PHAR3310 Lecture Notes: Introduction to Receptors

Important Notices

An overview of PHAR3310/PHAR3311 will be given at the beginning of the Introductory Practical this afternoon and tomorrow afternoon, commencing at 1:00 PM.
Format, lecturers, topics, and assessment will be covered.
Attendance is mandatory.
Unit Co-Coordinators:
- A/Prof Steve Mutsaers (steve.mutsaers@uwa.edu.au)
- Dr. Sai Seto (sai.seto@uwa.edu.au)

Molecular Pharmacology PHAR3310 - 2025: Introduction to Receptors

Lecturer: Dr. Liz Johnstone (Rm 1.37D, M Block, Pharmacology, liz.johnstone@uwa.edu.au).

Lecture Series Outline

Lecture 1: Ligand binding to receptors - Key concepts of affinity & selectivity.
Lecture 2: The concept of agonism - The key concept of efficacy – conventional and biased agonists.
Lecture 3: The concept of antagonism - Competitive, irreversible & allosteric antagonists, and inverse agonists.
Lectures 4-6: Quantitative receptor pharmacology
- Using ligand binding studies to determine receptor subtype density – use of Saturation and Competition binding approaches.
- Using functional studies to determine receptor subtype function – use of the Schild analysis.
Lectures & labs are integrated closely – applied pharmacology.

How Drugs Work

Drugs work by binding to drug targets.
Paul Ehrlich (1854-1915): “Entities (drugs) do not act unless attached.”
Drug + Target

Drug Targets

Drugs bind to a variety of cellular entities, including:
- Membrane receptors & transporters.
- Enzymes.
- Nuclear receptors.
- DNA.
- Ion channels.
Examples: salbutamol, ramipril, cyclophosphamide, verapamil, fluticasone.
Binding is a key concept in drug action.

Exceptions

Can you think of a drug that doesn’t need to be bound to work?

Receptor Prevalence

Of the ~1500 drugs currently approved:
- ~45% bind to Receptors.
- ~25% bind to Enzymes (e.g., anti-inflammatories like ibuprofen).
- ~8% bind to Ion Channels (e.g., local anaesthetics like lidocaine).
- ~7% bind to Transporters (e.g., antidepressants like fluoxetine (Prozac®)).
Focus will be on receptors.
Reference: Santos et al (2017).

Receptors as Sensing Elements

Receptors are the cell’s ‘sensing elements’ that recognize endogenous hormones, neurotransmitters, and mediators.
They transduce the binding signal into a change in cell activity.
- Hormone, neurotransmitter, etc. -> Receptor -> Signalling pathways -> Change in cell activity.
- Recognition -> Transduction (lecture 2).
Example: adrenaline binds to 1-adrenoceptor on cardiac cell, increasing heart rate & contraction and intracellular calcium concentration ( $\uparrow$ [Ca2+]i).
Also targeted by exogenous drugs.

Receptor Binding Sites for Ligands

Receptors are proteins (typically > 300 amino acids in length) with small cavities for drug binding, called ‘binding sites’.
Binding site: a special arrangement of amino acids (& their functional groups).
Forces of attraction: H bonds, ionic bonds, van der Waals forces.
Binding site & drug typically have:
- Complementary 3-D shapes.
- Chemical forces of attraction, both of which facilitate binding.
Chemical forces of attraction are typically weak chemical forces.
*Covalent bonds are rarely involved.

Strength of Drug Binding

The strength of drug binding depends on:
- Type of bond.
- Distance between the drug and binding site.
- Number of bonds.
Inverse relationship between distance and force.
*If the combined forces of attraction are strong such that drug binding occurs then the drug is said to have affinity for the binding site.
Affinity is the tendency of a drug to bind to its target.
Examples:
- Van der Waals: 1/d5-1/d8 (+)
- Hydrogen: 1/d4 (++)
- Ionic: 1/d (+++)
- Covalent: 1/d (++++) H bond (strong), ionic bond (stronger) van der Waals forces (weak)
- Polar amino acids: Ser, Thr, Tyr
- Hydrophobic amino acids: Phe, Trp, Tyr
- Acidic Charged amino acids: Asp, Glu. Asp, Glu

Affinity Defined

Affinity is the tendency of a drug to bind to its target.
Affinity depends on:
1. Complementary chemical forces of attraction.
2. Complementary 3-D shapes between the drug & target.
Affinity is what brings the drug to the drug target.
Without affinity, drugs do not bind and do not act.

