(1/19) Jaki Lecture: Cholinergic Nervous System Functions (2) Cholinergic Agonists: ACh is the prototype. It is a flexible molecule capable of different conformations. Agonists attempt to resemble ACh in more potent forms

- ACh Receptor Activity: Free rotation of the acetate group and quaternary ammonium group are conferred by the b and a carbon, respectively. Only in the (+ac) conformation, is ACh able to bind to the receptor. This specific requirement, amidst free rotation is by design; for if ACh were restricted to this conformation it would cause overstimulation

- Substrate Modifications (for the designing of experimental drugs) o Omnium Core Group: A positively charged functional group is critical for binding

§ N+ à S+: Sulfur exhibits identical activity to N § N+ à P+: Phosphorous is a much larger atom, inducing steric hindrance. 1/12th the activity. ß § N+ à C: Oh great, a tert-butyl. NOT. Inactive, no activity. This eliminates the basic function. Ø

o Omnium Substituents: A minimum of two methyl groups on the charged functional group is essential § Methyl à Ethyl: Each subsequent ethyl modification gradually decreases the activity.

• (1/3) à (1/50) à Inactive. As the TEC molecule, the false neurotransmitter, it is inactive § Remove all Methyls: Essentially have NH3, become less basic.

- Receptor-Specificity Modifications on the Ethylene Bridge o b-methyl Substitution: Selective muscarinic agonist o a-methyl Substitution: Selective nicotinic agonist o b+a-methyl Substitution: Inactive. Methyl substitutions act to block one side of the

molecule from attack, inducing specificity for the other. Substituting both side – inactive. Muscarinic Agonists (for mAChR)

- Modification of the Acetyl Group o Acetyl à Carbamoyl: Carbamates are less likely to be hydrolyzed and therefore are

more stable. Additionally, they are orally active. Example: Bethanechol - Muscarine: Natural alkaloid found in Inocybe and Clitocybe, but first discovered in the Mario

mushroom – Amanita muscaria. Muscarine is called a ‘Cholinomimetic’ alkaloid o ACh resemblance: Muscarine has a b-ethyl substitution completing the ring.

Muscarinic Antagonists: aka Anticholinergics, Antimuscarinics, Cholinergic blockers - Tropine Alkaloids: Traditionally known for their nitrogenous bicyclic 7-membered tropane

rings, these naturally occurring molecules bind very tightly to the muscarinic receptors. o Natural tropine alkaloids, Atropine and Scopolamine, have substituents that enhance binding activity,

making them very potent antagonists of the muscarinic receptors. o Atropa belladonna: Also known as Deadly Nightshade, this is a toxic tropine alkaloid of the Solanaceae

family. Historically known to be used by Roman females to induce mydriasis - Essential Chemical Features of Antimuscarinics: To be an active anticholinergic, must follow some rules

o (1) R1: Aromatic Ring, Typically Benzyl group. [Van der Waals interactions] o (2) R1: ‘Backwards Stereochemistry: Point “backwards” [Restrictive Active Site] o (3) N: Must have a basic amine N+ for the (-) active site [Ionic Interactions] o (4) Specific distance from R1 to N [Restrictive Active Site]

- Beneficial Chemical Features of Antimuscarinics: These features improve the affinity and efficacy of the drug o R2: Hydrophobic Ring, Typically Carbocyclic or H. [Van der Waals, Pi Stacking] o R3: Reactive EWG/EDG, Typically H, OH, Carboxamide, etc. [H-Bonding] o X: Ester, Ether, or hydrocarbon [H-Bonding]

- Receptor-bound Conformation of Atropine and its Interactions with mAChRs

- This section focused on the general basis of muscarinic anti-cholinergic activity and its skeletal structure. Next we will look at the drugs that have been designed based on this information

Notice the Nitrogen is charged (+). This is required for active site binding as the (F) “Anionic Binding Site” is negatively charged. In the charged state, these compounds are not orally available or capable of crossing the BBB. They will be given in the neutral state, and protonate in the stomach.

