1. Good and Bad AChE Inhibitors
PHM142 Fall 2015
PHM Fall 2015
Instructor: Dr. Jeffrey Henderson
Presented by
Sanielle Cole
Ingrid Lowe
Vincent Le
Vincent Nguyen

2. What is AChE?
AChE refers to the enzyme acetylcholinesterase. It is a serine hydrolase and a key enzyme in the CNS.
Location
Postsynaptic membrane of cholinergic synapses
Function
Terminate the action of acetylcholine (and some other choline esters) by the process of hydrolysis. Acetylcholine is hydrolysed to acetate and choline.

3. Overall Action of AChE

4. Mechanism of AChE
The active site of AChE consists of two subsites: the anionic subsite and esteratic subsite.
Anionic: Serves to bind a molecule of ACh to the enzyme
Esteratic: Location of hydrolytic reaction (contains the catalytic triad)
There is also a peripheral anionic site distinct from the choline binding pocket of the active site.
It serves to bind ACh and other quarternary ligands acting as non competitive inhbitors

5. Structure and Mechanism of AChE

6. Good AChE Inhibitors Inhibit cholinesterase enzyme
Reversible, competitive or non-competitive
Diagnosis/Treatment of diseases
Alzheimer’s Disease (AD)
Most common form of dementia
Loss of cholinergic neurons in the brain  Decreased ACh
Inhibit cholinesterase enzyme from breaking down ACh, increasing both level and duration of NT action.
Reversible, competitive/noncompetitive – therapeutic applications
Irreversible – toxic effects
Diagnosis/treatment of diseases such as myasthenia gravis, AD, post-operative ileus, bladder distention, glaucoma and antidote to anticholingergic overdose
Alzheimer’s Disease
Progressive neurological disorder – most common form of dementia
Loss of memory and other intellectual abilities interfering with daily life.
Loss of cholinergic neurons in the brain and the decreased level of ACh
Treatment inhibit AChE (no cure) – decrease the breakdown rate of ACh
Treats symtoms related to emmory, thinking, language, judgment and other thought proceses
Boost cholinergic neurotransmission in forebrain regions and compensate for loss of functioning brain cells.

7. Reversible Inhibitors
Piperidines
Donepezil (Aricept)
Carbamates
Rivastigmine (Exelon)
Phenanthrene Derivatives
Galantamine (Razadyne, Nivalin)
Others
Piperidines: characterized by the heterocyclic amine
Carbamates: characterized by the carbamic acid group
Also include other ones such as physotigmine (treats glaucoma, eye drops), neostigmine (treats MG orally, reverse neuromuscular block IV), pyridostigmine (treats MG orally), ambenonium, demecarium
Phenanthrene Derivatives: characterized by three fused benzene rings
Other reversible inhibitors include caffeine, tacrine, edrophonium (diagnosis of myasthenia gravis), huperzine A, ladostigil, ungeremine, lactucopicrin

8. Reversible Inhibitors
Donepezil
Selective, reversible
Binds to peripheral anionic site
Rivastigmine
Less selective, slowly reversible
Binds to the esteric site
Galantamine
Selective, rapidly reversible
Binds to anionic site
Donepezil
- available as disintegrating tablet and oral solution, being 100% oral bioavailability with ease crossing the blood-brain barrier and slow excretion
Better tolerated
Rivastigmine
- Powerful
- Not as selective – binds to BuChE and AChE.
- orally as capsules or liquid formulations, with good absorption and bioavailability of about 40% in the 3 mg dose
Galantamine
Nicotinic, cholinergric receptors! Enhances activity of nicrotinic receptors in presence of ACh.  Affects other neutrotransmitter systems leading to more beneficial effects
absorption is rapid and complete, with absolute oral bioavailability between 80 and 100% and seven hours half-life.

9. Irreversible Inhibitors
Ophthalmology
Glaucoma
Organophosphorus Compounds
Diisopropyl fluorophosphate
Echothiophate

10. Bad AChE Inhibitors Bind irreversibly to the enzyme
Mostly Organophosphorus compounds which are commonly Insecticides and nerve agent/gases
Esters or thiols of phosphate derivatives
Bind irreversibly to the enzyme (and exert undesired effects)
Mostly Organophosphorus compounds which are commonly Insecticides and nerve agent/gases
Esters or thiols of phosphate derivatives

11. General Structure of OPs
Organophosphorus Compounds
Important to note that thio forms are metabolised to more reactive oxo form before binding
Here you see that the phosphorus is double bonded to an oxygen or sulphur atom
R2 usually has an Oxygen or sulphur bonded to phosphorus
X may belong to a halogen, aliphatic, aromatic or heterocyclic groups.

12. E+PX ⇄ E•PX → EP+X OPs are substrate analogues to ACh
The OPs exert their main toxicological effects through non- reversible phosphorylation of esterases in the nervous system
Organophosphates are relatively toxic to both insects and man.
- OPs are substrate analogues to Ach and like natural substrate enter the active site covalently binding to serine –OH group
- The OPs exert their main toxicological effects through non-reversible phosphorylation of esterases in the central nervous system
- Organophosphates are relatively toxic to both insects and man. However, in environments they are often degraded through hydrolysis of the phosphate ester.
- The stabilization of the phosphorylated AChE is considered aging and at this point the enzyme cannot be regenerated. This step can take minutes to days to occur. This is dependent on the structure of the inhibitor as well as its metabolites.

