Download Nicotinic Acetylcholine Receptor

Nicotinic Acetylcholine Receptor
The
electrostatic
isopotential
surface--a surface representing
constant electric potential--for a
model
of
the
nicotinic
acetylcholine
receptor.
Some
members of this family of
receptors in the brain bind
nicotine and are targets for the
addictive activity of this drug. The
receptor is composed of five
subunits with similar amino acid
sequences encircling a central
channel. Neurotransmitters such
as acetylcholine bind at two of the
five subunit interfaces, gating, or
opening, the channel to allow sodium ions to enter from outside the cell. The
red surface corresponds to the isopotential contour -1 kT/e, where k is the
Boltzmann constant, T is temperature, and e is electronic charge. The contour
shows the electrostatic influence on the binding of acetylcholine. The model
was developed by Igor Tsigelny, Naoya Sugiyama, and Palmer Taylor at the
Departments of Pharmacology and Chemistry/Biochemistry at the University of
California, San Diego, in collaboration with Steven M. Sine at the Mayo
Foundation in Rochester, Minnesota. Visualized by Igor Tsigelny at SDSC.
MEDICINAL CHEMISTRY
Pharmaceutical Sciences 621& Chemistry 569
Professor C. M. Thompson (x4643) mailto:%[email protected]
Lecture 8
DRUGS ACTING ON NEUROTRANSMITTERS AND
THEIR RECEPTORS - ACETYLCHOLINE AND DOPAMINE (in part)
ACETYLCHOLINE
A. Acetylcholine and Cholinergic Receptors. The cholinergic system is
found in the central nervous system (CNS), in the autonomic nervous system
and the skeletomotor system. Acetylcholine (ACh) is the neurotransmitter of
interest:
CH3C(O)OCH2CH2N+(CH3)3
Cholinergic receptors show duality and have sites of differing binding
properties: "nicotinic" and "muscarinic." Acetylcholine bind to both types!
Nicotine Receptor: binds nicotine and are found in all autonomic
ganglia and at the neuromuscular endplate of striated muscle.
Muscarinic Receptor: bind muscarine and occur at post-ganglionic
parasympathetic terminals -> control peristalsis, glandular secretion, pupil
constriction, vasodilation and heart rate reduction.
Amanita muscaria mushroom. Euripedes lost his wife and 3 children to
poisoning by this mushroom. Also contains amanatins (octapeptides) which
cause death by inhibition of RNA polymerase leading to renal or hepatic failure
(nitrogen should bear a positive charge).
The degeneration of cholinergic pathways in the CNS results in "neuronal
tangles" -> neuron arrangement messed up. This process is hypothesized to be
a major contributor to Alzheimer's disease (not the same as senile dementia), a
premature and progressive memory loss. Drug treatment with choline
replacement, cholinergic agonists have shown marginal success.
A.1. Acetylcholine Metabolism
As ACh is synthesized, it is stored in the neuron "depot" (a.k.a. vesicles)
and is released by neuronal stimulation, which occurs by K+ depolarization
(below). Although the cholinesterase is shown "floating" in the synapse (for
effect),
it
is
actually
a
membrane
bound
protein.
Acetylcholine release is inhibited by botulinus toxin (LD50 = 1 ng / Kg; or 7 x 108
g to kill a 70 kg person). Produces muscle paralysis by blocking the active
zone of the presynaptic membrane. A protein exists that fuses the vesicle to the
membrane, anchoring it to release ACh. Botulism toxin binds this protein and
the vesicle can not get to the edge to release the ACh.
A.2. Nicotinic Acetylcholine Receptor.
The receptor was isolated with the help of the electric eel and electric
ray, which have a copious concentration of these receptors; much higher than
the human brain.
specie
ray
eel
human
ACh-R conc.
1000 nmol/kg tissue
50-100 nmol/kg tissue
brain
0.1-1.0
nol/kg
tissue
Electric ray can give a shock of 50-60 volts and an eel up to 600 volts
and 1 kilowatt energy. At any rate, these receptor rich tissues were isolated
with the help of the Siamese cobra.