Antimuscarinics in Therapy - Synthetic Derivatives: These compounds lack a tropine group

o Includes: Homatropine and Tropicamide. They have shorter durations of action compared to atropine - ‘Selective’ Synthetic Derivatives: This slide was kinda skipped, she just mentioned they were selective

o Includes: Pirenzepine, Darifenacin, Solifenacin - Charged Quats: More closely resemble atropine, with a quaternized Nitrogen that is charged.

o Includes: Methscopolamine, Oxitropium, Ipratropium (SAMA), and Tiotropium (LAMA) o Mechanism: Due to their charge, they do not cross the BBB. These compounds are specific

to the M3 receptor and thereby dilate the bronchial muscles. Used in Asthma treatment § Ipratropium (Atrovent) Tiotropium (Spiriva)

- Notable Antidepressants o Paroxetine (Paxil): An antidepressant with off-target high affinity for mAChRs.

These are unwanted anticholinergic side-effects. The R1 group is a fluorinated phenyl ring. Since fluorine is so small, does not inhibit mAChR binding.

o Venlafaxine (Effexor): An antidepressant without anticholinergic side-effects. This compound is being mentioned as a proof-of-principle example. Its phenyl ring has a methoxy group, making it too large for reception by mAChR, thereby not allowing for anticholinergic activity.

- Mydriatics and Antispasmodics - The anticholinergic activity is just a side-effect o Mydriatic: Homatropine methyl bromide, Oxyphenonium o Antispasmodic: Glycopyrrolate, Clidinium Bromide, Banthine

- Parkinson’s Disease (PD) Agents o Theory: ACh and DA are needed to carefully balance normal muscle control. PD patients frequently have

severely lowered DA levels, causing a chemical imbalance theoretically leading to tremors and rigidity. By using anticholinergics to similarly decrease ACh, balance is restored, however at a lower level.

o Compounds: Uncharged, such that they can work centrally § Trihexyphenidyl (Artane, Tremin, Pipanol), Benztropine (Cogentin), Orphenadrine (Norflex),

Biperiden (Akineton). - Compounds with potent anticholinergic AE

o Includes: Amitriptyline (antidepressant, insomnia), Disopyramide (antiarrythmic), Clozapine (antipsychotic), Diphenhydramine (anti-histamine)

Nicotinic Antagonists (for nAChR) - Site of Action: Preganglionic cholinergic synapses, NMJ - Ganglionic Receptor Blockers

o Competitive: Compete with nicotine for the receptor, without eliciting an effect. These compounds currently have no clinical use. Ex: Hexamethonium, Tetraethylammonium

o Non-Competitive: Bind to an allosteric site of the receptor, changing its conformation. These short-acting compounds are used during neurosurgical procedures to treat excessive bleeding.

§ Ex: Trimethaphan, Camsilate § MoA: Acting in the periphery, these compounds induce vasodilation, effectively lowering blood

pressure to reduce bleeding. - Neuromuscular Blockers

o MoA: The basal activity of nicotinic receptors involves repolarization and depolarization in the transmission of electric potentials. The neuromuscular blocker compounds bind tightly to the ACh binding site on the nAChR, causing a briefly intense depolarization-agonist effect, followed by a dominant antagonistic effect due to tight binding. This is overstimulation of the nAChR

§ The NMJs are very large and do not have a barrier (sheath), making them accessible to polarized compounds and otherwise large molecules. Big ‘n Charged.

o Competitive: Ex: Tubocurarine, Gallamine, Pancuronium § Clinical Use: Anesthetic used to facilitate endotracheal intubation

and to provide skeletal muscle relaxation during surgery or mechanical ventilation.

§ Tubocurarine is a naturally occurring alkaloid in Curare, the plant extract used as an arrow poison by SA Natives. It has poor oral bioavailability- meat is edible after the hunt!

§ Pancuronium: Longer-acting and more potent than d-Tubocurarine. It has lethal effects through action on cardiac muscarinic receptors, causing a major increase in heart rate. Notably, it is the second of three drugs administered in US Lethal Injection Protocol.

