NS - Cholinergic Receptor Systems

Describe the general structure of Cholinergic System

- Acetylcholinesterase (AChE) enzymes maintain the synapse by removing acetylcholine through hydrolysis into acetyl and choline
- Choline Acetyl Transferase (CAT) transfers an acetyl group from Acetyl Co-enzyme A to choline
- There are both ionotropic and metabotropic acetylcholine receptors on both pre-synaptic and post-synaptic membranes

Describe some characteristics of Acetylcholinesterase

- There are two types, Acetylcholinesterase found at neurone synapses and Butyryl-Cholinesterase found in blood plasma
- It is an efficient enzyme with a turnover rate of 25000/s
- It has an anionic binding site formed by Aspartate, and an ester binding site formed by Histidine and Serine, with Tyrosine to hold the ester in place

Describe the mechanism of catalysis for Acetylcholinesterase

- Acetylcholinesterase follows Nucleophilic Addition-Elimination mechanism, twice with active and inactive Serine
- Serine initially acts as a Nucleophile, while Histidine can either be an Acidic or Basic catalyst based on the protonation state of the serine intermediate
- When Serine is acetylated, the enzyme becomes inactivated, until the serine is regenerated
- Histidine aids the mechanism and leaving groups as water and serine are poor nucleophiles, and choline and serine are poor leaving groups
- Aspartate allows the carbonyl to be positioned within distance of the Serine+Histidine for hydrolysis

What are Cholinesterase inhibitors, giving examples of therapeutic classes?

- They are agents that prevent action of Cholinesterase enzymes on acetylcholine, increasing the concentration and duration of acetylcholine in a synapse
- They may be used to treat Glaucoma, Myasthenia Gravis and sometimes Alzheimer's disease
- There are four main therapeutic classes which are Tetra-alkylammonium ions, Quaternary Ammonium Alcohols, Carbamates and Organophosphates

What is transient inhibition, and which drugs follow this type of inhibition?

- Transient inhibition is when the target for inhibition is not directly interacted with
- For Cholinesterase inhibitors, transient inhibitors interact with the Aspartic acid, rather than the Serine
- Tetra-alkylammonium ions and Quaternary Ammonium Alcohols are transient inhibitors, acting non-covalently and reversibly, however the latter is used more in diagnosis rather than treatment

Describe the mechanism of action of Carbamates

- Carbamates inactivate the Cholinesterases by transferring their carbamoyl group to the Serine residue
- This prevents Acetylcholine binding (acting as a reversible competitive inhibitor), and the enzyme becomes unavailable for a prolonged time (minutes) while it hydrolyses the carbamoyl group
- This is due to the carbamoyl intermediate being more stable than the acetylcholine intermediate, so more energy/more time is needed for hydrolysis

What are Carbamates?

- Carbamates are the main therapeutic class of Acetylcholinesterase inhibitors
- They mainly act on post-ganglionic parasympathetic neurones and have a medium duration of action
- They are used in the treatment of Glaucoma, Myasthenia Gravis and Alzheimer's
- Protonated Carbamates will not pass the BBB for therapeutic effect (eg. Neostigmine), while more lipophilic carbamates can pass the BBB for treatment of Alzheimer's (eg. Pyridostigmine)

What are Organophosphates?

- Organophosphates are a class of Acetylcholinesterase inhibitors that are long lasting, used as poison/nerve gas, but nowadays used as insecticides
- They work by covalently bonding to the enzyme, as a result they are highly toxic and irreversible
- The phosphate group bonds strongly to the Serine residue and forms a highly stable intermediate, inactivating the enzyme indefinitely (irreversible phosphorylation)

Describe the design of Organophosphate Antidotes

- Compounds need a strong nucleophile to cleave the strong P-O of phosphate esters, regenerating Cholinesterase activity as a result
- Pralidoxime is most commonly used and it removes the phosphate group from the Serine residue
- ProPAM is a prodrug of Pralidoxime that is more lipid soluble so can pass the BBB and work on the CNS after metabolism into Pralidoxime

What are clinically used Cholinesterase inhibitors?

- Clinically used Cholinesterase inhibitors include the Carbamates, Quaternary Ammonium Alcohols and some Tetra-alkyl ammonium ions
- Polar compounds are used for treatment of PNS conditions
- Non-polar compounds are used for treatment of CNS conditions

How do Acetylcholinesterases inhibitors improve certain Glaucoma, Myasthenia Gravis and Alzheimer's?

