Cholinergics and Anticholinesterases

Acetylchlonine (Ach)

Our Nervous System

Because of its wide and important involvement, understanding Nervous system is important to treat many diseases

Functions – • To transmit signals to and from body organs or cells to carry out o Heartbeat, Respirationo Digestion, Hormone secretiono Movement, body pressure

• To process sensory information• Logic, Decision and Memory

Drugs based on Nervous System treat various clinical conditions

Cholinergic NS NS based on other Neurotransmitters

Impaired or excessive gastric/secretion

Epilepsy – irregular neuronal activity leading to muscle spasm

Glaucoma – increased pressure on eyeBradycardia – slow heart beat

Anxiety – feeling irrational fearRelief from pain

Alzheimer’s and –loss of memory

Parkinson disease – loss of movement control

Myasthenia gravis – muscle weakness

Cause unconsciousness during surgery

First lesson in Medicinal chemistry• Medicinal Chemistry aims to cure diseases. But to do

that we need to know what biological factor is causing disease. Then we create drugs that interact with that particular factor to either suppress it or stimulate it.

• These biological factors arise from alteration in the process of normal body functioning. Thus to find and understand what goes wrong during illness, we must first study the normal functioning of body. This allows us to recognize a central biological factor that is most involved in causing disease. Then we make drugs to act on it.

Neurons

• Neurons are individual cells of the Nervous System that process and transmit signals by electrical and chemical process.

• Adjacent neurons are physically separated by the each other. The gap region is called synapse.

Fig: Neurotransmitters moving through Synapse between two neurons

Pre-synaptic Neuron(sends signal)

Post-synaptic Neuron(receives signal)

• Neurotransmitters (NT) are endogenous (produced by body) chemicals that transmit signals across a synapse from sending presynaptic neuron to the target postsynaptic neuron

• They are synthesized and stored in neuron itself• There are many NTs eg Acetylcholine, Adrenaline,

serotonin, dopamine, GABA• The process of transmission of signal along a neuron

and over the synapse is called neurotransmission. Signal can pass over the synapse by either chemically or electrically

• One neurons interacts with many other neurons in all possible directions.

Our Nervous system

Types of peripheral NS

• Somatic NS- – controls voluntary muscle Movement– Transmits sensory information to brain

• Autonomic NS– Controls involuntary body functions such as Heart

beat, secretion (GI acid/insulin), fight or flight responses

Two types of Autonomic NSParasympathetic NSUses Acetylcholine

Sympathetic NS Uses Adrenaline

Makes body ready for fight or flight

Makes body ready for rest

Did you note the mono-directionality ?

• Instead of a single light switch that you can turn on/off or a single volume knob you can turn high/low…..

• In this case you have two independent control system for doing opposing things, eg sympathetic increases heart beat while parasympathetic is required to slow heart beat, there is no way neither NS can reverse or undo it’s action by itself.

Introduction

• Cholinergics refer to the part of Nervous system that utilize Acetylchlonine (Ach) as a neurotransmitter. It is key NT in the parasympathetic NS

• A unique feature of Ach is that the same molecule can bind with two different receptors (muscarinic and nicotinic receptor) using different conformation.

AcetylcholineAcetyl Choline

Physio-Chemical property of Ach• It is ester of acetic acid and choline• It is soluble in water due to salt form at Nitrogen • In solid form it is stable but in solution, the ester group gets hydrolyzed (ie

ester group turns into acid and alcohol). • If acid or base is present, as in stomach, then rate of hydrolysis is so fast

that it prevents oral dosing of Ach • Even if we prepare it’s solution in neutral water and inject in blood so as to

bypass the acidic stomach, an ester hydrolyzing enzyme called butrylcholinesterase significantly degrades it s that the pharmacological response is very weak

• Even if it was administered in stable form, It’s ionic ammonium group prevents good penetration across the lipophillic cell wall

• This chemical property makes it a weak agonist plus since it is non-selective agonist of Muscarinic and Nicotinic

• (Thus having no ester group and no ionic amine is an approach tp making more stable and strong Ach agonist. However selectivity is a different issue)

Based on above info we can make simple changes to Ach hopefully design derivatives to

improve stability and cell penetration.

