Chapter 3:  Receptors

3.1       Introduction

3.2       Drug-Receptor Interactions

3.2A    General Considerations

3.2B    Interactions (Forces) Involved in the Drug-Receptor Complex

·        Charge-Transfer Complexes

o       Charge donor (partially negative) group transferring charge to charge acceptor (partially positive)

o       Electron-rich aromatic p groups (tyrosine's  or tryptophan's side chain) attracted to electron-poor p groups.

o       -1 to -7 kcal/mol stability to drug-receptor complex

·        Hydrophobic Interactions

o       Believed to be an entropy effect in which nonpolar or lipophilic groups shed ordered water molecules, increasing entropy

o       Not believed by Hildebrand  --- not enough attraction between alkanes and water to begin with

o       -0.7 kcal/mol per carbon in a chain

·        Van der Waals or London Dispersion Forces

o       Instantaneous dipole-induced dipole interactions

o       Need close surface complementarity for good attraction

o       -0.5 kcal/mol per close contact

3.2C    Determination of Drug-Receptor Interactions

Schild analysis is used to determine the nature of antagonists to its receptor. Schild plots also give information on the potency of competitive antagonists and can hint at the presence of multiple binding sites. Information on whether the antagonist molecule binds with some form of cooperativity can also be inferred. The Schild equation is;

(conc. ratio - 1) = (antagonist conc.) / Ki

Conc. ratio = Conc. of agonist producing a defined response in the presence of an antagonist, divided by the concentration producing the same response in the absence of the antagonist. So antagonist EC50 / agonist EC50.

Ki = dissociation equilibrium constant for the antagonist

From the Schild plot a value known as the pA2 can be found. The pA2 is the negative logarithm of the concentration of antagonist which would produce a 2-fold shift in the concentration response curve for an agonist, and is a logarithmic measure of the potency of an antagonist.

The pA scale is useful as it performs as an empirical measure of antagonist potency, which theoretically could characterise activity, specificity, and time-action relationships. This scale allowed scientists to present findings empirically instead of describing their results as "very sensitive". The pA2 is calculated by extrapolating the value on the x-axis when y=0.

So in this example the pA2 is 6.  Note: pA2 = -log Ki.

The slope of the Schild plot gives information about the nature of the antagonist i.e. whether or not it is competitive binding and information on the cooperativity. The steepness of slope depends upon both the equilibration time and the degree of antagonism.

When the slope of the Schild plot is not 1 a number of possibilities arise: (1) the antagonist is not competitive, (2) a multimolecular interaction between drugs and receptors is being observed, (3) equilibrium conditions have not been attained in the experimental procedure.

The third condition is important as dynamic equilibrium in contrast to true equilibrium conditions may distort the nature of the antagonism; i.e. a competitive antagonist could appear to be noncompetitive. This could occur in isolated tissues, which possess uptake and/or degradative mechanisms for either the agonist or antagonist.

When the Schild slope is equal to 1 this indicates that the antagonism is competitive and reversible. It also indicates that the agonist is acting at a single receptor subtype, and that the tissue has no uptake mechanism for the agonist. It can also be concluded that the antagonist causes a parallel rightward shift of the log agonist concentration response curve with no loss of maximal response.

Active Transport

Active transport requires the expenditure of energy to transport the molecule from one side of the membrane to the other, but active transport is the only type of transport that can actually take molecules up their concentration gradient as well as down. Similarly to facilitated transport, active transport is limited by the number of protein transporters present. We are interested in two general categories of active transport, primary and secondary. Primary active transport involves using energy (usually through ATP hydrolysis) at the membrane protein itself to cause a conformational change that results in the transport of the molecule through the protein. The most well-known example of this is the Na+-K+ pump. The Na+-K+ pump is an antiport, it transports K+ into the cell and Na+ out of the cell at the same time, with the expenditure of ATP.

