Classification of neurons

Transcription

1 محمد نورالدين محمود

2 Classification of neurons Nervous system Central Peripheral Brain Spinal cord Sensory Motor Somatic Autonomic Cholinergic Adrenergic Nicotinic Muscarinic α Β N1-N10 M1-M5 α1, α2 β1, β2

3 Peripheral NS The peripheral nervous system (PNS) is so called because it is peripheral to the central nervous system (CNS; the brain and spinal column). Sensory nerves take messages from the body to the CNS Motor nerves carry messages from the CNS to the rest of the body. Some neurons takes the message from CNS to skeletal muscles (voluntary) through single neuron with NO synapses, therefore, the signal propagation is fast and short lived Other neurons takes the massage from CNS to smooth and cardiac muscles (involuntary) through two neurons with a synapse, therefore the signal propagation is bit slower and longer lived. The chemicals are effectively carry a message from a neuron to muscle or from neuron to another through the synapses. Those chemical messengers are called neurotransmitters. There are a large number of neurotransmitters in the body, but the important ones in the peripheral nervous system are acetylcholine and noradrenaline

4 Neural synapse provides Refreshment and amplification of signal Extend duration of signal Spread of signal Signal transmission at a synapse.

5 For voluntary muscles we have one possible way to control For voluntary muscles we have two possible ways to control Cell membrane Enteric NS Intestine Enzyme

6 1. Sympathetic Neurons: Located in smooth and cardiac muscles and respond to noradrenaline At the heart, the action of noradrenaline leads to contraction of cardiac muscle and an increase in heart rate. Elsewhere, it relaxes smooth muscle and reduces the contractions of the gastrointestinal and urinary tracts. It also reduces salivation and the dilatation of the peripheral blood vessels. In general, the sympathetic nervous system promotes the fight or flight response by shutting down the body's housekeeping roles (digestion, defecation, urination, etc.), while stimulating the heart. 2. Parasympathetic neurons Located in smooth and cardiac muscles and respond to acetylcholine The effects are opposite to those of sympathetic neurons activation. For example, cardiac muscle is relaxed, whereas the smooth muscle of the digestive and urinary tracts is contracted. 3. Enteric Neurons: Located in the wall of intestine and respond to serotonin, neuropeptide, Acetylcholine and Dopamine are among many other neurotransmitters take part in this system It receives messages from sympathetic and parasympathetic nerves, but it also responds to local effects to provide local reflex pathways which are important in the control of GIT function.

7 Signal transduction at Ach neuronal synapse 1. The first stage involves the biosynthesis of acetylcholine. Acetylcholine is synthesized from choline and acetyl coenzyme A at the end of the presynaptic neuron. The reaction is catalysed by the enzyme choline acetyltransferase. 2. Acetylcholine is incorporated into membrane-bound vesicles by means of a specifi c transport protein. 3. The arrival of a nerve signal leads to an opening of calcium ion channels and an increase in intracellular calcium concentration. This induces the vesicles to fuse with the cell membrane and release the transmitter into the synaptic gap. 4. Acetylcholine crosses the synaptic gap and binds to the cholinergic receptor, resulting in stimulation of the second neuron. 5. Acetylcholine moves to an enzyme called acetylcholinesterase, which is situated on the postsynaptic neuron, and which catalyses the hydrolysis of acetylcholine to produce choline and acetic acid (ethanoic acid). 6. Choline is taken up into the presynaptic neuron by a transport protein to continue the cycle. 7. The presynaptic neuron also contains receptors for acetylcholine and noradrenaline, which act as another control system for acetylcholine release

8 Which of the ligands below act through specific receptor and which act through non-specific receptor? Synapse with acetylcholine acting as the neurotransmitter. Presynaptic control systems.

9 Co-transmitters Co-transmitters are messenger molecules released along with Ach. The particular co-transmitter released depends on the location and target cell of the neurons. Each co-transmitter interacts with its own receptor on the postsynaptic cell. Co-transmitters have a variety of structures and include peptides, such as vasoactive intestinal peptide (VIP), gonadotrophin-releasing hormone (GnRH), and substance P. The roles of these agents appear to be as follows: 1. they are longer-lasting and reach more distant targets than acetylcholine, resulting in longer-lasting effects 2. the balance of co-transmitters released varies under different circumstances (e.g. presynaptic control) and so can produce different effects.

