatly increased greatly increased greatly increased ≈80 ≈50 ≈20 ≈80 ≈80 ≈50 ≈20 ≈60 ≈60 0 ≈60 ≈40 ≈20 ≈40 ≈60 ≈40 ≈20 ≈20 ≈20 0 94 (96.2) 4 (3.8) 0.036 0.5 0.5 0.02 0.02 0.0025 0.0025 0.0025
DN: %inhibition of plasma cholinesterase by dibucaine10-5 mmol/l FN: %inhibition of plasma cholinesterase by fluoride 10-5 mmol/l
Acetylcholinesterase inhibitors •prevents hydrolysis of acetylcholine by acetylcholinesterase at sites of cholinergic transmission •although butyrylcholinesterase is inhibited, acetycholinesterase is the primary target of these drugs •inhibition can be •reversible, by competitively blocking the substrate reaching the active site: edrophonium, carbamates •quasi-irreversible, by covalent reaction with the active site serine, inactivating the catalytic ability of the enzyme (quasi = seemingly): organophosphate inhibitors •competitive inhibition takes place by blocking substrate at the active site (tacrine, edrophonium, carbamates), •non competitive inhibition occurs by binding to the peripheral site (propidium, gallamine). •bis-quaternary ligand decamethonium bind across both active and peripheral sites •3 main types •simple alcohols bearing quaternary ammonium group •edrophonium •carbamate esters of alcohols bearing quaternary or tertiary ammonium groups •neostigmine, physostigmine •organic derivatives of phosphoric acid or organophosphates: •insecticides: parathion, malathion •nerve gases: diisopropylflurophosphonate, sarin
•Structure activity •reversible carbamate inhibitors •addition of carbamoyl group (O-C-O-NH) increases potency and duration of action •edrophonium, lacking carbamoyl group, is less potent and short-acting •presence of quaternary nitrogen inceases potency and affinity to the enzyme •both neostigmine and physostigmine exist as cations at physiological pH, thus enhancing their association with the active site •potency of reversible carbamate inhibitors contributed by •addition of carbamoyl group (O-C-ONH) •presence of quaternary nitrogen •Mechanism of action •quaternary compounds inhibit the enzyme reversibly by •either binding with the esteratic site (Glu-HisSer), •or with the peripheral anionic site •quaternary alcohols (edrophonium) •bind reversibly to enzyme preventing access of acetylcholine •enzyme-inhibitor complex does not involve a covalent bond •this reversible binding and its rapid renal elimination result in its short duration of action (2-10 minutes) •carbamate esters (neostigmine and physostigmine) •by serving as alternate substrates with a similar binding orientation as acetylcholine, undergo 2step hydrolysis sequence analogous to that described for acetylcholine •carbamyl-ester linkage is hydrolysed by acetylcholinesterase, but at a much slower rate •attack by the active serine centre gives rise to carbamoylated enzyme •covalent bond of carbamoylated enzyme is more resistant to the second (hydration) process, •t½ for hydrolysis of the dimethycarbamoyl enzyme is 15-30 minutes •duration of inhibition of the carbamoylating agent is 3-4 hours
•organophosphates •organic derivatives of phosphoric acid organophosphates e.g. di-isopropyl flurophosphate •the tetrahedral geometry of the organophosphates resembling the transition state for the acetyl-ester hydrolysis, serve as true hemisubstrates and act at the esteratic site (GluHis-Ser) •undergo initial binding and hydrolysis by the enzyme, resulting in phosphorylated active site •covalent phosphorus-enzyme bond is extremely stable and hydrolyses in water at a very slow rate (hundreds of hours) •if the alkyl groups in the phosphorylated enzyme are methyl or ethyl, spontaneous regeneration of the enzyme requires several hours •secondary or tertiary alkyl groups further enhance the stability of the phosphorylated enzyme and significant regeneration of the enzyme is not observed and return of acetylcholinesterase activity depends on synthesis of new enzyme •stability is further enhanced through ageing which results from the loss of one of the phosphonate alkyl groups •strong nucleophiles (pralidoxime) able to split phosphorus-enzyme bond before ageing has occurred, can be used as cholinesterase regenerator for organophosphorus insecticide poisoning •once ageing has occurred, enzyme-inhibitor complex is stable and resistant to split even with oxime regenerator compounds •Pharmacokinetics •absorption •quaternary compounds •neostigmine absorb poorly from conjunctiva, skin, and lungs since their permanent charge renders them relatively insoluble in lipids •larger doses are required for absorption via oral route than via parenteral routes •tertiary ammonium group •physostigmine, is well absorbed from all sites and can be used topically in the eye •distributes into central nervous system and is more toxic than the more polar quaternary ammonium carbamates •non-polar carbamate insecticides •poorly absorbed across skin •dermal:oral lethal doses higher than ratios for organophosphate pesticides •organophosphates •well absorbed from the skin, lung, gut, and conjunctiva •very effective insecticides but dangerous to humans