Secondary active transport involves using energy to establish a gradient across the cell membrane, and then utilizing that gradient to transport a molecule of interest up its concentration gradient. An example of this mechanism is as follows: E. coli establishes a proton (H+) gradient across the cell membrane by using energy to pump protons out of the cell. Then those protons are coupled to lactose at the lactose permease transmembrane protein. The lactose permease uses the energy of the proton moving down its concentration gradient to transport lactose into the cell. This coupled transport in the same direction across the cell membrane is known as a symport. E. coli uses similar proton driven symports to transport ribose, arabinose, and several amino acids.

 

Facilitated Diffusion

Facilitated diffusion utilizes membrane protein channels to allow charged molecules (which otherwise could not diffuse across the cell membrane) to freely diffuse in a nd out of the cell. These channels comes into greatest use with small ions like K+, Na+, and Cl-. The speed of facilitated transport is limited by the number of protein channels available, whereas the speed of diffusion is dependent only on the concentration gradient.

Facilitated diffusion or active transport vs. passive diffusion

The Na+-glucose secondary transport mechanism

Another secondary active transport system uses the Na+-K+ pump as the first step, generating a strong Na+ gradient across the cell membrane. Then the glucose-Na+ symport protein uses that Na+ gradient to transport glucose into the cell.

 

This system is used in a novel way in human gut epithelial cells. These cells take in glucose and Na+ from the intestines and transport them through to the blood stream using the concerted actions of Na+-glucose symports, glucose permeases (a glucose facilitated diffusion protein), and Na+-K+ pumps. Note that the epithelial cells are joined together by tight junctions to prevent anything from leaking through from the intestines to the blood stream without first being filtered by the epithelial cells.

 

o       G-coupled Protein Receptors (GCPRs)

§         Serpentine membrane-spanning proteins winding back and forth 7 times:  from the outer aqueous environment, through the membrane, into the cytoplasm, and back

§         Binding of ligand to receptor activates adjacent G protein, leading to further signaling downstream

b adrenergic receptor

 

This receptor binds the ligand epinephrine, also known as adrenaline, which is released by the adrenal glands above the kidneys in response to very stressful stimuli. Once released, epinephrine courses throughout our blood stream and adsorbs to specific receptors on the surfaces of cells in various tissues throughout the body. The result is the establishment of the primitive mammalian fight / flight reaction. This reaction increases heart rate, decreases blood flow to gut, increases blood flow to skeletal muscles, and increases blood glucose by causing liver and muscle cells to break down glycogen and release resulting glucose into the circulation. How does epinephrine/adrenaline evoke all these responses? Acting as a ligand, it binds to its own receptor displayed on the surface of a variety of cell types throughout the body. This beta adrenergic receptor is a 7 membrane-spanning, serpentine receptor embedded in the plasma membranes of these cells. Epinephrine ligand is not internalized into the cell. Instead, while bound for a short period of time to its receptor, it causes the latter to release biochemical signals into the cell cytoplasm. Single receptor molecules will change their conformation in response to ligand binding. This conformation change affects the configuration of the cytoplasmic domains of the receptor, that is, the loops of receptor protein that protrude into the cytoplasm.

 

Cytoplasmic signal transduction

 

The b adrenergic receptor communicates with the cytoplasm by stimulating a second protein, which is known as a G protein for reasons that will become clear. The G protein normally lies near the receptor in an inactive, quiet state. When the receptor is activated by ligand binding, it will rapidly poke the G protein. The G protein responds by switching itself on into an active state. Once in the active state, the G protein will send signals further into the cell. However, the G protein will remain in the active state for only a brief period of time, after which it will shut itself off. In effect, the G protein acts like a binary switch, a light switch which, once turned on, will remain on for a limited period of time before it flips itself off. The G protein's two states (ON or OFF) are determined by the guanine nucleotide that it binds (whence the term G protein). When it is inactive it binds GDP; when active, it binds GTP. Accordingly, the resting, OFF form of the G protein sits around with its bound GDP. When a ligand- activated receptor pokes it, the G protein releases its bound GDP and allows a GTP molecule to jump aboard. This GTP-bound form of the G protein represents the active ON configuration of the G protein. While in the ON state, it releases downstream signals. After a short period of time (seconds or less), the G protein will then hydrolyze its own GTP down to GDP, thereby shutting itself off. This hydrolysis represents a negative feedback mechanism which ensures that the G protein is only in the active, signal- emitting ON mode for a short period of time.