10 Drug targets to control autonomic and somatic NS Q) How many types of targets are available here? A) two, enzyme and switch Acetylcholinestrase (enzyme) Ach receptor (switch) So during this chapter we will study compounds which control the autonomic and somatic nervous systems by acting on those two targets.

11 Structure-Activity Relationship (SAR) Structure-activity relationship (SAR): is the relation ship between changes in structure and biological activity. It NOT MEASURES the value of change in the activity. Quantitative structure-activity relationship (QSAR): is SAR which MEASURES the amount of change in activity. Three-dimensional quantitative structure-activity relationship QSAR (3DQSAR): is QSAR which considers the 3D structure and configuration of the molecule. Four-dimensional quantitative structure-activity relationship QSAR (4DQSAR): is 3DQSAR with other dimension represent the possible ligand conformations.

12 SAR for Ach As we said previously, the easiest approach to design a drug against certain receptor is to use the normal substrate for that receptor as a lead. In drug development the lead compound is studied to find out which parts of the molecule are important for activity, thus should be preserved in the designed drug. The previous approach is used usually when the receptor structure is unknown. According to copious studies, any compound similar to Ach will be active against both Nicotininc and Muscarinic receptors if it has: 1. +ve charged nitrogen: neutral nitrogen cause loss of activity. 2. Precise distance between nitrogen and ester group: ethylene bridge is optimal. It is this distance which determines the selectivity to muscarinic or nicotinic receptors 3. The ester functional group is important 4. The overall size of the molecule should not be bigger than Ach. 5. The N-substitutions should at least - include 2 methyl groups. A third large alkyl group is tolerated 6. The size of ester group should be small. Pharmacophore of acetylcholine.

13 SAR for Ach (Cont.) Distance is important (ethylene bridge) The size of N-substitutions should be small except one The size of ester is important (Acetoxy group) +ve charge is important (Quaternary nitrogen)

14 SAR for Ach (Cont.) Quaternary nitrogen is essential Bad for activity

15 SAR for Ach (Cont.) Distance from quaternary nitrogen to ester is important Ethylene bridge must be retained Bad for activity

16 SAR for Ach (Cont.) Methyl group of acetoxy group cannot be extended Methyl group can be replaced with group od similar size Bad for activity Active

17 SAR for Ach (Cont.) Ester is important Bad for activity

18 SAR for Ach (Cont.) Minimum of two methyl groups on quaternary nitrogen Bad for activity Active

19 SAR for Ach (Cont.) Conclusions of SAR: Tight fit between Ach and binding site The 3 methyl groups fit into small hydrophobic pockets, one of the pockets is big Ester interacting by H-bonding. The methyl group of ester fit into small hydrophobic pocket Quaternary nitrogen interacting by ionic bonding Me Acetoxy O O Ethylene bridge NMe 3 4 o Nitrogen

20 Subtypes of cholinergic receptors Receptor types Not all cholinergic receptors are identical Two types of cholinergic receptor - nicotinic and muscarinic Named after natural products showing receptor selectivity Activates cholinergic receptors on smooth muscle and cardiac muscle Activates cholinergic receptors at nerve synapses and on skeletal muscle Acetylcholine is natural messenger for both receptor types

21 Subtypes of cholinergic receptors (Cont.) Nicotine showed selectivity for cholinergic receptors present on skeletal muscle or at the synapses between different neurons. Whereas muscarine showed selectivity for cholinergic receptors present on smooth muscle and cardiac muscle. Both of nicotine and muscarine are more rigid, thus more selective to particular subtype of cholinergic receptors More flexible Muscarine Acetylcholine Nicotine - Less flexible - Can fit only muscarinic rec - Affects only smooth m. More flexible Can fit both receptors - Less flexible - Can fit only nicotininc rec - Affects only skeletal m. Muscarinic receptor Nicotinic receptor

22 Subtypes of cholinergic receptors (Cont.) The more rigid is the molecule, the higher is the selectivity. But the rigid molecule is selective for which of the receptor subtypes, muscarinic or nicotinic? This is frequently can be estimated from the distance between the atoms which are equivalent to ester oxygen and quaternary nitrogen.