•distribution •lipid solubility •increases distribution into the central nervous system •tertiary ammonium agents – physostigmine (for glaucoma), duration of action 0.5-2 hours •carbamate insecticide - carbaryl •organophosphate – •echothiopate, moderate lipid solubility, duration of action 2-7 days, •parathion, highly lipid soluble, duration of action 7-30 days •quaternary ammonium compound •limits entry of agent into central nervous system •synthetic compounds •edrophonium, not orally active, duration of action 5-15 minutes •neostigmine, orally active, duration of action 0.5-2 hours •pyridostigmine (for myasthenia gravis), orally active, duration of action 4-8 hours •metabolism •carbamates •metabolized by plasma esterases in the body and cholinesterase •the quaternary alcohol moiety is cleaved, giving rise to the carbamoylated enzyme •t½b is 1-2 hours but duration of enzyme inhibition is 3-4 hours •duration of effect is chiefly determined by the stability of the inhibitor-enzyme complex and not by metabolism •organophosphates •hydrolysed by plasma and tissue esterases to corresponding phosphoric and phosphonic acids •some cytochrome P450 mixed function oxidases play a role in deactivation of certain organophosphorus, are responsible for converting thiophosphate insecticides (parathion, malathion) containing P=S bond to phosphorates with a P=O bond, resulting in their inactivation •malathion rapidly metabolised to inactive metabolites •elimination •via urine •edrophonium, •neostigmine •pyridostigmine •hydrolysed products of organophosphorus compounds •renal excretion plays a minor role in the elimination of physostigmine
•Pharmacodynamics •effects depend on relative degree of muscarinic and nicotinic stimulation •stimulation of muscarinic receptors at autonomic effector organs •stimulation, followed by paralysis, of all autonomic ganglia and skeletal muscle (nicotinic actions) •stimulation, with occasional subsequent depression, of cholinergic receptor sites in the central nervous system (mainly muscarinic) •effects on CNS •low concentrations: diffuse activation of EEG and subjective alerting response •higher concentration: cause generalised convulsions, which may be followed by coma and respiratory arrest •effects on CVS •increase activation of •both sympathetic and parasympathetic ganglia supplying the heart •the acetylcholine receptors on the cardiac and smooth muscle •in the heart, parasympathetic effects predominate •vagal response, negative chronotropic, inotropic, dromotropic effects with fall in cardiac output •reduction in ventricular conduction as a result of prejunctional modulation of sympathetic discharge (inhibition of noradrenaline release) as well as inhibition of postjunctional cellular sympathetic effects •modification of the tone of vessels innervated by the cholinergic nerves •the net effects on vascular tone is a balance of activation of both sympathetic and parasympathetic systems •activation of sympathetic ganglia would increase vascular resistance •effects on neuromuscular junction •at low concentrations, moderately prolong the actions of acetylcholine and increased in strength in myasthenia gravis, or after neuromuscular blockade with muscle relaxants •at higher concentrations, accumulation of acetylcholine can result in •fibrillation of muscle fibres •antidromic firing of motor neuron, resulting in fasciculation that involves entire motor unit •initial phase of depolarising neuromuscular blockade may be followed by a phase of nondepolarising blockade •direct nicotinic agonist effect •some quaternary carbamate cholinesterase inhibitors (neostigmine) have an additional direct nicotinic agonist effect at the neuromuscular junction, may contribute to effectiveness of agent in therapy of myasthenia gravis
•effect potentiation by 4-aminopyridine •not an acetylcholinesterase •increases both spontaneous and evoked release of acetylcholine from presynaptic nerve terminal, thereby increasing the force of muscle contraction •antagonises neuromuscular blocking action of aminoglycosides •effects on other organs •effect on the eye, respiratory tract, gastrointestinal tract, urinary tract are similar to the effects of direct-acting cholinomimetics because all the organs are innervated by parasympathetic system •Reversing “irreversible inhibition of acetylcholinesterase •pralidoxime •designed to have the right size and charge to bind to the enzyme active site, next to the serine group •a special side group (hydroxylamine) was added to pluck the organophosphate from the serine •can reverse some nerve gas inhibitions •other oximes such as obidoxime have similar effects •other acetylcholinesterase inhibitors •tacrine (tetrahydroaminoacridine) •crosses the blood-brain barrier •used as a respiratory stimulant, and in the treatment of Alzheimer’s disease and other central nervous system disorders •has been used to counter the effects of muscle relaxants •galanthamine •crosses the blood-brain barrier •used in the treatment of Alzheimer’s disease and dementia •has been used to counter the effects of gallamine and tubocurarine •donepezil and rivastigmine •crosses the blood-brain barrier •used in the treatment of Alzheimer’s disease
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