AchConceptual Agonist drugs

Drug design is a conceptual thing. We don’t really know if drug will really work as expected until we synthesize and test them. To improve chances of success the design part should not be random but have some rational

Muscarinic receptor

• They are agonized by a poisonous mushroom derived compound called muscarine

• They occur primarily in the CNS and in Autonomic NS, and are part of a large family of G-protein-coupled receptors

• Physiological functions include heart rate and force, contraction of smooth muscles and the release of other neurotransmitters.

• There are five subtypes of muscarinic AChRs based on pharmacological activity: M1-M5

Nicotinic receptor

• They are agonized by nicotine• They also occur in the CNS and Autonomic NS plus are

exclusive in neuromuscular junction, and are part of a ligand gated ion channel receptors

• Physiological functions depend upon muscle-type or neuronal-type

• Muscle-type nicotinic AChRs are localized at neuromuscular junctions and allow muscle contraction and maintain muscle tone; (thus these are targets for muscle relaxants)

• Neuronal type are involved in cognitive function, learning and memory, arousal, reward, motor control and analgesia.

H3C O

O

CH2 CH2 N(CH3)3

QuaternaryAmmonum group

Ethylenegroup

Acyloxygroup

SAR of cholinergics as Muscarinic agonist• Cholinergic drugs mimic action of Ach on

Muscarinic or nicotinic receptors and produce the same effect as Ach but in greater magnitude

• A general strategy of making an agonist is to use the original compound, in this case Ach, as a framework

1) Modification of quaternary Ammonium groupa) Presence of nitrogen in quaternary ionic form is

important for agonist activity. Replacement of Nitrogen with other elements such as Sulphur, Arsenic and Phosphorous reduces activity

b) Presence of three methyl group is needed for agonist activity. Changing the three methyl group by higher alkanes or Hydrogen also causes loss of activity

H3C O

O

CH2 CH2 N(CH3)3

QuaternaryAmmonum group

Ethylenegroup

Acyloxygroup

Replacement with Arsenic or Phosphorous maintains + charge but still reduces activity

Replacement with sulphur removes the + charge and causes reduced activity

Only this has good potency

Conclusion 1) positive charge needed 2) Positive charge should be on Nitrogen only3) Charge on Nitrogen only possible when it bonded to 4 atoms ie quaternary form needed

Valency S = 4P = 3Ar = 3N =3

• If R = methyl,(CH3) --> active• If R = ethyl (C2H5) --> antagonist!• If R = propyl (C3H9) and higher alkyls --> inactive• If only one of the R = ethyl or propyl active but

less potent than Ach• If any or all R = H activity goes on decreasing

2) Change in the ethylene groupc) A “rule of five” idea states that there should

be no more than 5 atoms between the Nitrogen and the terminal Hydrogen

As the chain length increased from two, activity is rapidly lost.

C O

N

O

H

H

H

1

2

34

5

This tells us about the

relative size of binding site

d) Inclusion of methyl group in the ethylene carbons can alter selectivity

Methyl inclusion in β carbon relative to N retains potency of Ach and more selective to muscarinic receptor. This compound is called methacholine and used clinically

Methyl inclusion in α carbon relative to N reduces potency but makes more selective to nicotinic receptor. This is not used clinically

•Methyl group in Beta carbon•As potent as Ach•Selective to Muscarinic receptor•Used clinically

•Methyl group in alpha carbon•Not As potent as Ach•Selective to Nicotinic receptor•Not Used clinically

Methacholine

H3C O

O

CH2 CH N(CH3)3

CH3

H3C O

O

CH CH2 N(CH3)3

CH3

3) Modifications to the Acetoxy groupe) Substituting the Acetyl with higher homologous group such as propionyl or butyryl Reduces activity.