• MoA: It has 2 ACh-like moieties, allowing it to bind to 2 receptors simultaneously

Targets for Drug Action: ----- The Cholinergic Synapse ----- - Acetylcholine Esterase (AChE): In the synaptic cleft, AChE catabolizes ACh to its constitutive components,

choline and acetic acid. Inhibition of this enzyme will cause an increase in [ACh] in the cleft, prolonging the action of ACh at postsynaptic receptors. Compounds capable of this activity are called Indirect Cholinomimetics

o Enzyme Activity: [ACh + AChE è Choline + AChE-Acetyl] then rapid hydrolysis à AChE. o Acetyl group is passed to Ser-200, the enzyme intermediate T1/2 is microseconds.

- Indirect Cholinomimetics: Indirect, because there is not receptor binding, but still induce a cholinergic effect o Reversible AChE Inhibitors: Instead of an Acetyl group, these drugs donate a

carbamoyl group to AChE, which is far more stable (less-reactive) taking the enzyme-intermediate longer to regenerate (T1/2 = 15-20mins)

§ Carbamate-Ester Type Reversible AChE-I: Physostigmine, Carbaryl, Neostigmine, Pyridostigmine, Rivastigmine

• The Aryl carbamates are more efficient than alkyl! § Physostigmine is the lead compound isolated from the Calabar bean. It is used

to treat: Glaucoma, CPD Overdose, and exhibits TCA CNS effects. § Neostigmine, due to its charged quat nitrogen, lacks central activity. It is used

as a: Prophyaxis of postoperative abdominal distention, urinary retention, and myasthenia gravis

§ Pyridostigmine: Similarly lacks CNS effects due to quat Nitrogen. It is orally effective, and used to treat: Myasthenia Gravis, and works for prophylaxis of nerve gas exposure. Longer-acting.

§ Rivastigmine (Excelon): A Pseudo-irreversible and CNS-acting AChE, it is used to treat Alzheimer’s Disease (AD). Slowly dissociating carbamylated enzyme - ~10hours

o Irreversible AChE Inhibitors: Donation of Phosphate Ester groups to AChE will cause irreversible inhibition. Following phosphorylation, the propyl groups dissociate leaving just the phosphate. This process is called aging, and it completely inactivates AChE. (T1/2 = hours to days). Ex: Diisopropylphosphorofluoridate (DFP)

§ Organophosphates: Capable of forming phosphateesters with AChE, which are very stable to hydrolysis. They require activation at the ‘A’ group.

• A: O or S. Oxygen (Active-form), Sulfur (Pro-drug) • X: Good leaving group (F, CN, Thiomalate, etc). Enzyme kicks it off • R1: Alkoxy R2: Alkoxyl, alkyl, or tert-amine

§ DFP and Echothiopate are used to treat Glaucoma, for at maximum 4 weeks. However, they have fall out of favor and are rarely used.

§ Nerve Gas/Chemical Warfare Agents: Organophosphates are more commonly know as potent nerve gases, like Tabun, Sarin, and Paraoxon.

§ Insecticides: Organophosphates can be used as insecticides. To make them less toxic to humans, the sulfur (pro-drug) variation are used. These thiophosphates are bioactivated by CYP450 present only in insects, making them selectively toxic. (Ex: Malathion à Malaoxon)

• Schrader (drug): This is a phosphoramide organophosphate compound that is inactive due to a poor leaving group. However, it insects, it is oxidized into a toxic metabolite

Organophosphate Poisoning - Poisoning: Ingestion of an organophosphate will result in AChE inactivation. AChE(Ser-200) Nucleophilic attack

on the phosphate will form a phosphoester, which can be hydrolyzed by water, though is relatively well-shielded. - Aging: Since the Ser-P phosphoester bond is well-shielded, the organophosphate R2 group is more prone to

hydrolysis. If hydrolyzed, the serine bond strengthens, making the AChE inactivation permanent. - Antidote Rescue: IF AGING HAS NOT YET OCCURRED, Pralidoxime chloride can attack the phosphoester

bond via nucleophilic attack and substitute out the phosphate group, thereby regenerating AChE.