23 Muscarinic receptors Muscarinic receptors are switches belongs to G-protein coupled receptors Receptor binds messenger leading to an induced fit Opens a binding site for a signal protein (G-protein messenger induced fit closed open G-protein bound G-protein split

24 Muscarinic receptors (Cont.) Activation of membrane bound enzyme G-Protein is split and subunit activates a membrane bound enzyme Subunit binds to an allosteric binding site on enzyme Induced fit results in opening of an active site Intracellular reaction is catalysed Enzyme Enzyme subunit active site (closed) active site (open) Intracellular reaction

25 Muscarinic receptors (Cont.) Muscarininc receptor is a switch receptor belong to class of G-protein coupled receptors (GPCRs) At present, five subtypes of the muscarinic receptor have been discovered (M1 M5). Cell membrane Muscarinic receptor Ockenga, Wymke, et al. "Non-neuronal functions of the M2 muscarinic acetylcholine receptor." Genes 4.2 (2013):

26 Muscarinic receptors (Cont.) Subtypes of M- receptor M 1 Neural M 2 Cardiac M 3 Glandular, smooth muscles M 4 M 5 Main location CNS, Gastric & salivary glands Heart, GIT, CNS Gastric & salivary glands, GIT, Eye CNS CNS Cellular response Increase IP 3, DAG, Depolarization, excitation, increase potassium conductance a) Decrease in camp inhibition, b) Decrease in Ca ++ conductance, c) Increase K + conductance Increase IP 3, Ca ++ conductance, As M 2 As M 3 Functional responses CNS excitation, gastric secretion Cardiac inhibition, neural inhibition Gastric & saliva secretion, GI smooth muscle contraction, occular accomodation Enhanced locomotion Not known

27 Muscarinic receptors (Cont.) d + R d + d - NMe 3 d - Possible induced dipole dipole interaction between quaternary nitrogen and hydrophobic aromatic rings in binding site The N+ induces dipole in aromatic rings One molecule of Ach binds to the receptor Muscarinic receptor binding site.

28 Nicotinic receptors Nicotinic receptors are switches belong to the group of ligandgated ion channels At present, ten subtypes of the nicotinic receptor (α1 α10). Receptor Binding site Messenger Cell membrane Five glycoprotein subunits traversing cell membrane Induced fit Gating (ion channel opens) Cell membrane Cell membrane

29 Nicotinic receptors (Cont.) The binding sites at nicotinic receptors Binding sites Ion channel b a a g Cell membrane d a g b d a 2xa, b, g, d subunits Two ligand binding sites mainly on a-subunits

30 Nicotinic receptors (Cont.) Each nicotinic receptor has two receptive sites and activation of the receptor requires binding to both of them. Each receptor site is located at the interface between α-subunit and the near by ε or δ subunits. Each receptive site has an anionic site that binds to the cationic ammonium head (ε or δ subunits) and a site that binds to the blocking agent by donating a hydrogen bond (α-subunit) Shorter molecules like acetylcholine need two molecules to activate the receptor, one at each receptive site. W. C. Bowman (2006). "Neuromuscular block". British Journal of Pharmacology. 147 (S1): S277 S286. doi: /sj.bjp PMC PMID

31 Cholinergic agonists Acetylcholine as an agonist Advantages - Natural messenger - Easily synthesized Disadvantages - Easily hydrolysed in stomach (acid catalysed hydrolysis) - Easily hydrolysed in blood (esterases) - No selectivity between receptor types - No selectivity between different target organs Cell membrane ON (agonist) Enzyme

32 Cholinergic agonists (Cont.) Nicotine and muscarine as cholinergic agonists Advantages - More stable than Ach - Selective for main cholinergic receptor types - Selective for different organs Muscarine Disadvantages - Activate receptors for other chemical messengers - Side effects Nicotine

33 Active conformation of acetylcholine Several freely rotatable single bonds Large number of possible conformations Active conformation does not necessarily equal the most stable conformation Bond rotations in acetylcholine leading to different conformations.