The venom of certain snakes contain peptides called bungarotoxin
(peptide of about 61-75 amino acids containing lots of arginines and lysines cationic amine residues). They block cholinergic neurotransmission by direct
binding to the receptor. Can you now think of how they used the snake to trap
the eel [receptor]? Sure you can?
A.3. Muscarinic Acetylcholine Receptor.
Far more stereospecific and structure specific than the nicotinic receptor.
In fact, only as recently as 1980 have scientists studied the differences between
nicotine and muscarinic receptors. As a result, the structure, biochemistry, and
operation of the muscarinic receptor is still vague; especially since there are no
organs "rich" in this particular receptor.
Two subtypes of receptor, M1 and M2 have been identified:
M1: closes K+ channels
M2: located in heart muscle, cerebellum and hindbrain and is regulated by GTP.
That is, a second messenger is usually interactive with the M2.
The breakthrough came when an agonist called pirenazepine, was
found that selectively binds, or rather has a high affinity for the M1.
A.4. Cholinergic Agonists.
You can generally increase stimulation of the ACh-receptor two ways:
1. by direct binding of an agonist to the receptor - direct acting.
2. by inhibition of acetylcholinesterase (AChE), prolonging the agonist
action because the protein responsible for its breakdown is rendered inactive. It
is called - indirect acting.
A.4.a. Design of
Modification of Acetylcholine.
Cholinergic
Agonists:
Structural
1. Ammonium Group. The replacement of the ammonium moiety with
either a sulfonium or phosphonium results in a complete loss of activity.
Increasing one methyl group to a larger alkyl (e.g., ethyl) results in 25%
activity. Increase two methyl groups in size -> lose all activity. Replacement of
ammonium group with tert-butyl retains 0.003% of activity. Implies that the
charge distribution and size are important factors to ACh-R action.
2. Ethylene bridge. Acts as a "perfect spacer" or ruler between the
carbonyl and the quaternary ammonium moiety and ensures the proper
distance for receptor binding. Rule of Five: Should be no more than four atoms
between the ammonium and the terminal methyl group, otherwise a loss of
activity. Branching on the ethylene bridge tolerates methyl only.
3. Ester Group. Not very amenable to modification and a change from a
methyl to a phenyl makes a good antagonist! Some activity can be maintained
by replacement with a ketone/ether and carbamate (carbachol).
4. Cyclic analogs. Many have good activity including muscarine itself. The
acetoxy cyclopropyls were used to probe the receptor. Trans isomer had same
effect as muscarine.
A.5. Mode of Cholinergic Binding.
ACh is very flexible. Two major conformations. Eclipsed not considered
but could be a possible contributor once inside the receptor.
Gauche conformer = muscarinic
Anti conformer = nicotinic
It is generally accepted that the ammonium group binds a carboxylate
(glutamate or aspartate) aided by van der Waals interactions of a hydrophobic
pocket. Approximately 5.9 Å away a hydrogen donor forms a H-bond with the
carbonyl. Some speculation over the acetoxy-methyl binding a smaller pocket
also has been forwarded.
Requirements for nicotinic and muscarinic receptors are similar except
the muscarinic has a methyl group to complement a pocket and the nicotinic a
carbonyl group. Recheck the two structures.
A.5.a. Cholinergic Blocking Agents.
Drugs that inhibit the interaction of ACh with its receptor are called
"cholinergic blocking agents." [Note: peripheral cholinergic synapses are
muscarinic.] Anticholinergic agents decrease the secretion of saliva (dry-mouth)
and gastric juice, decrease G.I and urinary peristalsis and pupil dilation. Some
are used in the treatment of peptic ulcer, ophthalmology and Parkinson's
disease.
Some of the oldest anti-cholinergics are the "belladonna alkaloids" also
known as deadly nightshade, which has excitatory and hallucinogenic effects.
Story time: Belladonna was used by the ancient Hindu's, Roman Empire
and the Middle Ages to induce prolonged poisoning. Much of the poison uses
were quite fun to the poisoner, watching the victim fall to memory loss,
disorientation, destruction of mental faculties prior to death. Thus, they could
be viewed as possessed by demons! Hippocrates indicated its use in the
treatment of gastric disturbances. Belladonna gets its name (Atropa belladonna)
from Carolus Linnaeus, the founder of modern botanical nomenclature, who
named it after Atropos, the oldest of the Three Fates, who was said to cut the
thread of life after her sisters had spun and measured it.