R O

O

CH2 CH2 N(CH3)3

If R = propionyl (C3H7), butyrl (C4H9) or higher than activity is reduced

f) The ester group isn’t mandatory as quanternary amine group but an oxygen atom is required in this region

g) Since ester group makes it liable to hydrolysis, alternate groups were included and found that replacing the ester with carbamate, ether or ketone function resists hydrolysis while maintaining activity

H3C O CH2 CH2 N(CH3)3

H3C CH2 CH2 N(CH3)3

O

H2N O

O

CH2 CH2 N(CH3)3carbamates

Ethers

Ketone

(Carbamates are resistant enough to Gastric acid to be given orally)

Modification to reduce hydrolysis

SAR of cholinergics/Muscarinic

agonist1. Presence of nitrogen in quaternary ionic form is important for

agonist activity2. Presence of three methyl group in Nitrogen is needed for

agonist activity3. A “rule of five” idea states that there should be no more than

5 atoms between the Nitrogen and the terminal Hydrogen4. Inclusion of methyl group in beta carbon to N makes

muscarinic selective in alpha carbon to N makes nicotinic seelctive

5. The ester group isn’t mandatory as quanternary amine group but an oxygen atom is required in this region

6. Replacing the ester with carbamate, ether or ketone function resists hydrolysis while maintaining activity

H3C O

O

CH2 CH2 N(CH3)3

QuaternaryAmmonum group

Ethylenegroup

Acyloxygroup

Designing a better drug by using SAR

• Problem with Ach was it’s instability and unselective activity

• Combine point 4 and point 6 into a new structure and see what you get?

• How does it compare with Ach?

Pharmacological action of AchA) Through the muscarinic receptorCardiac effects • Bradycardia, • decrease of atrioventricular conduction. • decrease of the strength of atrium

contractions. Blood vessels• Acetylcholine injection causes release of nitric

oxide (NO) which dilates blood veins

• Effects on smooth muscles• intestine: an increase in tone with sometimes an increase

in the peristaltic contractions. This can lead to Nausea and vomiting.

• ureters: increase in tone. • bronchi: bronchoconstriction. (An aerosol of acetylcholine

can cause an attack of asthma)• Effects on secretions • Acetylcholine increases digestive (abundant saliva),

bronchial, cutaneous (sweat) and lacrimal (tears) secretions.

• Effects on the eye• Acetylcholine induces a decrease of iris diameter or miosis

which can lower the intra-ocular pressure

• Through the nicotinic receptor• In Autonomic NS, Ach allows nerve transmission• In neuromuscular junctions– At low dose Ach allow skeletal muscle movement

(important for breathing)– At high does, it causes muscle paralysis!

• In Brain, cholinergic deficiency causes Alzheimer disease

Bio-synthesis of Ach

Specific Muscarinic agonist

Drugs other than Ach that produce the same effects As Ach at Muscarinic receptor but for longer time and greater intensity– Methacholone Chloride– Carbachol Chloride– Bethanecol Chloride– Pilocarpine Hydrochloride

Mechanism of Action (MOA) : They act directly by binding to muscarinic receptor as a agonist and produce the same effects as Ach

Methacholone Chloride

• It is a muscarinic selective cholinergic agonist.• It’s S enantiomer is 240 times more potent than R

enantiomer. Howeever, R iosmer is a weak inhibitor of Ach degrading enzyme called Acetylcholinesterases.

• Thus this is given as a racemic mixture• Use – It is used to induce bronchospam in asthma

patients for purpose of verifying the diagnosing asthma

• MOA

Carbachol Chloride

• It is carbamate analog of Ach. This feature makes it very resistant to hydrolysis by both GI acid and Acetylcholinesterase such that it can be given orally. But it is not selective to muscarinic or nicotinic receptor.