34 Active conformation of acetylcholine Rigid Analogues incorporating the acetylcholine skeleton (C C O C C N) Rotatable bonds locked within ring Restricts number of possible conformations Defines separation of ester and N Pharmacophore of acetylcholine

35 Requirements for cholinergic agonists Stability to stomach acids and esterases Selectivity for cholinergic receptors Selectivity between muscarinic and nicotinic receptors Knowledge of binding site SAR for acetylcholine

36 Instability of ACh The instability of Ach is due to the high flexibility of the linker which connects quaternary amine and ester group Therefore, the +vely charged quaternary amine can interacts with carbonyl oxygen and further reduces electron density at carbonyl carbon. Thus increases electrophilicity of carbonyl group Thus Increases sensitivity to nucleophiles such as water. Neighbouring group participation. The arrow indicates the inductive pull of oxygen which increases the electrophilicity of the carbonyl carbon

37 Design of cholinergic agonists Requirements 1. Correct size 2. Correct pharmacophore - ester and quaternary nitrogen 3. Increased stability to acid and esterases 4. Increased selectivity

38 Design of cholinergic agonists (Cont.) Two approaches are used to increased stability to acid and esterases: 1. Prevent quaternary amine from approaching the carbonyl oxygen (steric approach): - Shields protect ester from nucleophiles and enzymes - Shield size is important - Must be large enough to hinder hydrolysis - Must be small enough to fit binding site 2. Feed electrons to carbonyl carbon (electronic approach): - Replace ester with urethane - Stabilizes the carbonyl group

39 Design of cholinergic agonists (Cont.) Properties Three times more stable than acetylcholine Increasing the shield size increases stability but decreases activity Selective for muscarinic receptors over nicotinic receptors S-enantiomer is more active than the R-enantiomer Stereochemistry matches muscarine Not used clinically hinders binding to esterases and provides a shield to nucleophilic attack the methyl group of methacholine occupies the same position as a methylene group in muscarine. This is only possible for the S - enantiomer of methacholine Methacholine (racemic mixture). Comparison of muscarine and the R - and S -enantiomers of methacholine

40 Design of cholinergic agonists (Cont.) Use of electronic factors Properties Resistant to hydrolysis Long lasting NH2 and CH3 are equal sizes. Both fit the hydrophobic pocket NH2 = bio-isostere Muscarinic activity = nicotinic activity Used topically for glaucoma Carbachol (contains NO steric effect. Do you expect it is also non-selective?) Resonance structures of carbachol.

41 Design of cholinergic agonists (Cont.) Use steric + electronic factors Properties Very stable Orally active Selective for the muscarinic receptor Used to stimulate GI tract and urinary bladder after surgery Bethanechol (contains steric effect. Do you expect it is also non-selective?) Resonance structures of carbachol.

42 Muscarinic selective agonists Used to activate smooth muscles linked to parasympathetic NS (involuntary muscles) For treatment of glaucoma; switching on the GIT and urinary tract after surgery; For treatment of certain heart defects by decreasing heart muscle activity and heart rate.

43 Muscarinic selective agonists (Cont.) Although there is no quaternary ammonium group present in pilocarpine, it is assumed that the drug is protonated before it interacts with the muscarinic receptor. Pilocarpine can adopt a conformation having the correct pharmacophore for the muscarinic receptor; i.e. a separation between nitrogen and oxygen of 4.4 A. Pilocarpine is also being considered for the treatment of Alzheimer's disease, as are other muscarinic agonists such as oxotremorine and various arecoline analogues

44 Nicotinic selective agonists Nicotinic selective agonists Used to activate skeletal muscles connected to somatic NS (voluntary muscles) Mainly used for treating mysthenia gravis (reduction in nicotinic receptors in muscles) This agent is very similar in structure to methacholine, and differs only in the position of the methyl substituent. Varenicline is used clinically, however. It is a partial agonist at nicotinic receptors

45 Muscarinic selective antagonists Selective muscarinic antagonists can 1. bind to cholinergic receptor and stabilizes the inactive form of the receptor (OFF position) 2. Prevent acetylcholine (and other agonists) from binding Postsynaptic nerve Postsynaptic nerve Ach Ach Ach Antagonist OFF (antagonist) Cell membrane Enzyme

46 Muscarinic selective antagonists (Cont.) Muscarininc receptor is a switch receptor belong to class of G-protein coupled receptors (GPCRs) At present, five subtypes of the muscarinic receptor have been discovered (M1 M5). Cell membrane Muscarinic receptor Ockenga, Wymke, et al. "Non-neuronal functions of the M2 muscarinic acetylcholine receptor." Genes 4.2 (2013):

