Week 4: Pharmacodynamics and Pharmacogenetics

Question 1

Types of Drug Receptor Interactions

Answer 1

Agonists - Full or Partial

Antagonists - Competitive, non-competitive, non-receptor

Non-competitive antagonists - Irreversible, Allosteric

Non-receptor antagonists - chemical/functional

Question 2

Agonists

Answer 2

Binds to a receptor and produces a measurable biological effect.

• An agonist may mimic the response of an endogenous ligand.
• When an agonist binds to a receptor it stabilizes the receptor in a particular conformation (usually the active conformation).
• -> DR does not happen very often
• For most drug receptor pairs DR and R* (activated receptor) are unstable and exist only briefly

Question 3

Full Agonists

Answer 3

Drugs that bind to their receptor and are able to produce the maximal biological response

• Full agonists have high intrinsic activity (i.e. they are able to maximally activate the receptor).
• Full agonists stabilize receptors in their active conformation.

Question 4

Intrinsic Activity

Answer 4

Ability of drug to activate the receptor

Question 5

Partial Agonists

Answer 5

Molecules that bind to the receptor but only produce a partial response, even when all the receptors are bound.

• Unable to activate the receptor to the same degree as a full agonist
• Even if you keep increasing drug concentrations, it will not increase the receptor activity
• Partial agonists have an intrinsic activity less than that of full agonists.
• Partial agonists can also act as antagonists since they may block the binding of full agonist ligands if given together
• Example: Tamoxifen is a drug used to treat breast cancer.
• It is a partial agonist of the estrogen receptor - binds to estrogen receptor and produces a small effect
• Blocks binding to the estrogen receptor in estrogen-dependent breast cancer

Question 6

Antagonists

Answer 6

Inhibit the action of agonists.

• Antagonists have no effect in the absence of an agonist.
• Antagonists that bind to receptors have affinity but no intrinsic activity (don’t activate that receptor)
• Binds to receptor
• Has affinity to receptor
• Cannot activate the receptor (no intrinsic activity) - usually endogenous ligands (e.g. NTs)

Question 7

Competitive Antagonist

Answer 7

Bind to the same site on the receptor as the agonist.

• Has affinity but no intrinsic activity.
• Binding is reversible
• Adding more agonist will overcome the effect of a competitive antagonist.
• Notice that the presence of an antagonist causes a parallel rightward shift in the dose response curve.
• Competitive antagonists increase the EC50 but do not affect the maximal efficacy.
• Many drugs act clinically as competitive antagonists.
• Examples include acetaminophen (tylenol), statins (lower cholesterol), and beta receptor blockers (blood pressure)

Agonist produces an effect (S shaped curve)
Agonist + antagonist produces a parallel rightward shift (same slopes)
Agonist conc. changes
Antagonist conc. is the same
Antagonist alone produces no effect

Question 8

Measuring Competitive Antagonism

Answer 8

Use a single concentration of agonist and vary the concentration of competitive antagonist.

This allows you to determine the IC50, the concentration of antagonist required to produce 50% inhibition (i.e. the functional strength of the inhibitor).

The IC50 can be misleading because it is dependent on the experimental conditions.

Different agonists have different effects

Concentration of agonist is also a factor

We can calculate the Ki using the ChengPrusoff equation.

The Ki represents the binding affinity of the inhibitor (better than IC50 for measuring competitive antagonism)

Question 9

Cheng Prusoff Equation

Answer 9

Ki = IC50 / 1+[S}/Kd

[S] = agonist
Kd = antagonist binding affinity

The Ki represents the binding affinity of the inhibitor (better than IC50 for measuring competitive antagonism)

Question 10

Non-Competitive Antagonists

Answer 10

Can occur either at the agonist binding site (irreversible antagonist) or at an allosteric site (allosteric antagonism).

Non-competitive antagonists decrease the maximal efficacy.

Note that the EC50 does not change when an non-competitive antagonist is added.

The apparent maximal efficacy DOES change (decreases)

You can’t knock the noncompetitive antagonist out of the binding site by increasing agonist concentrations

Question 11

Irreversible Antagonists

Answer 11

Bind to the receptor with very high affinity (usually a covalent or ionic bond).