Signaling Cascades

We will make a brief excursion into the downstream signaling pathway (often called a signal cascade) that is triggered by the active G protein. In fact the G protein is formed from 3 distinct protein subunits, termed alpha, beta, and gamma. When in its inactive OFF state, 3 subunits are bound together; the a subunit has the job of binding the guanine nucleotide, in this case GDP. When the beta adrenergic receptor activates the G protein, the alpha subunit releases GDP, binds GTP and falls away from the beta and gamma subunits.

Once this happens, the GTP-bound a subunit also loses affinity for the receptor, dissociates from it, and moves over and pokes yet another nearby protein, the enzyme adenylate cyclase, which until this time has been inactive. Once it is poked by the active, GTP-binding a subunit of the G protein, the adenylate cyclase enzyme gets activated and does its job: it cyclizes ATP into 3'5' cyclic AMP. This reaction involves the release of the beta and gamma phosphates from the ATP and the linking of the surviving a phosphate (still attached to the 5' hydroxyl of ribose) to the 3' hydroxyl as well, forming a circular or cyclic structure, whence the term ''cyclic adenosine monophosphate'' or simply cAMP.  

After a several second encounter with the adenyl cyclase enzyme, the alpha subunit of the G protein will hydrolyze its bound GTP and release the adenyl cyclase, thereby reverting to an inactive OFF signalling state. It will then rejoin the beta and gamma subunits that it deserted earlier in the game. The adenyl cyclase, no longer being poked by the activated a subunit of the G protein, will shut down and stop making cAMP from ATP. The whole cycle has resulted in only a brief pulse of signaling, in this case the production of several hundred cAMP molecules made by the adenylate cyclase during its brief period of activity.

Once made, the cAMP molecules act as intracellular glycogen, the high cAMP concentrations enable a kinase to

  1. phosphorylate and thereby activate an enzyme that activates glycogen phosphorylase which in turn breaks down glycogen into glucose-l-phosphate molecules; and
  2. it phosphorylates glycogen synthase, and in this way turns it off, thereby preventing the reconversion of the released glucose to glycogen. These two changes together ensure the mobilization of glucose through the breakdown of glycogen stored in the liver. A number of other reactions are triggered as well that together contribute to the fight/flight response.

There is enormous signal amplification in this cascade. A single epinephrine molecule (present at 10-10M) may cause the activation of dozens of a subunits of G proteins. Each of these in turn will activate the synthesis of a single adenylate cyclase, and each of these in turn will synthesize hundreds of cAMP molecules. Each of these in turn can activate a cAMP-dependent kinase that will on its own right modify hundreds of target molecules in the cell.

 

Introduction to GERD (gastroesophageal reflux disease)

http://www.gerd.com/intro/frame/frame.htm

Acetylcholine receptors

Acetylcholine was one of the first neurotransmitters to be discovered.

AChsynth

Acetylcholine is produced by the synthetic enzyme choline acetyltransferase which uses acetyl coenzyme A and choline as substrates for the formation of acetylcholine. Dietary choline and phosphatidylcholine serve as the sources of free choline for acetylcholine synthesis. Upon release, acetylcholine is metabolized into choline and acetate by acetylcholinesterase, and other nonspecific esterases. Acetylcholine release can be excitatory or inhibitory depending on the type of tissue and the nature of the receptor with which it interacts.

Cholinergic receptors can be divided into two types, muscarinic and nicotinic, based on the pharmacological action of various agonists and antagonists. Muscarinic receptors originally were distinguished from nicotinic receptors by the selectivity of the agonists muscarine and nicotine respectively.