47 Muscarinic selective antagonists (Cont.) Probable mechanism which explains why antagonists have bulky groups and agonists have not. Sojka, Amy. G Protein-Coupled Receptor Guidebook. Agonist binding (1) induces inward motions (2) of the extracellular side of TM5-7 resembling a clothes pin-like motion. This is followed by an outward movement of the cytosolic side of TM5-7 (3), allowing the G protein to bind (4) and become activated(5). Right: metaphor used to describe GPCR activation. G G-protein can get activated

48 Muscarinic selective antagonists (Cont.) Clinical Effects Decrease of saliva and gastric secretions Relaxation of smooth muscle Decrease in motility of GIT and urinary tract Dilation of pupils Uses Shutting down digestion for surgery Ophthalmic examinations Relief of peptic ulcers Treatment of Parkinson s Disease Anticholinesterase poisoning Motion sickness

49 Muscarinic selective antagonists (Cont.) 1. Atropine Racemic form of hyoscyamine Source - roots of belladonna (1831) (deadly nightshade) Used as a poison Used as a medicine: - decreases GIT motility - antidote for anticholinesterase poisoning - dilation of eye pupils CNS side effects - hallucinations 2. Hyoscine (scopolamine) Source - thorn apple Medical use - treatment of motion sickness Used as a truth drug to get info from spies (CNS effects) LogP=1.5 pka=8.9 The asymmetric center in atropine is easily racemized as it is next to a carbonyl group and an aromatic ring. This makes the proton attached to the asymmetric center acidic and easily removed. Q) Why adminstration of atropine and hyoscine give CNS side effects while Ach does not? A) Look at logp of each. LogP=0.5 pka=6.4 LogP= -3.5 pka=6.4

50 Muscarinic selective antagonists (Cont.) Q) Why administration of atropine and hyoscine give CNS side effects while Ach and atropine methionate do not? A) Look at logp of each. As both atropine and hyoscine are tertiary amines rather than quaternary salts, they are able to cross the blood brain barrier as the free base. Once they are in the brain, they can become protonated and antagonize muscarinic receptors which causes CNS effects such as hallucination, restlessness, agitation, and hyperactivity. LogP = 1.5 pka = 8.9 LogP = -3 pka = 13 LogP = -3.5 pka = 6.4 LogP = 0.5 pka = 6.4

51 Therefore, to reduce CNS side effects quaternary salts of atropine and atropine analogues are used clinically. ipratropium is used as a bronchodilator in chronic obstructive pulmonary disease. Atropine methonitrate acts at the intestine to relieve spasm. LogP = -3 pka = 13 The combination preparation ipratropium/salbutamol is a formulation containing ipratropium bromide and salbutamol sulfate (albuterol sulfate) used in the management of chronic obstructive pulmonary disease (COPD) and asthma (Combivent ).

52 Muscarinic selective antagonists (Cont.) Relative positions of ester and nitrogen similar in both molecules. The Nitrogen in atropine is ionized at physiological ph Amine and ester are important binding groups which forms ionic and H-bonds (similar to Ach) Aromatic ring of atropine is an extra binding group which forms vdw interactions (not present in Ach), therefore atropine binds more strongly than acetylcholine The bulky aromatic ring induce different fit. In other word, atropine stabilizes the OFF conformation of receptor. the distance between the ester and the nitrogen groups is similar in both molecules Acetylcholine skeleton superimposed on to the atropine skeleton ClogP=1.3

53 Muscarinic selective antagonists (Cont.) IMPORTANT STRUCTURAL FEATURES FOR MUSCARINIC ANTAGONISM: 1. aromatic ring, 2. ester group, 3. basic nitrogen (which is ionized) NON- IMPORTANT STRUCTURAL FEATURES FOR MUSCARINIC ANTAGONISM: Complex ring system was not necessary for antagonist activity. Chain contraction to two carbon atoms can be carried out without loss of activity,

54 Muscarinic selective antagonists (Cont.) Therefore, a simplified model for muscarininc antagonists Pharmacophore = ester + basic amine + aromatic ring

55 Muscarinic selective antagonists (Cont.) Examples of Muscarinic antagonists (ophthalmics) (ophthalmics) (Parkinson s disease) (Parkinson s disease) (Anti-ulcer)