- For Glaucoma, inhibiting the enzyme means enhanced effect of Acetylcholine on ciliary body, so increases eye fluid drainage, relieving intracellular-ocular pressure
- For Myasthenia Gravis, inhibiting the enzyme increases lifetime of Acetylcholine at neuromuscular junctions, enhancing stimulation of muscle fibres
- For Alzheimer's, inhibiting the enzyme leads to more Acetylcholine in the CNS, improving cognition, and symptomatic relief at early stages of Alzheimer's

Describe the characteristics of Nicotinic Acetylcholine receptors

- Nicotinic Acetylcholine Receptors are ligand gates ion channels, with 5 subunits: alpha, beta, gamma, theta and epsilon
- The subunit combination is what determines the ligand binding properties
- Subunit combination varies from location to location so making a universal Nicotinic receptor antagonist is difficult
- They are named after discovery that Nicotine is an agonist for all Nicotinic Acetylcholine receptors

What are challenges of designing Nicotinic drugs and what are the templates used for drug design?

- Challenge for design arises from the diversity of Nicotinic Acetylcholine receptors, and the pharmacological differences of these receptors in different species (for pre-clinical testing)
- The templates used for drug design are Nicotine, Epibatidine and Cytisine

What are the clinical uses of Nicotinic Agonists?

- Nicotine = Full agonist for all receptors, used as a smoking cessation aid
- Vareniciline = Partial agonist, used as a smoking cessation aid
- Suxamethonium = Neuromuscular blocking agent, used as muscle relaxant during surgery (prevents muscle twitching)
- Galantamine = Allosteric agonist (causes conformational change in receptor), used for Alzheimer's

What are Nicotinic Antagonists and their clinical uses?

- Nicotinic Antagonists prevent binding of agonists and target post-ganglion neurones and neuromuscular receptors
- Trimetaphan = limited used in Pulmonary Oedema and Hypertension
- Pancuronium = used as a muscle relaxant in general anaesthesia (steroidal)
- Atracurium = used as a muscle relaxant in general anaesthesia (non-steroidal)

Describe the characteristics of Muscarinic Acetylcholine Receptors

- Muscarinic Acetylcholine receptors are G-Protein coupled receptors, following a second messenger model
- They have three targets for drug compounds: Orthosteric (defined binding site), Allosteric (undefined binding site) and Bitopic (properties of both)
- There are 5 different subtypes, split by their response: Excitatory response = M1, M3 and M5, Inhibitory response = M2 and M4
- They are named after the discovery of Muscarine binding to every Muscarinic Acetylcholine receptor

What is the effect of Muscarinic Receptor activation on the Heart?

- The heart has M2 Acetylcholine receptors found in the atria and nodes
- The M2 receptors follow a Gi pathway (inhibitory), inhibiting production of cAMP, by inhibiting the Adenyl Cyclase enzyme
- This leads to decreased Ca2+ influx, but increased K+ influx
- This causes decreased Cardiac Output and decreased Heart Rate
- Drugs are screened for interaction with M2 and M4 receptors due to involvement with heart activity

Describe the properties of Pilocarpine

- Pilocarpine is a non-selective muscarinic agonist used to treat Glaucoma
- It acts on the M3 receptor in the Iris Sphincter muscle, causing constriction of the pupil and promoting intra-ocular fluid drainage (half life ~ 3/4hrs)

What are the Clinical Uses of Muscarinic Agonists?

- Carbachol = Non-selective muscarinic agonist, does not pass the BBB and administered through the ocular route
- Xanomeline = M1 and M4 selective receptor agonist, clinical trial for Schizophrenia
- Cevimeline = M3 selective receptor agonist, used in dry mouth

What are Muscarinic Antagonists and their clinical uses?

- Muscarinic Antagonists prevent binding of agonists and target post-ganglionic neurones only
- Benzatropine = used in Parkinson's disease
- Scopolamine = used in motion sickness, and treating drug addictions
- Atropine = non-selective muscarinic antagonist, produces mydriasis and cycloplegia (both causes dilation of the pupil, latter involves paralysis of ciliary muscles)

Give examples of conditions that may be treated using Antimuscarinic agents

- Antimuscarinic agents = Muscarinic antagonists
- Oxybutynin = treating urinary incontinence using a Muscarinic antagonist
- Ipratorium = acts as bronchodilator in asthma, lowering intracellular cGMP concentration (anti-secretory)
- Methylscopolamine = acts as an anti-ulcer agent (anti-secretory)

What is the difference in the Pharmacophore of Nicotinic and Muscarinic agents?

- Muscarine has a 4.4 Angstrom distance between N and C-O (compared with Acetylcholine)
- Nicotine has a 5.9 Angstrom distance between N and C=O (compared with Acetylcholine)