• Uses: It’s use is limited to treatment of glaucoma and constrict the pupils during eye surgery.

• MOA

Bethanecol Chloride• It is an carbamate derivative of Ach which contains

a methyl group in beta carbon to Nitrogen. This makes the molecule very stable to hydrolysis and selective to muscarine too.

• Uses: – treat urinary retention resulting from general

anesthetic – treat gastrointestinal atony (muscles lose their

peristalic ability)• MOA

PilocarpineChloride

• It is a plant derived alkaloid whose structure does not match the established SAR but still acts like an cholinomimetic

• It is not selective to muscarine. Unlike other muscarine agonists, it can penetrate the eye well following topical application

• Uses: – Treat dryness of mouth caused by radiation therapy in the head

or neck– Glaucoma– constrict the pupils during cataract surgery

• MOA

• Tell why carbamates resist AchE

Anticholinesterase

• Acetylcholinesterase (AchE) is a enzyme that hydrolyzes Ach into Acetic acid and Choline

• MOA: Anticholinesterase drugs work by inhibiting the enzyme Acetylcholinesterase which prevents hydrolysis of Ach thus increasing their concentration in the synapse which promotes more Ach action. Since they promote Ach activity without binding to any receptor they are also called indirectly acting cholinergic agonists

• Note: There is enough Acetylcholinesterase in the synapse to hydrolyze 3 X 108 molecules of Ach in 1 millisecond (10-

3sec). Normally only 3 X 106 Ach are released into synapse

Applications• Improve muscle strength in Myasthenia gravis• Glaucoma • Alzheimer's• Insecticides • Chemical weapon (serine gas) • There are two types: Reversible and Irreversible

Theory of AchE inhibitors

If instead of acetyl group there is carbamate group then hydrolysis will be resisted. The AchE which is not hydrolyzed cannot be used again. Thus goal of AchE inhibitor is to provide such hydrolysis resistant functional group such as carbamates or phosphate ester

During hydrolysis of Ach, the AchE gets acylated. It needs to be hydrolyzed by water to be regenerated in free from or else it can’t function again.

Reversible Anticholinesterase

• These are compounds can act by two ways:• A) They bind but don’t react with AchE with greater

affinity than Ach like Ach does or • B)these compounds that bind and react with AchE to

form acylate AchE which is mores table form but still capable of being easily hydrolyzed

• Reversible means that they inhibit AchE for short time (only few mins)

• These compounds have more therapeutic uses than irreversible ones eg Physostigmine and Neostigmine

Physostigmine

• It is an alkaloid type anticholinesterase obtained from the seeds of calabar beans

• It has no charged amine and is more lippohillic and can thus penetrate the blood brain barrier

• It has very great affinity for AchE but that can only in charged form. Thus there is pH limitation in it’s activity.

• Uses – Glaucoma– Counter CNS poisoning by atropine and tricyclic depressents

• MOA – It inhibits AchE by binding and reacting to it and carbamylating it. This carbamylated enzyme is slow to hydrolysis

Neostigmine

• It is a synthetic anticholinesterase based on Physostigmine.• It resembles the aromatic features of physostigmine and also

the distance between the ester and ammonium is same• But since it has charge on Nitrogen, it cannot cross the CNS like

physostigmine does• Also its half life is shorter than physostigmine• Uses

– Myasthenia gravis– To counter Urinary retention

• MOA – It inhibits AchE by binding and reacting to it and carbamylating it. This carbamylated enzyme is slow to hydrolysis

Irreversible Anticholinesterase• These compounds act by only one way: that bind and

react with AchE to AchE with greater affinity than Ach to form acylated enzyme which strongly resist hydrolysis

• Irreversible means that they inhibit AchE for very long time(many hours)

• These compounds have less therapeutic uses than reversible ones.

• Serine gas and organophosphate insecticides are based on this concept.