56 Muscarinic selective antagonists (Cont.) SAR for muscarinic antagonists Important features for muscarinic antagonism: 1. Tertiary amine (ionised) or a quaternary nitrogen 2. Aromatic ring 3. Ester 4. N-Alkyl groups (R) can be larger than methyl (unlike agonists) 5. Large branched acyl group 6. R = aromatic or heteroaromatic ring 7. Branching of aromatic/heteroaromatic rings is important

57 Muscarinic selective antagonists (Cont.) SAR for Agonists 1. Quaternary nitrogen 2. Aromatic ring 3. Ester 4. N-Alkyl groups = methyl 5. R = H SAR for Antagonists 1. Tertiary amine (ionised) or quaternary nitrogen 2. Aromatic ring 3. Ester 4. N-Alkyl groups (R) can be larger than methyl 5. R = aromatic or heteroaromatic 6. Branching of Ar rings important Message and address theory is applied for muscarinic ligands Address Message Where to bind? Cholinergic, adrenergic,..etc What to do? Agonist, antagonist,..etc Portoghese, Philip S., et al. "Application of the message-address concept in the design of highly potent and selective non-peptide. delta. opioid receptor antagonists." Journal of medicinal chemistry 31.2 (1988):

58 Muscarinic selective antagonists (Cont.) 12.7 Binding Site for Antagonists There is a hydrophobic binding regions next to the normal acetylcholine binding site. The overall shape of the acetylcholine binding site plus the extra binding regions may have to be T- or Y-shaped in order. Therefore, propantheline, which contains the complete acetylcholine skeleton, as well as the hydrophobic acyl side chain binds more strongly to the receptor than acetylcholine itself. Propantheline stabilizes the OFF conformation of the receptor. s on its message. Inactive (Not T shaped) The binding of propantheline to the muscarinic receptor.

59 Home works The relationship between structural differences of muscarinic receptors subtypes and selectivity of ligands The relationship between structural differences of nicotinic receptors subtypes and selectivity of ligands. Other receptors where message and address theory works

60 Nicotinic selective antagonists (Cont.) Extract from Curare plant Used for poison arrows causes paralysis (blocks acetylcholine signals to muscles), used in surgery to reduce muscle tone. Active principle = tubocurarine The molecule has two positively charged nitrogen atoms (one tertiary, which is protonated, and one quaternary). The distance between the two nitrogen atoms is crucial for activity. Possibly one of the positively charged nitrogens on tubocurarine binds to the anionic binding region of an acetylcholine binding site, while the other binds to a nearby cysteine residue Å Curare Interaction of tubocurarine to nicotinic receptor Tubocurarine.

61 Nicotinic selective antagonists (Cont.) Each nicotinic receptor has two receptive sites and activation of the receptor requires binding to both of them. Each receptor site is located at the interface between α-subunit and the near by ε or δ subunits. Each receptive site has an anionic site that binds to the cationic ammonium head (ε or δ subunits) and a site that binds to the blocking agent by donating a hydrogen bond (α-subunit) Shorter molecules like acetylcholine need two molecules to activate the receptor, one at each receptive site. W. C. Bowman (2006). "Neuromuscular block". British Journal of Pharmacology. 147 (S1): S277 S286. doi: /sj.bjp PMC PMID

62 Nicotinic selective antagonists (Cont.) Nicotinic antagonism produced by two type of compounds: 1. Antagonists: Switch the receptor into OFF (induce no depolarization) and prevent binding of Ach (Nondepolarizing agents). 2. Inverse agonists: Switch the receptor into ON (induce depolarization) and prevent binding of Ach (Depolarizing agents).

63 Inverse agonists Non-depolarizing agents A decrease in binding of acetylcholine leads to a decrease in its effect and neuron transmission to the muscle is less likely to occur. It is generally accepted that nondepolarizing agents block by acting as reversible competitive inhibitors. That is, they bind to the receptor as antagonists and that leaves fewer receptors available for acetylcholine to bind. Decamethonium congeners, which prefer straight line conformations (their lowest energy state), usually span the two receptive sites with one molecule (binding inter-site). Longer congeners must bend further when fitting receptive sites Å 11.4 Å