• Even at high agonist concentrations, they can’t be out-competed.
• Example: Aspirin irreversibly inhibits the enzyme COX1 in platelets.
• It does this by acetylating a serine residue which hampers access of substrates.
• This decreases clotting and helps prevent heart attack and stroke.
• In order for new COX1 activity to reappear, new platelets must be synthesized

Question 12

Allosteric Antagonists

Answer 12

Allosteric antagonists bind to a site on the receptor other than the agonist binding site.
They can either change the conformation of the agonist binding site or prevent the receptor from being activated even when the agonist is bound

Non-competitive: increasing agonist does not displace antagonist at the active site

Acts the same way on the dose-response curve as the irreversible

This is reversible but it binds at a completely distinct site

Question 13

Non-Receptor (other) Antagonists

Answer 13

Chemical or Functional

Question 14

Functional Antagonists

Answer 14

Bind and sequester the agonist so it is unavailable to act on its receptor (inactivates the agonist)

Example: Your patient has overdosed on heparin, an acidic negatively charged drug. You administer protamine sulfate to prevent the toxic effects of heparin overdose. Protamine sulfate is positively charged and binds to heparin.

ACTS ON THE AGONIST

Question 15

Functional Antagonists

Answer 15

Have the opposite physiological effects to the agonist.

Example: Patients taking thyroid hormones to treat hypothyroidism may experience the side effect of tachycardia. Although this is independent of the beta receptor activity, beta blockers are often given to treat this side effect (binds to a totally different receptor)

ACTS ON A DIFFERENT RECEPTOR

Question 16

Major Types of Receptor Families

Answer 16

Ligand-gated ion channels

G-protein coupled receptors

Enzyme-linked receptors

Intracellular receptors

Question 17

Ligand-gated ion channels

Answer 17

Control the flow of ions across the cell membrane.

• Pore through the middle
• Binding of agonist allows the receptor to open
• Change in membrane potential OR change in ion potential mediates the effect
• There is some selectivity in the type of ion that passes through (e.g. Na, Cl)
• Ions flux across the cell membrane plays a role in neurotransmission, cardiac conduction, muscle contraction, and secretion making these important drug targets.
• Response to these receptors occurs exceptionally rapidly (milliseconds).
• Ligand gated ions show some degree of selectivity in terms of which ions can pass through their pore.

Question 18

Nicotinic Cholinergic Receptor

Answer 18

Type of ligand gated ion channel.

• 5 subunits
• 2 ligand binding sites (neurotransmitter ACh binds to each of the subunits)
• Allows Na+ to move into the cell
• When two molecules of acetylcholine (an endogenous agonist) bind to the receptor, the ion channel opens and sodium enters the cell.
• The result of sodium entering the cell is the generation of action potentials and the contraction of skeletal muscle.
• Can you think of another agonist for this receptor? —-NICOTINE

Question 19

GABAa Receptor

Answer 19

Type of ligand gated ion channel.

• Binding of ϒ-aminobutyric acid (GABA) to its receptor opens a chloride ion channel.
• When chloride ions rush into the cell, the membrane potential is driven further away from its threshold for activation (hyperpolarization)
• We are making the inside of the cell more negative with respect to the outside of the cell
• Therefore, agents that activate the GABA receptor cause CNS depression (e.g. sedation).
• Benzodiazepines (valium) potentiate the actions of GABA (drugs end in -pam).
• Does not bind to the channel but allows more GABA to bind to the channel (acts together with GABA)
• They are used clinically in the treatment of anxiety, as sedatives, anti-epileptics, muscle relaxants and in the treatment of ethanol withdrawal

Question 20

G-Protein Coupled Receptors

Answer 20

GPCRs are thought to be the most abundant type of receptor and are the target of ~50% of all drugs.

• GPCRs have three major components:
  1. 7 transmembrane spanning receptor with an extracellular ligand binding domain.
  2. G-protein that has three subunits (α,β,ϒ).
• There are many subtypes of the α subunit including Gαs, Gαi, Gαq and others (mediate different effects)
  3. Effector, usually an enzyme, ions channel or other protein that mediates activity of GPCR
• Stimulation of GPCRs results in responses that last seconds to minutes.

Question 21

GPCR Activation

Answer 21

1. Receptor is not interacting with the G-protein
   - Alpha subunit of G-protein is bound to GDP
   - Receptor is unoccupied
2. When a drug/hormone/NT binds to the ligand binding domain of the GPCR, it stabilizes the receptor and the receptor changes shape
   - Conformational change of the receptor causes the interaction of the receptor with the G-protein
   - G-protein releases GDP, binds GTP in its place
3. Alpha subunit of G-protein dissociating from beta/gamma subunits
   - Binds to the effector and activates the effector (adenylyl cyclase) to hydrolyze ATP → cAMP + PPi
   - Beta/gamma subunit can also have some activity
4. Hormone/drugs/NT dissociates and receptor goes back to resting state
   - GTP → GDP
   - Adenylyl cyclase is deactivated

Question 22

Second Messengers

Answer 22

One of the most important roles of GPCRs is to activate the production of second messengers.