• They cause cholinergic crisis• Their common structural feature is the presence of

Phosphate ester bond which strongly resist hydrolysis

Cholinergic crisis

• A cholinergic crisis is an over-stimulation at a neuromuscular junction due to an excess of acetylcholine (ACh). This happens due to inactivity (perhaps even inhibition) of the AChE enzyme, which normally breaks down acetylcholine. This is a consequence of some types of nerve gas, (e.g. sarin gas) or insecticides.

• It causes muscle paralysis and respiratory failure

Aging

Irreversible Anticholinesterase containing Phosphate ester resist hydrolysis very strongly. They also undergo a feature called aging which increases this resistance even more. When AchE becomes acylated for a long time with, one of the ester bond is broken. This creates a negative charge that oppose nucleophillic attack on phosphorous and in this state it resists hydrolysis even more. At this stage antidotes against Irreversible anticholinesterase such as PAM doesn’t

work. Thus regeneration of AchE is blocked for even longer periods leading to cholinergic crisis

Organophosphate poisoning

Most insecticides use concept of irreversible AchE inhibition to kill pesticides. These compounds contain Phosphate ester bond that strongly resist hydrolysis. They are very lipophillic and volatile also. They can quickly enter the blood stream and inhibit AchE for a many hours. This promotes Ach activity in the synapse which leads to ‘cholinergic crisis’. When this happens muscles stop responding to Ach causing paralysis and respiratory failure (death)

Antidote to insecticidesBecause insecticides can lead to chloinergic crisis, antidotes were designed to hydrolyze the acylated AchE. A successful antidote was PAMIt has a quaternary ammonium and a very strong nucleophile called oxime group and both work together to free AchE from the phosphate ester compounds

Pralidoxime(PAM)

How PAM reverses poisoning by organophosphate or serine gas ?

There are two sites in the binding pocket of AchE. One is a anionic site which is empty and other is the Esteric site where the phosphate compound sits. 3 event follows• First the charged ammonium of PAM binds to the anionic site. • From there PAM’s strong nucleophillic oxime group can be in close

distance to attack the ester bond between Phosphorous and serine amino acid of AchE

• This form free AchE and phosphorylated PAMLimitation: For PAM to work, it must be used immediately following exposure to insecticides or serine gas or no more than 36 hrs of exposure or else aging will occur and it wont be able to dislocate the phosphate form the receptor

The search is on for stronger nucleophilic molecules that effectively displaces phosphate even in aged form. This is a problem entirely of pure chemistry. No receptor/enzyme considerationBut enzymes are very powerful in catalyzing any reaction. What if we could engineer a protein to specifically cleave this bond? Protein engineering is a established science!

Thank You

RevisionAutonomic NS/

Parasympathetic

Anatomical Location

Subtype and Anatomical location

Subtype

Muscarinic receptor Nicotinic receptor

Named after it’s agonist Muscarine Named after it’s agonist Nicotine

It is a GPCR It is a Ligand gated ion channel

Subtypes : M1-M5 Muscle type or neuronal type

Location CNS, Autonomic NS Location : CNS, Autonomic, Neuromuscular junctions

Allow Smooth Muscle contraction Allow Skeletal muscle contraction

Acetylcholine

2 problems prevent it from being a good drug– Unselective– Unstable (Easy to hydrolyze)

Solution– Ethylene group control selectivity– Acyloxy group controls stability

H3C O

O

CH2 CH2 N(CH3)3

QuaternaryAmmonum group

Ethylenegroup

Acyloxygroup

(maintains activity)

(controls selectivity)

(modify stability)

2 ways of promoting Ach activity

• 1) Directly: Agonize muscarinic receptor• 2) Indirectly : Inhibit Acetylcholinesterase

(AchE) ---> Ach not hydrolyzed ---> more Ach to bind to receptors for longer time(But This also promotes nicotinic activity!)