64 Inverse agonists Decamethonium: - binds stronger than Ach to receptor. - It acts as agonist, activated receptor, cause persistent depolarization and prevent Ach binding. - Highly stable (longer duration of action) Similar molecules which act as inverse agonist include suxamethonium has a 10-atom distance between its N atoms, like decamethonium. Yet it has been reported that it takes two molecules, as with acetylcholine to open one nicotinic ion channel. The presence of ester render the molecule easily hydrolyzed by estrases (metabolism varies among individuals) Thus it has a fast onset and short duration of action (5 10 minutes), but suffers from various side effects. It is used in surgery and tracheal intubation. Succinic acid linker Both compounds have effects on the autonomic ganglia, thus having side effects. They may bind to Ach receptors in heart and thus increase heart-rate and fall in blood pressure

65 Nicotinic selective antagonists (Cont.) Antagonists The design of pancuronium and vecuronium was based on tubocurarine, but involved a steroid nucleus acting as a spacer (10.9 Å) between the two nitrogen groups. - Fast onset then tubocurarine but slower than suxamethonium - Long duration of action than suxamethonium (45 min) - Pure antagonists (no agonistic depolarizing effect). - Thus, have lower side-effects including on heart Steroid acts as a spacer for the quaternary centres (10.9 Å) The design of atracurium was based on the structures of tubocurarine and suxamethonium. - Short duration of action (30 min) due to rapid hydrolysis by Hofmann degradation and plasma cholinesterases. - Avoids patient variation in metabolic enzyme - Lacks cardiac side effects

66 Antagonists Atracurium is stable under acid ph But unstable under blood ph due to Hofmann elimination The important features of atracurium are: 1. the spacer a 13-atom chain connects the two quaternary centres; 2. the blocking units the cyclic structures at either end of the molecule which block the binding site from acetylcholine; 3. the quaternary centres these are essential for receptor binding. If one is lost through Hofmann elimination, the binding interaction is too weak and the antagonist leaves the binding site; 4. the Hofmann elimination the ester group adjacent to quaternary amine facilitate its elimination as tertiary amine specially at physiological ph due to the acidity of the beta-hydrogen. The electron withdrawing ester group increases the acidity of the hydrogen on the beta -carbon such that it is easily lost

67 Antagonists - Similar speed of onset (about 2 minutes) compared to - Shorter duration of action (15 min) - action (about 15 minutes), although the duration is longer - Hydrolysis by Hofmann degradation and plasma cholinesterases.

68 Inverse agonists Can fit both Ach binding sites Stabilize ON conformation (agonist) Prevent Ach from binding (antagonist) Ach flexible Ach Have address and message part (similar to muscarinic agonists), thus have side effects Similar to muscarinic agonists which are not T-shaped Pure antagonists Can fit both Ach binding sites Stabilize OFF conformation and prevent Ach from binding (antagonist) Ach Less flexible Ach No obvious address and message (not similar to muscarininc agonists), thus have no side effects

69 Acetylcholinestrase (AchE)is present attached to post-synaptic neurons and can hydrolyse and deactivate acetylcholine AchE prevents acetylcholine reactivating receptor Inhibitor of AchE blocks Ach hydrolysis thus extending the life of the signal (similar effect to cholinergic agonist) Signal Nerve 1... Nerve 2 Acetylcholinesterase enzyme

70 The enzyme concentration at synaptic space is almost stable due to the anchor of enzyme cluster. The enzyme cluster is similar to a tree with the trunk and three branches being collagen. Each branch holds 4 AchE units by disulfide bridges. Thus, total 12 AchE units in the tree. The trees are rooted immediately next to the cholinergic receptors such that they efficiently capture acetylcholine as it departs the receptor. AchE is most specific for Ach, although another enzyme called A soluble cholinesterase enzyme called butyrylcholinesterase is also present in various tissues and plasma was found in plasma and tissue which can hydrolyse various esters Active Inactive

71 Some amino acids are non-catalytic (e.g. Tryptophane) and only drag Ach to the active site. Other amino acids are catalytic and involved in ester hydrolysis like (Serine, Histidine and Glutamic acid). The mechanism of Ach hydrolysis include: 1. The histidine residue acts as an acid base catalyst which can give and take H+ from serine residue. 2. After loosing H+, serine becomes highly nucleophile and can strongly attack the ester of Ach. The hydrolysis of Ach is very efficient x 10 6 faster than uncatalysed hydrolysis, thus acetylcholine hydrolysed within 100 msecs of reaching active site.