13. Most Common Examples Insecticides:
ethyl parathion
malathion
methyl parathion
Nerve Agents: Used in Chemical Warfare
Sarin Gas
Tabun, Soman
VX - VX is the most toxic and long lasting of the nerve gases
These compounds, as irreversible cholinesterase inhibitors, are effective in very low concentrations and are capable of causing death within minutes of exposure.
The inhibition of the enzyme leads to accumulation of ACh in the synaptic cleft resulting in over-stimulation of nicotinic and muscarinic ACh receptors and impeded neurotransmission. The typical symptoms of acute poisoning are agitation, muscle weakness, muscle twitching, hypersalivation, sweating. Severe poisonings may cause respiratory failure due to asphyxia, unconsciousness, confusion, convulsions and/or death.

14. Treatment of Organophosphate Intoxication
Organophosphates irreversibly inhibit AChE at serine residues
Leads to muscarinic, nicotinic or central systems crisis
Non-pharmacological treatment
Resuscitation, oxygen supply or decontamination
Pharmacological treatment
Symptomatic
Parasympatolytics (ex. Atropine)
Anticonvulsives (ex. Diazepam)
Causal
Oxime reactivators (ex. pralidoxime, trimedoxime, asoxime, obidoxime)
Parasympatolytics – atropine – reduce effects of accumulated ACh
Anticonvulsives – diazepam – diminish neuromuscular seizures

15. Detoxification of Carbamate
Increase water solubility of carbamate to remove in urine
Carboxylesterases (CESs)
Hydrolyze carboxyl esters
Carbamates are structurally similar to carboxyl esters
Dependent on the chemical structure
Differs with animals
Mechanism:
acylates serine residue in the protein, releasing the alcohol moiety of the ester
Water comes in and attacks the intermediate and forms carboxylic acid and free active CES
***carbamic acid gets rapid degraded into carbon dioxide and its respective amine
Differs with animals in terms of hydrolyzing activity

16. Detoxification of Organophosphorus
Oxidation and hydrolysis
Carboxylesterases
Naturally present CESs in mammals called B-esterases
Same process as carbamates except final step
The phosphorylated enzyme cannot be reactivated by water
One CES molecule per OP molecule
Phosphotriesterases (PTEs)
Bond cleavage of phosphorus and leaving group of OPs
The final products are easily eliminated
One PTE molecule can degrade multiple OP molecules
B-esterases are inhibited by Ops
Known PTEs include DFPase (Diisopropyl fluorophosphates)and paraoxonase
PTE products are more polar so they accumulate less in fat and are easily excreted. They are also less toxic than the parent OP.
PTEs are usually found in the serum and liver of many biological life forms including mammals, birds, fish, molluscs, and bacteria

17. Summary
AChE: Terminates the action of acetylcholine (and some other choline esters) by the process of hydrolysis. Acetylcholine is hydrolysed to acetate and choline.
Good AChE Inhibitors
Diagnosis/Treatment of diseases  Alzheimer’s Disease
Mostly reversible
Piperidines; Donepezil (Aricept)
Carbamates: Rivastigmine (Exelon)
Phenanthrene Derivatives: Galantamine (Razadyne, Nivalin)
Few irreversible
Organophosphorus Compounds: Diisopropyl fluorophosphate, echothiophate
Bad AChE Inhibitors
Insecticides: Ethyl parathion, malathion, methyl parathion
Nerve Agents: Used in Chemical Warfare: Sarin gas, Tabun, Soman, VX – VX
Detoxification
Carbamates: Carboxylesterases
Organophosphorus compounds: Carboxylesterases, phosphotriesterases (PTEs)

18. Summary continued.
Organophosphates irreversibly inhibit AChE at serine residues
Leads to muscarinic, nicotinic or central systems crisis
Non-pharmacological treatment
Resuscitation, oxygen supply or decontamination
Pharmacological treatment
Symptomatic
Parasympatolytics (ex. Atropine)
Anticonvulsives (ex. Diazepam)
Causal
Oxime reactivators (ex. pralidoxime, trimedoxime, asoxime, obidoxime)
Parasympatolytics – atropine – reduce effects of accumulated ACh
Anticonvulsives – diazepam – diminish neuromuscular seizures

19. References
Čolović, M. B., Krstić, D. Z., Lazarević-Pašti, T. D., Bondžić, A. M., & Vasić, V. M. (2013). Acetylcholinesterase Inhibitors: Pharmacology and Toxicology. Current Neuropharmacology, 11(3), 315–335.
Hermona S., Shlomo S. (2001). Acetylcholinesterase — new roles for an old actor. Nature Reviews Neuroscience 2,   .
Katzung BG. Introduction to autonomic pharmacology. In: Basic and clinical pharmacology, 8th edition. USA: The McGraw Hill Companies, Inc, 2001:75–91.
Elersek, T. and Filipic, M. (2011). Organophosphorous Pesticides- Mechanism of Their Toxicity. Stoytcheva, M (Ed.) Pesticides - The Impacts of Pesticides Exposure (Pages ). Croatia: Intech
Prins, J. M., Chao, C.-K., Jacobson, S. M., Thompson, C. M., & George, K. M. (2014). Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: an in vitro model for organophosphate oral exposure.Toxicology in Vitro : An International Journal Published in Association with BIBRA, 28(5), 715–