Example: Isoprenaline Binding

Isoprenaline binding to 1-adrenoceptor (a G protein-coupled receptor).
Isoprenaline forms many hydrogen bonds and van der Waals interactions, creating a strong, specific interaction between ligand & receptor.
Warne (2011) Nature.

Drug Affinity & Concentration

Affinity enables drug binding (i.e., no affinity, no binding).
How much drug binding occurs also depends on drug concentration.
According to the Law of Mass Action, as the concentration of drug is increased, the equilibrium is driven to the right and a greater fraction of targets are bound by the drug.
Fraction of target bound by drug = $\frac{[drug]}{[drug] + K_A}$ , which is a rectangular hyperbolic function.

Receptor Binding

No drug, no receptors bound (e.g., adrenaline).
As [drug] increases, receptors bound increase.
Drug binding is saturable.
The [drug] at which 50% of receptors is bound is called the $K_A$ & is a measure of affinity.

KA Value

The [drug] at which 50% of a target is bound is called $K_A$ (equilibrium dissociation constant).
$KA$ is a measure of affinity (inverse relationship; a low $KA$ value indicates high affinity).
$K_A$ is constant for a given drug and drug target combination (e.g., adrenaline & -AR), but varies markedly between drug-drug target combination pairs (e.g., adrenaline has lower affinity for -ARs, and no affinity for most other potential drug targets).
Differences in affinity ( $K_A$ ) explain:
1. Why drugs vary in potency.
2. Why drugs display selectivity (act on some drug targets but not others).

Affinity and Selectivity

If a drug has a high affinity for one target (e.g., A), then it can bind extensively to that target at low [drug] without binding significantly to other lower affinity targets (B, C & D).
At low [drug], the drug will selectively bind to high affinity drug targets (A) with minimal binding to other targets (B, C & D).

Loss of Selectivity

As [drug] increases, it will bind more to A, but also bind to lower affinity drug targets such as B & C.
Thus, selectivity is reduced as [drug] is increased.
Binding to these additional binding sites may cause unwanted side-effects.
The vast majority of drugs lose selectivity at high doses.
Selectivity is a concentration-dependent property of a drug that enables drugs to bind preferentially to one drug target over other drug targets.

Selectivity Context

All drugs are to some extent ‘selective’, as no drug can bind to all drug targets.
The term ‘selective’ is relative.
Example: for adrenoceptor (ADR) agonists previously mentioned: adrenaline has high affinity for ADRs ( and ) & lower affinity for other receptors (e.g., opioid Rs, cholinoceptors, etc.) and is ADR-selective agonist. isoprenaline has high affinity for -ADRs  & 2) and lower affinity for -ADR ( &  and is -ADR-selective agonist. salbutamol is 2-ADR-selective agonist.
When using the term ‘selective’, need to specify the target for which the drug is selective.

Key Revision Points

To work, drugs must bind to drug targets, which include receptors, enzymes, transporters, ion channels, DNA, etc.
Ligand binding depends on key chemical forces: van der Waals forces and ionic & H bonds which become weaker as the distance between the ligand and binding site is increased
The fraction of receptors bound (y) depends upon two factors, ligand concentration & equilibrium dissociation constant ( $KA$ ) , according to the following relationship $y = \frac{[DR]}{[RT]} = \frac{[D]}{[D] + KA}$ .
The equilibrium dissociation constant ( $K_A$ ) is a measure of drug affinity for the receptor (inverse relationship)
High affinity may confer selectivity, which may be lost at high [drug], as drug binding occurs to lower affinity targets.

Lecture Outcomes

From this lecture, you should be able to:
- Explain the key factors involved in ligand binding.
- Understand the relative roles of different chemical forces in ligand binding.
- Explain the influence of $K_A$ and [A] on fractional receptor occupancy (y).
- Describe the relationship between $K_A$ and drug affinity.
- Explain the concept of selectivity, and its relationship with drug affinity and ligand concentration.

Extra Info – Non-examinable

The following three slides explain the derivation of the Hill equation on slide 14, which describes the relationship between fractional receptor occupancy, ligand concentration and the equilibrium dissociation constant. This content is non-examinable.