Nicotinic cholinergic receptors

Nicotinic receptors produce pharmacologically and physiologically distinct responses from muscarinic receptors, although acetylcholine (and other agonists such as carbamylcholine) stimulates each type of response. Nicotinic responses are of fast onset, short duration and excitatory in nature. The pharmacology of nicotinic receptors has been studied in great detail and our understanding of how ion channel-coupled neurotransmitter receptors work is based largely on the study of this class of proteins.

Nicotinic receptors are found in a variety of tissues, including the autonomic nervous system, the neuromuscular junction and the brain in vertebrates. They also are found in high quantities in the electric organs of various electric eels and rays. The high quantities of receptors in these tissues and the use of neurotoxins from snake venom (e.g., cobra venom) that bind specifically to the nicotinic receptor aided the purification of the receptor protein (see below).

 

nicotine

 

Agonists such as acetylcholine, carbamylcholine and nicotine produce the physiological responses associated with nicotinic cholinergic activation. Acetylcholine produces an influx of sodium through a ligand-gated ion channel. Acetylcholine and carbamylcholine also stimulate muscarinic receptors and therefore should be considered mixed cholinergic agonists.

Nicotinic receptor

a-Bungarotoxin binds to the a and b subunits and probably blocks both the channel and the ACh binding site. Local anesthetics and other compounds such as phencyclidine bind to the receptor, apparently at the site of the sodium channel and modulate the binding of acetylcholine to the active site. Local anesthetics also prevent ion conductance through a direct action at the channel.

 

In general terms, acetylcholine binds to the a-subunits of the receptor making the membrane more permeable to cations and causing a local depolarization. The local depolarization spreads to an action potential or leads to muscle contraction when summed with the action of other receptors. Nicotinic receptors possess a relatively low affinity for acetylcholine at rest. The affinity for acetylcholine is increased during activation (through an allosteric mechanism which increases the likelihood of another molecule of acetylcholine binding to the other a subunit). At high concentrations of acetylcholine, the affinity for acetylcholine becomes higher and the receptor subsequently becomes desensitized. The ionophore (ion channel) is open during the active state and local anesthetics may bind to the open channel.

The subunit composition of nicotinic receptors differs in skeletal muscle, autonomic ganglia and brain. The table below lists some of the properties of receptors found in different tissues. Note that multiple subunit compositions are possible, which may permit the development of compounds selective for a particular combination. Within the CNS, the a4b2 combination predominates.

 

Nicotinic Acetylcholine Receptors

Receptor

Skeletal muscle

Autonomic ganglion

CNS

CNS

Subunits

a1,b1,d,g(e)

a3,a5,a7,b2,b4

a3,a4,b2,b4

a7,a8,a9

a-Bungarotoxin

+

+/-

-

+

Antagonists

a-Bungarotoxin

Hexamethonium

Dihydro-b-erythroidine
Mecamylamine

a-Bungarotoxin
Mecamylamine

Agonists

Epibatidine

Epibatidine

Epibatidine
ABT-418

 

 

Nicotinic antagonists

Antagonists for nicotinic receptors include such diverse compounds as curare, a-bungarotoxin and gallamine. Nicotinic receptors found at the neuromuscular junction differ from the receptors found in autonomic ganglia and can be distinguished both pharmacologically and biochemically.

 

gallamine

succinylcholine.gif

 

Gallamine (a mixed muscarinic and nicotinic antagonist) and decamethonium are more effective antagonists at the neuromuscular junction than at the autonomic ganglia. The spacing of the charged nitrogens seems to be of critical importance in the selectivity of the drugs. Gallamine and succinylcholine are used during surgery to block nmj receptors and produce paralysis. Succinylcholine is used more often because it can be metabolized by acetylcholinesterase to produce inactive compounds. Note the structural similarity to acetylcholine. Decamethonium is another nicotinic antagonist with some selectivity for the neuromuscular junction

 

decamethonium

hexamethonium

 

Ganglionic blockers include the quaternary compounds hexamethonium and tetraethylammonium as well as the tertiary and secondary amines mecamylamine and pempidine. While quaternary amines competitively inhibit cholinergic responses in autonomic ganglia, tertiary and secondary amines also have a noncompetitive component.