• During AchE mediated hydrolysis of Ach, AchE gets gets attached with acyl group

• This acylated AchE needs to be hydrolyzed by water to be free and function again

• Anticholinesterase inhibitor, both reversible and irrversible contain functional groups which makes this step very difficult for water since water is not a strong nucloephile

• Order of Resistance to hydrolysis• AchE-Phosphate ester>>>>>> AchE-carbamate

>> AchE-ester• Reversivle Anticholinesterase block AchE for

few min----> thus have therapeutic value• Irreversivle Anticholinesterase block AchE for

many hours---> thus have toxic effect --> cholinergic crisis --> respiratory failure

Q) How Ach gives only specific Muc or Nic response if it can bind to both of them?

Design criteria of good AchE inhibitor

• All AchE inhibitor based insecticide below Phosphorylate the enzyme and have a peculiar group in the right side of compound. Figure out the organic chemistry.

• Remember: Rational drug design is not limited by not having software to look at binding site or do any fancy docking, QSAR. In this case the fact that acylated AchE needs to be hydrolyzed and and hydrolysis is a nucleophilic attack that is affected by leaving group is all that you need

• But there is no rationality for making an irreversible AchE inhibitor unless you are making a chemical weapon.

• Our focus should be on making a better PAM alternative. All existing drugs are based on oximes because it is a powerful nucleophile. Do you know any stronger nucleophilies? How about sulphur based compounds?

All these are useless in aging and the charge on Nitrogen makes it difficult to reverse poisoning in CNS.We need a antidote that• Has stronger nucleophilic group than oxime• Enough lipophilicity to penetrate into brain•You will later see that charge in nitrogen isn’t mandatory

First lesson in Drug design

• When we say a drug or endogenous ligand interacts with its target receptor or enzymes we mean that there is formation of chemical bond such as– H-Bond– Dipole bond– Ionic bond– Covalent bond – Van der wall bond– Hydrophobic– Pi-pi bond, pi-cation bond (you won’t find this in books but

this is important too as you will see in next slide)

Introduction to Structure based drug design (SBDD)Implementation of knowledge of chemical bonding and binding site to design drugs

Notice how enzmye catalysis works.

1) In AchE binding site, Hydrogen from a Histidine protonates O of Ach and OH of erine attacks as a nucleophile, just like what happens in lab: first C=O protonation by some acidic catalyst then OH of water attacks as a nucleophillic. Difference is in enzyme both functionality is built in by various amino acids, one acidic type (Histidine – it is basic in neutral form but because of it’s basicity it mostly remains in conjugated acid form) and other nucleophilic type (Serine)

Amino Acids grouped by chemical property

2) The charged Nitrogen is used to anchor Ach into binding site, where it forms not the expected ionic bond (cation of Ach and anion in enzyme) but cation-pi bond. The pi refers to aromatic rings provided by tryptophan and phenylalanine of the enzyme.

What this implies is that positive charge on PAM, which makes brain penetration difficult, is not really required. Instead we can include functional groups that do pi-pi bonding with the enzyme ie aromatic rings. This increases lipophilicity too!

Thus, In theory………N

OH

this should also work

N

OH

this should also work and penetrate brain more

We made specific changes in PAM in response to knowledge of how Ach chemically binds to AchE. This change has advantage of penetration brain better. Because we used the binding site as a reference, this is called Structure based Drug Design and is one of the principle of Rational drug design.

Assignment: Watch this video and learn about the Structure based Drug Design of the first Anti-influenza drug, Zamanavir and bring written report Youtube/How protein crystallography changed drug design

N

N

OH

instead of this

How chemical interaction dictates conformation?Ach When bound to nicotine

receptor Ach When in aqueous solution

trans form Gauche form

Ref : Conformation of acetylcholine bound to the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 85 (1988)

Q)Normally, gauche is more stable than trans form in solution. However when bound to nicotineAch readily adopts such Trans conformation. Why?Ans) At this trans form the total entropy ie chemical interaction with receptor overcomes any entropy loss to finally result a large change in gibbs free energy ie thermodynamics of binding matters