Belladonna (translation anyone?) contains atropine and scopolamine, the
major active ingredients found in the "witches brew." They are esters of tertiary
amines with a bulky ester component and are mixed M1 and M2 antagonists.
[NOTE: the amine group is protonated at physiological pH.] Fit to the receptor
is good and the phenyl moiety "bends out" to possibly block incoming ACh to
displace it.
Atropine. 10 mg can kill a child. The diagnosis: widespread paralysis,
convulsions, circulatory collapse, blood pressure drop, inadequate
respiration
and coma.
Scopolamine: truth serum and sleep inducer
Cocaine: note only the similarity in structure to these other two
alkaloids.
We are all pretty familiar with the effects of cocaine - note that atropine
and scopalamine are down regulating drugs (sleep, blood pressure lowering,
etc.) whereas cocaine is an up regulating stimulant. What is the basic difference
in the structures - STEREOCHEMISTRY!
A.5.b. Ganglionic Blocking Agents. interfere with nicotinic ACh
receptors. Usually simple ammonium groups. Used in the control of
hypertension by decreasing vasoconstriction.
Et4N+
Me3N+
+
(CH2)6
NMe3
Because they have no "second" binding interaction, they are non-specific
and have many side effects. They also fit so "loosely" that they are in/out of the
receptor rapidly. Basically abandoned.
A.5.b.2. Neuromuscular Blocking Agents. Widely used in surgery
because they can help relax abdominal muscles without the use of deep (and
risky) anesthesia. Two classes of these agents exist.
1. Competitive Agents. Curare (tubocurarine: active ingredient): known
as the arrow poison of South American Indian. Competitive agents are usually
bulky rigid molecules with an optimum distance of 10 +/- 0.1 Å.
Tubocurarine:
intra-ammonium
distance
10.3
Å
by
x-ray.
Story Time Again!Curare has a long and romantic history. Used
primarily for killing wild animals, it is a generic term for arrow poisons, the most
widely used is tubocurarine. Studied as early as 1805, it was brought to the
attention of medical practitioners between 1932-1940 in Europe, when it was
used to treat specific disorders and tetanus (lockjaw: can't move muscle) to
relax muscles at a very low concentration. Biomechanistically, curare combines
with the cholinergic receptor sites at the post-junctional membrane (surface)
and thereby, competitively blocks the transmitter action of ACh. The receptor
becomes "insensitive" to ACh and thus, the motor nerve impulse and the
endplate potential (K+ flux) falls dramatically. However, and this is a big
"however," the end-plate region of the remainder of the muscle fiber retains
their normal sensitivity to K+ and the muscle fiber still responds to direct
electrical stimulation. In summary, you are unable to move your muscles but
you still very much feel the pain.
2. Depolarizing Agents. flexible structures with free bond rotation. They were
devised (not natural products) through mimicry of the N+ - N+ distance, but
they act by a different mechanism.
Me3N+ (CH2)10+NMe3
decamethonium
Me3N+ CH2CH2-OC(O)CH2CH2C(O)OCH2CH2+NMe3
succinylcholine
These molecules bind normally to the ACh-receptor and triggers the
same response as ACh - a brief contraction of the muscle - however, it is
followed by a prolonged period of transmission blockage leading to muscular
paralysis. Succinylcholine is far shorter lived than decamethonium because it is
a substrate for acetylcholinesterase (AChE), the enzyme responsible for
hydrolyzing esters in the neural synapse. For medical use, an anesthetic is coadministered.
NOREPINEPHRINE AND THE ADRENERGIC RECEPTOR
A. Adrenergic System:
Also known as the sympathetic nervous system and is involved in
homeostatic regulation, increased heart rate, cardiac contraction, blood
pressure, bronchial airway tone, carbohydrate and fatty acid metabolism.