Second messengers are crucial in conveying and amplifying signals from GPCRs.

Different G-proteins act on different effector molecules and produce different second messengers.
A: alpha molecule (s) is activating the adenylyl cyclase → PKA → protein phosphorylation
B: different alpha molecule (q) targets different effector molecule → DAG or IP3 → PKC or Ca2+

Remember spare receptors from PD lecture 1?

• Binding of one agonist drug to a GPCR can activate adenylyl cyclase.
• Adenylyl cyclase can form dozens of molecules of cAMP.
• This is an example of signal amplification.

Question 23

Enzyme-linked receptors

Answer 23

Transmembrane receptors that translate an extracellular ligand binding event to activate (or inhibit) an intracellular enzyme domain.

Most of these receptor enzymes act by adding or removing phosphate groups to or from specific amino acid residues on proteins.

Phosphorylation of proteins is a ubiquitous signalling pathway as it can dramatically alter the structure and function of many proteins.

Stimulation of these receptors results in duration of effect from minutes to hours.

Question 24

Insulin Receptor

Answer 24

Type of Enzyme-linked receptor

Receptor tyrosine kinase.

• Binding of insulin results in autophosphorylation of tyrosine residues on the cytoplasmic side of the receptor.
• The receptor then phosphorylates other target proteins known as insulin receptor substrates (IRS).
• IRS go one to activate other other intracellular signalling molecules such as MAP kinase and inositol 3 phosphate which lead to production of the biological actions of insulin → increases glucose uptake, glycolysis

Question 25

Intracellular Receptors

Answer 25

Reside in the cytosol of the cell (challenge for the drug to pass through the cell membrane)

• In order to access intracellular receptors, drugs must be sufficiently lipid soluble to cross the plasma membrane or be a substrate for an uptake transporter.
• Most intracellular receptors are nuclear receptors/ transcription factors
• Ligand binding causes changes in the structure of the nuclear receptor
• Translocation of drug to the nucleus and activation of transcription.
• Effects of activation of these receptors can take hours or days to develop

Question 26

Regulation of Receptors

Answer 26

Receptor activation is tightly regulated to prevent overstimulation which could be damaging.

Repeated administration of some drugs over time results in reduced effect.

Question 27

Tachyphylaxis

Answer 27

Occurs due to receptor desensitization (application too many times, the drug loses effect)

Question 28

Desensitization

Answer 28

Desensitization can be defined as the decreased ability of a receptor to respond to stimulation by a drug.

Desensitization can be mediated by:

1. Inactivation
2. A refractory period
3. Down-regulation

Question 29

Receptor Desensitization

Answer 29

Repeated exposure of agonist can desensitize a receptor in three ways:

1. Phosphorylation - the enzymes protein kinase A (PKA) or β-adrenergic receptor kinase (βARK) phosphorylate the cytoplasmic part of the receptor.
   - This causes the binding of β-arrestin which blocks binding to the G-protein (blocks effector)
   - This blocks activation of the effector (adenylyl cyclase).
2. Sequestration - Some receptors are sequestered in endosomes.
3. Degradation - Some receptors are degraded in lysosomes

Question 30

Drugs that do not act on receptors and enzymes

Answer 30

Antacids relieve the symptoms of heartburn by neutralizing stomach acid.
-This is a chemical effect.

Mannitol is a sugar-alcohol (osmotic diuretic) that draws water into the lumen of the nephron.

• Mannitol helps promote water elimination from the body.
• This is an osmotic effect.

Mesna prevents the toxicity of some chemotherapeutic drugs.

• Mesna has a thiol (-SH group) that allows it to bind reactive and toxic metabolites of some chemotherapeutic drugs.
• This is a chelation effect (drug chelates the toxic drug).

Question 31

Pharmacogenetics

Answer 31

Study of the genetic basis for variation in drug response.

• The first studies of relevance to pharmacogenetics occurred in the 1930’s.
• These related not to drugs but the genetic control of a taste response (genetics could influence how we taste).

Question 32

PTC Tasting

Answer 32

The ability to taste PTC is determined by a single gene that codes for a taste receptor on the tongue.
The “PTC gene” was discovered in 2003. It is called TAS2R38.

GPCR that detects bitter taste
With both tasting receptors they can taste PTC

Question 33

Antipyrine Metabolism

Answer 33

Antipyrine: An old drug that used to be used as an analgesic and antipyretic agent.
- Metabolized by a lot of CYPs so it’s a “sloppy” probe drug but a good indicator for general CYP mediated metabolism.