Ganglionic blockers are used to treat hypertension in some cases. Because they block both sympathetic and parasympathetic responses, their use is restricted to emergency situations or circumstances where the patient can be monitored (orthostatic hypotension is one of the common side effects).

Succinylcholine and decamethonium are both depolarizing blockers of nicotinic receptors, in that they initially mimic the action of acetylcholine. Following the initial depolarization, the depolarizing blockers exert a long-acting blockade of the receptor, thereby preventing further activation by acetylcholine. The trimethylammonium group seems to be important for action as a depolarizing blocker since compounds with a triethylammonium group do not cause the depolarization but do block the action of acetylcholine (see gallamine for instance).

Nicotinic agonists

Over the past several years, a variety of research groups have focused on the development of selective nicotinic agonists. Nicotinic agonists could be useful in the treatment of a variety of neurological disorders including Alzheimer's disease, Parkinson's disease and chronic pain. Epibatidine is a nicotinic agonist isolated from the skin of an Ecuadoran frog Epipedobates tricolor that displays potent analgesic properties.

 

nicotine

ABT-418

 

Another nicotinic agonist, ABT-418, exhibits some cognition enhancing properties. Note its similarity to nicotine, with an ixoxazole moiety replacing the pyridyl group of nicotine. Epiboxidine is a structural analogue that combines elements of both epibatidine and ABT-418. It also is a potent nicotinic agonist.

 

epibatidine

epiboxidine

 

Two other derivatives are worth noting. The azetidine analogue of epibatidine, ABT-594, is a potent analgesic with significantly fewer side effects than epibatidine. SIB-1508 (altinicline) is another nicotinic agonist with potential utility in the treatment of Parkinson's disease.

ABT-594

altinicline

 

Muscarinic receptors

Acetylcholine and carbamylcholine can bind to both muscarinic and nicotinic receptors, yet the responses elicited by activating each receptor differ in several ways. Muscarinic responses are slower, may produce excitation or inhibition and involve second messenger systems, rather than the direct opening of an ion channel. Muscarinic receptors are G protein-coupled receptors and mediate their responses by activating a cascade of intracellular pathways. Muscarine is the prototypical muscarinic agonist and derives from the fly agaric mushroom Amanita muscaria. Like acetylcholine, muscarine contains a quaternary nitrogen important for action at the anionic site of the receptor (an aspartate residue in transmembrane domain III). Most muscarinic agonists obey the "rule of five" atoms from the quaternary ammonium moiety to the terminal atom.

Amanita muscaria

muscarine

Muscarinic receptors are found in the parasympathetic nervous system. Muscarinic receptors in smooth muscle regulate cardiac contractions, gut motility and bronchial constriction. Muscarinic receptors in exocrine glands stimulate gastric acid secretion, salivation and lacrimation. Muscarinic receptors also are found in the superior cervical ganglion where they can produce at least two physiologically distinct responses. In addition, muscarinic receptors are found throughout the brain, including the cerebral cortex, the striatum, the hippocampus, thalamus and brainstem.

In general the classical muscarinic antagonists such as atropine recognize a single class of binding sites as determined in binding assays. In the 1980's, several selective muscarinic antagonists were identified. Pirenzepine was very useful in the characterization of M1 muscarinic receptors, while AF-DX 116 was used to identify M2 receptors in the heart. M3 receptors are found in smooth muscle and in both exocrine glands (e.g., lacrimal glands) and endocrine glands (e.g., pancreas). Muscarinic agonists bind heterogeneously to receptors in both the brain and peripheral nervous system.