Classical Receptor Theory

The Hill equation, also called the occupation model of drug action.
The binding of a ligand (A) to an unbound receptor (free receptor, R) to form a ligand-receptor complex (bound receptor, AR) follows the law of mass action, [D] + [R]
Where:
- [D] = concentration of ligand
- [R] = concentration of free receptors
- [DR] = concentration of bound receptors
- $k_1$ = association rate constant
- $k_2$ = dissociation rate constant

Law of Mass Action

[D] + [R]
Rate of forward reaction (association) = $k1$ .[D].[R], and Rate of reverse reaction (dissociation) = $k2$ .[DR]
At equilibrium, rate of dissociation = rate of association
Thus, $k2$ .[DR] = $k1$ .[D].[R]
Rearranging, $k2/k1$ .[DR] = [D].[R]
The ratio $k2/k1$ is called the equilibrium dissociation constant, $K_A$ , a ligand-specific value related to affinity (see later)
Substituting, $K_A$ .[DR] = [D].[R] …..(i)
Total concentration of receptors [RT] = [R] + [DR]
Rearranging, [R] = [RT] – [DR] …..(ii)

Fractional Receptor Occupancy

$K_A$ .[DR] = [D].[R] …..(i)
[R] = [RT] – [DR] …..(ii)
Substituting (ii) into (i), $K_A$ .[DR] = [D]. ([RT] – [DR])
Rearranging, $\frac{[DR]}{[RT]} = \frac{[D]}{[D] + K_A}$ ……(iii)
Now, the ratio [DR]/[RT] is simply the fraction of receptors occupied by the ligand, and is called y.
Fractional receptor occupancy, y = $\frac{[DR]}{[RT]} = \frac{[D]}{[D] + K_A}$ …..(iv)

Multiple Choice Questions

Section 1: Ligand Binding to Receptors

What is the definition of affinity in drug-receptor interactions?
- A) The ability of a drug to induce a cellular response
- B) The ability of a drug to bind to its receptor
- C) The rate at which a drug dissociates from its receptor
- D) The ability of a drug to prevent receptor activation
Which of the following bonds is the weakest in drug-receptor interactions?
- A) Covalent
- B) Ionic
- C) Hydrogen
- D) Van der Waals
The equilibrium dissociation constant (KA) is a measure of:
- A) Drug potency
- B) Drug efficacy
- C) Drug affinity
- D) Drug solubility
A drug with high affinity for a receptor will have:
- A) A high KA value
- B) A low KA value
- C) No effect on KA
- D) High selectivity at all concentrations

Section 2: Agonism and Efficacy

Efficacy refers to:
- A) The binding strength between a drug and receptor
- B) The ability of a drug to activate a receptor and induce a response
- C) The ability of a drug to prevent receptor activation
- D) The rate of drug elimination from the body
Which of the following best describes a biased agonist?
- A) An agonist that binds irreversibly to its receptor
- B) An agonist that selectively activates specific signaling pathways
- C) An agonist that activates all receptor pathways equally
- D) An agonist that only binds to one receptor subtype

Section 3: Antagonism

A competitive antagonist:
- A) Binds irreversibly to the receptor
- B) Binds at a site distinct from the agonist binding site
- C) Competes with the agonist for the same binding site
- D) Reduces receptor numbers on the cell surface
Which type of antagonist cannot be overcome by increasing agonist concentration?
- A) Competitive antagonist
- B) Irreversible antagonist
- C) Partial agonist
- D) Inverse agonist
An inverse agonist:
- A) Blocks the receptor without affecting its activity
- B) Inhibits agonist binding but has no intrinsic activity
- C) Produces the opposite effect of an agonist by reducing baseline receptor activity
- D) Activates the receptor similarly to a full agonist

Section 4: Quantitative Receptor Pharmacology

Saturation binding studies are primarily used to determine:
- A) Drug efficacy
- B) Receptor density and affinity
- C) Drug metabolism
- D) Agonist potency
The Schild analysis is used to evaluate:
- A) The equilibrium dissociation constant of an antagonist
- B) The selectivity of a receptor subtype
- C) The efficacy of an agonist
- D) The rate of drug elimination

Section 5: Selectivity

A drug is said to be selective when it:
- A) Binds only to one receptor type at all concentrations
- B) Has a high affinity for its primary target compared to others
- C) Binds to all receptor types equally
- D) Is non-reversible in binding
Drug selectivity is lost at:
- A) Low concentrations
- B) High concentrations
- C) Equilibrium
- D) The point of receptor saturation

Answers

B) The ability of a drug to bind to its receptor
D) Van der Waals
C) Drug affinity
B) A low KA value
B) The ability of a drug to activate a receptor and induce a response
B) An agonist that selectively activates specific signaling pathways
C) Competes with the agonist for the same binding site
B) Irreversible antagonist
C) Produces the opposite effect of an agonist by reducing baseline receptor activity
B) Receptor density and affinity
A) The equilibrium dissociation constant of an antagonist
B) Has a high affinity for its primary target compared to others
B) High concentrations