72 AchE inhibitors Anticholinestrase drugs act as inhibitors for acetylcholinestrase. All the known inhibitors interact through covalent bonds to AchE Some inhibitors (e.g. carbamate) interact through weak covalent bond (reversible) Some inhibitors (e.g. organophospohorous) interact through stronmg covalent bond (irreversible)

73 1. Carbamates as AchE inhibitors The lead compound of carbamates was plant product physostigmine. Natural product from the African calabar bean Carbamate is essential (equivalent to ester of Ach) Aromatic ring is important Pyrrolidine N is important (ionised at blood ph) Pyrrolidine N is equivalent to the quaternary nitrogen of Ach Nitrogen of carbamate feeds electron to ester bond (Phys-O-Ser) and make it long lived Extra hydrophobic bonding

74 1. Carbamates as AchE inhibitors (Cont.) The benzene ring may be involved in some extra hydrophobic bonding and it provides a good leaving group during catalysis. The carbamate group is the crucial group responsible for physostigmine's inhibitory properties because N feeds Ser-O-acetyl group with electrons, thus stabilize it. - Histidine catalyses the nucleophilic attack of the serine residue on Physostigmine - The leaving group (this time a phenol) is expelled with the aid of acid catalysis from histidine and departs the active site to be replaced by a water molecule. - Water finds it difficult to attack the carbamoyl intermediate. Thus enzyme functionality is reduced by times Nitrogen of carbamate feeds electron to ester bond and make it long lived

75 1. Carbamates as AchE inhibitors (Cont.) Physostigmine has limited medicinal use because of serious side effects, and it has only been used in the treatment of glaucoma or as an antidote for atropine poisoning. Simpler analogues, however, have been used in the treatment of myasthenia gravis and as an antidote to curare poisoning. Miotine has the necessary carbamate, aromatic, and tertiary aliphatic nitrogen groups. It is active as an antagonist. It can cross BBB and produce CNS side effects. It undergoes chemical hydrolysis. Neostigmine and pyridostigmine were designed to deal with both these problems. Can be given intravenously to reverse the actions of neuromuscular blockers or used orally in the treatment of myasthenia gravis. Dimethyl carbamate N reduces chemical hydrolysis and extend half-life Ionized N reduces CNS side effects - The distance between quaternary N and ester O is almost 4.7 (similar to Ach) - The ring system reduces the flexibility thus increase affinity (lower loss of entropy after binding)

76 2. Organophosphorous comp. as AchE inhibitors (Cont.) Used mainly as chemical weapon (in wars) and insecticides. Developed in World War 2 Form strong bond with serine residue of AchE (irreversibly inhibit AchE) Lead to permanent activation of cholinergic receptors by Ach which results in death Insecticide Irreversible phosphorylation P-O bond very stable Used as chemical weapons (nerve gases). All compounds contain halogen as good leaving group

77 2. Organophosphorous comp. as AchE inhibitors (Cont.) The medicinally used organophosphorous compounds contains a quaternary N to provide similarity Ach increase interaction with the AchE reduce dose required and side-effects Used to treat glaucoma Topical application

78 2. Organophosphorous comp. as AchE inhibitors (Cont.) Parathion, malathion, and chlorpyrifos are used to inhibit AchE of insect, thus used as insecticides. The compounds have P=S (cannot interact with Ser) instead of P=O (can interact with Ser). P=S can be metabolically converted to P=O inside insect but not human. Parathion may cause other toxicities and has high lipid solubility and is absorbed easily through mucous membranes Malathion are used medicinally for the treatment of head lice, crab lice, and scabies.

79 2. Organophosphorous comp. as AchE inhibitors (Cont.) Antidotes for organophosphorous compounds were developed. The antidote should be strong nucelophil to cleave the P-O-Ser bond. Hydoxylamine is a stronger nucleophile, however it is too toxic for human Pralidoxime was rationally designed to mimic Ach, thus can bind to AchE at lower doses to reduce possible toxic effect of hydroxylamine. Pralidoxime is 1 million times more effective than hydroxylamine Cannot antagonize AchE inhibitors in the brain, since it is unable to cross BBB Converted by brain oxidases Can cross BBB