 

pirenzepine

AF-DX 116

 

In the late 1980's, molecular cloning techniques identified five different subtypes of muscarinic receptors. Each receptor shares common features including specificity of binding for the agonists acetylcholine and carbamylcholine and the classical antagonists atropine and quinuclidinyl benzilate. Each receptor subtype couples to a second messenger system through an intervening G-protein. M1, M3 and M5 receptors stimulate phosphoinositide metabolism while M2 and M4 receptors inhibit adenylate cyclase. The tissue distribution differs for each subtype. M1 receptors are found in the forebrain, especially in the hippocampus and cerebral cortex. M2 receptors are found in the heart and brainstem while M3 receptors are found in smooth muscle, exocrine glands and the cerebral cortex. M4 receptors are found in the neostriatum and M5 receptor mRNA is found in the substantia nigra, usggesting that M5 receptors may regulate dopamine release at terminals within the striatum. The structural requirements for activation of each subtype remain to be elucidated.

 

Muscarinic Acetylcholine Receptors

 

M1

M2

M3

M4

M5

Distribution

Cortex, hippocampus

Heart

Exocrine glands, GI tract

Neostriatum

Substantia nigra

Antagonists

Pirenzepine

AF-DX 116

pF-HHSiD

 

 

Agonists

Xanomeline, CDD-0097

 

 

 

 

G protein

Gaq/11

Gai/o

Gaq/11

Gai/o

Gaq/11

Intracellular response

Phospholipase Cb

Adenylyl cyclase inhibition

Phospholipase Cb

Adenylyl cyclase inhibition

Phospholipase Cb

Muscarinic antagonists

Muscarinic antagonists such as scopolamine and atropine are among the oldest known molecules, originally derived from natural sources. They are both alkaloids (natural, nitrogenous organic bases, usually containing tertiary amines) from the nightshade plant Atropa belladonna. The presence of an N-methyl group on atropine or scopolamine changes the activity of the ligand, possibly by preventing a close interaction between the ligand and the membrane or lipophilic sites on the receptor. The methyl group also prevents the penetration into the brain.

 

atropine

methyl-atropine

 

The potent anticholinergics are used to control the secretion of saliva and gastric acid, slow gut motility, and prevent vomiting. They also have a limited therapeutic use for the treatment of Parkinson's disease. In large doses however, the muscarinic antagonists with tertiary amines have severe central effects, including hallucinations and memory disturbances.

In recent years, the quaternary muscarinic antagonist ipratroprium has been used in the treatment of chronically obstructed pulmonary disorder as an adjunct to 2 agonist therapy. M3 muscarinic receptors mediate bronchoconstriction in the airways. Muscarinic antagonists such as ipratropium and the long-lasting tiotropium are effective bronchodilators.

 

ipratropium

tiotropium

 

The possible use of presynaptic antagonists to increase acetylcholine levels has attracted some attention recently. Muscarinic autoreceptors resemble pharmacologically the M2 receptor found in the heart. M2 antagonists enhance acetylcholine release by blocking the feedback inhibition produced by the action of acetylcholine on presynaptic terminals.

Muscarinic agonists

The ability for the quaternary ammonium group to fit into an anionic site on muscarinic receptors may be an important factor for the binding of a ligand to muscarinic receptors. For an example of the requirement of the quaternary amine moiety, consider that dimethylaminoethylacetate (the tertiary form of acetylcholine) is 1000-fold less than acetylcholine, in part due to a lower affinity for the receptor.

The molecule of acetylcholine is flexible and may form an infinite number of conformations from the extended to the quasi-ring structure. The three-membered ring of acetoxycyclopropyl-trimethylammonium iodide demonstrates the concept that the extended form of acetylcholine contains the highest intrinsic activity. The trans isomer has much higher activity than the cis isomer which orients the ester and the quaternary amine together.

cyclopropyl

While the quaternary nitrogen is essential for eliciting full muscarinic responses with muscarinic agonists, there are a few potent muscarinic agents which contain tertiary amines (e.g., arecoline, oxotremorine and pilocarpine). They are potent both peripherally and centrally although they are of limited therapeutic value because of the wide range of cholinergic responses that they elicit. Oxotremorine is of interest because of its ability to produce tremors, thereby providing an early model for Parkinson's disease.