Question 34

SNPs

Answer 34

SNPs (pronounced SNiPs) are base pair substitutions that occur in the population at an allele frequency of 1% or greater.

• Individuals differ from each other every 300 - 1000 nucleotides.
• There are approximately 10 million SNPs in the human genome.
• SNPs are not “abnormal”.
• They are part of natural genetic variation that creates diversity in a population.

Question 35

Coding SNPs

Answer 35

• SNPs that occur in exons are termed coding SNPs.
• If the base pair substitution results in an amino acid change, it is further classified as nonsynonymous (or missense).
• If the base pair substitution does not alter the encoded amino acid it is classified as synonymous (or sense).
• Note that substitution in the third base pair of a codon (the wobble position) usually does not alter the encoded amino acid.

Nonsynonymous: proline → glutamine
Synonymous: proline → proline

Question 36

SNPs in non-coding regions

Answer 36

Aside from coding SNPs (i.e. SNPs in exons), polymorphisms may occur in the 5’ or 3’ untranslated regions, in promoter regions, in introns and in the large intergenic regions.

Can still be important

1. SNPs in the regulatory regions (i.e. promoter, 5‘UTR and 3’UTR) may affect the level of mRNA by affecting the rate of transcription or the stability of the transcript.
2. SNPs in the introns, especially those near intron/exon boundaries can affect splicing.

Most intergenic SNPs have no known functional effect.

Question 37

Insertions/Deletions

Answer 37

In/del mutation: When a one or more nucleotides are added or deleted from a gene.

If they occur in exons, they may cause a frameshift may cause a change in protein structure and/or function

Insertions/deletions can range from 1 nucleotide to thousands of nucleotides in length.

Question 38

CNV

Answer 38

• Traditional genetics teaches us that we should have two copies of every gene in our body.
• Recent discoveries have shown that large sections of DNA (thousands to millions of bases) can vary in copy number.
• Some patients have been found to have 13 copies of a single gene!
• Try and think about this in terms of a gene dose effect (more later).
• Current estimates suggest that up to 12% of the human genome is subject to copy number variation.

Examples:

• Succinylcholine metabolism by butyrylcholinesterase (1950’s).
• Isoniazid metabolism by NAT2 (1950‘s and 1960’s).
• Debrisoquine metabolism (1970’s)

Question 39

Succinylcholine

Answer 39

Succinylcholine is a short acting neuromuscular blocking agent used as a muscle relaxant during procedures of short duration.

• It is rapidly inactivated (hydrolyzed) by the enzyme butyrylcholinesterase (also called plasma cholinesterase)
• 1 in 2000 Caucasians have a point mutation (position 209 A → G) in butyrylcholinesterase.
• The mutation results in an aspartate → glycine amino acid substitution and substantially decreases the ability of the enzyme to bind succinylcholine.
• The therapeutic consequence is prolonged apnea of anywhere from 1-6 hours.
• Patients without the mutation usually experience apnea for ~3 minutes.
• Patient FO - low succinylcholine = long apnea time
• Plasma cholinesterase rapidly inactivates succinylcholine.
• Patients with a point mutation in plasma cholinesterase are not able to inactivate succinylcholine effectively.

Question 40

Isoniazid

Answer 40

Isoniazid is an antibiotic used to eradicate tuberculosis.

• It is acetylated by the phase II drug metabolizing enzyme N-acetyltransferase 2 (NAT2).
• Studies in the 50‘s and 60’s identified that there appeared to be two distinct groups of patients based on plasma isoniazid concentrations.
• If slow acetylators are given too high of a dose, they may get peripheral neuropathy.

Question 41

NAT2

Answer 41

NAT2 has at least 26 known allelic variants.

• The most common polymorphism to result in the slow acetylator phenotype in Caucasians is position 341 T → C
• Results in an isoleucine → threonine amino acid substitution.
• The amino acid substitution causes a decreased Vmax without altering the Km.
• In Asians a position 857 G → A polymorphism is the most common polymorphism resulting in the slow acetylator phenotype.
• This polymorphism causes a glycine → glutamic acid amino acid substitution.
• The position 857 G → A polymorphism results in the production of an enzyme with decreased stability.

Question 42

Debrisoquine Metabolism

Answer 42

Debrisoquine is an antihypertensive agent that is metabolized by CYP2D6.
-Variability in metabolism has been linked to SNPs, gene deletion and gene duplication.