Stimulation of the sympathetic nervous system normally occurs in
response to physical activity, stress, allergic reactions, and provocation. In
many cases the "adrenergic reaction" is opposite to cholinergic effects. There
are two sub-systems:
1. Noradrenergic: controls behavior, mood and sleep
2. Adrenergic: uses epinephrine (a.k.a. adrenaline) as neurotransmitter.
B. Biosynthesis of Catecholamine Neurotransmitters: Why the name
catecholamines
All come from tyrosine, an essential amino acid!
A biosynthesis of catecholamines is presented:
C. Adrenergic Drugs:
Drugs acting at these neuron centers are called adrenergic drugs and
may be separated into two types; presynaptic and post-synaptic effects.
Pre-synaptic effects may be separated into the five following distinct categories,
although we shall only discuss two types:
1.
2.
3.
4.
5.
Drugs
Drugs
Drugs
Drugs
Drugs
acting
acting
acting
acting
acting
on
on
on
on
on
catecholamine synthesis...
catecholamine metabolism...
catecholamine storage...
catecholamine reuptake...
presynaptic receptors...
1. Drugs acting on catecholamine synthesis...
These are mostly enzyme inhibitors that interact/interfere with the steps
shown on the previous page. One example is -methyldopa, which is a
competitive inhibitor of dopa decarboxylase.
2. Drugs acting on catecholamine metabolism... Not discussed
3. Drugs acting on catecholamine storage... Probably the most important and
most widely studied of these drugs is (+)-amphetamine. Understanding the
mechanism of action of this drug allows for interpretation of an array of
analogs.
AMPHETAMINE: Amphetamine is used as a mood elevator,
psychomotor stimulant and appetite suppressant. It has a multiple neuronal
effect, inhibiting neurotransmitter uptake while increasing neurotransmitter
(dopamine) release. It is a direct receptor agonist-the receptor in this case
being a vesicle. If amphetamine is used continually to release dopamine,
paranoid schizophrenia may occur (due to neurochemical imbalances and sleep
disorders). So, how does amphetamine act on the norepinephrine neurons and
[dopamine] receptors? It is worthy to note that both amphetamine and cocaine
share this neurochemical system as their primary site of action, although
different mechanisms account for their physiologic effects.
First, compare the structure of amphetamine ("speed" "crank" "uppers")
and methamphetamine ("meth" "crystal") to the actual neurotransmitters:
Dopamine and norepinephrine are two major transmitters contained in
the neurons that regulate emotional behavior. Thus, stimulants may act on this
dopamine pathway by enhancing neuronal function by release of
dopamine/norepinephrine from "storage vesicles" (similar to ACh). Due to the
structural similarity, the illicit stimulants displace neurotransmitters from their
storage sites. The neurotransmitters are therefore "pushed out" into the
synaptic cleft where they stimulate receptors until the stimulant is metabolized
or removed from the storage site. How much amphetamine can your neurons
take? Unknown. Since this is a displacement reaction rather than an
equilibrium, there is no precise dose - effect curve. Dangerous. A cartoon
(loosely constructed) representing the effect is shown below.
4. Drugs acting on catecholamine reuptake...
The principal mechanism for the deactivation of released catecholamines
is not enzymic destruction (like the ACh - AChE system). Rather, "reuptake" of
catecholamine into the nerve ending occurs. The presynaptic membrane
contains an "amine pump" - an active transport system that moves the
catecholamines from the membrane to the vesicles. Therefore, drugs may
interact with the reuptake at several points in the process. For example;
a. The drug can be taken up by the pump (mistaken for
neurotransmitter) and transported to the storage site where it displaces the
neurotransmitter.
b. The drug can block the pump by binding to the membrane site where
the pump is "attached." That is, the drug acts to prevent reuptake by clogging
the holes. One drug that acts by mechanism b is cocaine. Cocaine acts by
preventing dopamine from being reabsorbed. Thus, the neurotransmitter
remains in the synaptic cleft where it continues to enervate the receptor long
after the normal activation time has lapsed. Although this process is
concentration dependent (dose-effect curve possible), this is only one site of
action for cocaine, and ill effects come from a combination of processes.