 

arecoline

oxotremorine

 

Simple tertiary amines do not show considerable potency for the receptor, but this can be counteracted if the rest of the molecule binds potently to the receptor (e.g., through an ester bioisostere). Oxotremorine fills this role with an amide group in a pyrrolidone ring as the nitrogen replaces oxygen in a hydrogen bond acceptor role. Arecoline (isolated originally from the betel nut) has a reversed ester acetylcholine profile, while pilocarpine has its ester in the cyclic form of a lactam ring, which may help increase the binding interaction. In general, it is important to have two sites for hydrogen bond acceptance in the ester isostere. The orientation of the ester isostere may be important for selective action as well.

pilocarpine

The events associated with G protein-coupled receptor activation are as follows.

  1. Agonist binds to the receptor, which has a high affinity for agonists at rest.
  2. The binding of the agonist stabilizes a receptor conformation promotes receptor/ G protein coupling and allows GTP to exchange for GDP on the G protein a subunit.
  3. The binding of GTP leads to the dissociation of the G protein from the receptor, thereby lowering agonist affinity. The agonist then dissociates from the activated receptor.
  4. The G protein consists of three subunits (a, b, and g) which also dissociate. The a subunit activates the appropriate second messenger system (e.g., phospholipase C for M1 receptors). The b and g subunits can exert independent actions.
  5. The a subunit is inactivated by the hydrolysis of GTP to form GDP by a GTPase intrinsic to the G protein (GTPase activity may be activated by other intracellular proteins called GTPase activating proteins [GAPs]).
  6. The a subunit (with GDP bound) can then recombine with the b and g subunits. The receptor is then in a high affinity state and ready for the binding of another agonist.

Alzheimer's disease

Alzheimer's disease is characterized by amyloid plaques and neurofibrillary tangles. Amyloid plaques contain deposits of b-amyloid, which is a 40-42 amino acid peptide derived from amyloid precursor protein. Neurofibrillary tangles contain a hyperphosphorylated t protein, which forms paired helical filaments. Alzheimer's disease is associated with a loss of cholinergic neurons which project from the basal forebrain to the cerebral cortex and the hippocampus. The loss of cholinergic neurons is progressive and results in profound memory disturbances and irreversible impairment of cognitive function.

The cause of Alzheimer's disease is unknown, yet several genes and gene products (proteins) have been implicated.

Drug development

Recent efforts have focused on the development of centrally active muscarinic receptor agonists for the treatment of Alzheimer's disease. The rationale for therapy involves replacement of acetylcholine, which is depleted in Alzheimer's patients as the basal forebrain neurons degenerate. An ideal candidate for a drug would have several features including high CNS penetrance, high efficacy and selectivity for forebrain receptors and a low incidence of side effects. The muscarinic agonist xanomeline is an arecoline derivative with very high affinity and selectivity for M1 muscarinic receptors. It contains a 1,2,5-thiadiazole ring, which is more stable than the ester found in arecoline. In CDD-0102 a 1,2,4-oxadiazole moiety serves as a suitable ester isostere.

xanomeline

CDD-0102

 

D.6      The Two-State (Multistate) Model of Receptor Activation

·        Conformational change occurs in the receptor, even in the absence of ligand, causing two or more states to exist.

·        D = drug, R = receptor, L = equilibrium between the resting ( R ) and active (R*) states of the receptor

·         

·        Full agonist: equilibrium shifts completely to R* upon ligand binding to R*.

·        Partial agonist:  binds to R* but not as strongly as  full agonist

·        Full inverse agonist:  binds completely to R, causing a 100% decrease in basal activity (activity in the absence of any agonist)

·        Partial inverse agonist:  binds less strongly to R than a full inverse agonist

·        Antagonist:  equal affinity for R and R*, so zero efficacy

o       Competitive:  displaces any agonist type from the receptor

o       Noncompetitive:  does not displace any agonist type from the receptor

·        Three-state receptor model:  two active conformations, one inactive

o       Helps to explain different agonist, inverse agonist behavior and affinity/efficacy among different agonists toward same receptor type

 

3.2.E    Topographical and Stereochemical Considerations

 

E.1       Spatial Arrangements of Atoms

·        Antagonists of H1 histamine receptor, different from histamine (agonist)

 

E.2       Drug and Receptor Chirality

 

·        Pfeiffer's rule:  when high receptor complementarity for one drug enantiomer (eutomer) exists, then a high eudismic ratio (ratio of eutomer to opposite enantiomer (distomer) means high eutomer potency

·        Only receptors with a minimum of three binding sites to the chiral ligand can have different enantiomer affinities:

 

·        Effects of distomers

o       Nothing (isomeric ballast)         

o       Toxin

o       Different therapeutic activity (binds to a different receptor): racemate is a hybrid drug if the enantiomers have different therapeutic activities; 2 or more chiral centers means enantiomers and diastereomers exist, and if the drug have 2 or more activities and is a stereoisomeric mixture, it's a pseudo hybrid drug

 

 

 

o       Needed for optimal therapeutic index

d enantiomer is the eutomer but increases uric acid;

l decreases uric acid levels

o       Inverse or partial agonist

o       Antagonist of the eutomer

·        FDA guidelines for chiral drugs

o       Since 1992, drug companies must justify development of racemate

o       Racemic switch allowed: new patent on eutomer of the racemate

 

E.3       Geometric Isomers (Diastereomers)

 

·        Diastereomers have different distances between atoms, so different receptor binding is expected.

 

 

E.4       Conformational Isomers

 

·        Need bioactive conformer

·        Making conformational constrained analogs to search for pharmacophore conformation

·        Bioactive conformer may not be lowest energy conformer of unbound ligand

 

E.5       Ring Topology

 

·        Tricyclic psychomimetic drugs have a spectrum of activities

o       Angles within groups of the compounds define activity

o       Angle a:  bending of ring planes

o       Angle b:  annelation angle of ring axes passing through carbon 1 and 4 of each aromatic ring

o       Angle g:  torsional angle of the aromatic rings viewed from the side

o       Tranquilizers:  a only

 

o       Mixed tranquilizer-antidepressants:  a and b only

 

o       Pure antidepressants:  a, b, and g

 

 

3.2.F    Ion Channel Blockers

 

·        Receptors can have allosteric sites

o       Calcium channel blockers bind to the same receptor but at different allosteric sites, so they have different structures

 

3.2.G   Case History of Rational Drug Design of a Receptor Antagonist:  Cimetidine

 

·        Cimetidine (Tagamet), antiulcer drug, selective H2 receptor antagonist, inhibiting HCl secetion in the stomach lining

 

 

·        2-methylhistamine (agonist) prefers H1 receptor

·        4-methylhistamine (agonist) prefers H2 receptor

·        Keep imidazole ring, but get rid of positive charge to get competitive antagonist, not agonist

·        Homologation and methylation to get a highly specific H2 antagonist

Result:

 

·        Problem:  Poor oral potency

·        Solution: Use isosteric electron-withdrawing group (S) near ring to keep positive charge off imidazole ring and to prefer a tautomer proposed to be needed for H2 receptor binding, but not screw up desired conformation (no intramolecular H-bonding, as O or NH would cause)

·        Result: 3 times more potent than burimamide as H2 antagonist

 

 

·        Further improvement: add 4-methyl group to improve population of desired imidazole tautomer

·        Result:  8-9 times more potent than burimamide, increase in healing rate on duodenal ulcers, fewer symptoms

·        Problem: Thiourea causes granulocytopenia (white blood cell reduction) in some patients; replace C=S with something else

·        Replacing C=S by C=O no good because of poor -log(H2 antagonist activity) vs. log P compared to N-substituted guanidines with electron-withdrawing groups

·        Result: Smith, Kline, and French marketed cimetidine (1st in UK in 1976):

·        Later H2-receptor antagonists showd that no imidazole ring is required and a positive charge near heterocyclic ring is OK