L02 Clinical Pharmacology Pharmacodynamics introduction

Overview

This lecture introduces Pharmacodynamics, the relationship between drug concentration at the site of action and the resulting effect. It works through the four protein classes drugs act on (receptors, enzymes, transporters/carrier molecules, ion channels), then builds the quantitative language used to describe drug action at receptors (affinity, potency, efficacy, and the concentration response curve), classifies drugs by whether they have efficacy (agonist, partial agonist, inverse agonist, antagonist), distinguishes reversible from irreversible competitive antagonism and from allosteric and non-receptor antagonism, and finishes with the mechanisms at ion channels, enzymes and transporters, plus selectivity at drug targets and loss of drug effect over time.

Drug targets: the four protein classes

• Pharmacodynamics is defined as “the relationship between drug concentration at the site of action and the resulting effect”.
• Sites of drug action are usually proteins in one of four main classes: receptors, enzymes, transporters/carrier molecules, ion channels.
• Current drug targets, by proportion of drugs acting on each class (pie chart, adapted from Nature Reviews Drug Discovery 10:579-590, 2011):
  • Receptors 53%
  • Enzymes 22%
  • Transporters 9%
  • Ion channels 8%
  • Other 8%
• Receptors are therefore the most common drug target.

Receptor superfamilies and their signalling

Four types of receptor-effector linkage, in order of increasing timescale:

1. Ligand-gated ion channels (ionotropic receptors): ligand binding directly opens or closes the channel, producing hyperpolarisation or depolarisation, then cellular effects. Timescale milliseconds. Example: nicotinic ACh receptor.
2. G-protein-coupled receptors (metabotropic): ligand binds the receptor, which couples via a G-protein (activating or inhibiting), producing a change in excitability and/or second messengers, leading to Ca2+ release, protein phosphorylation or other pathways, then cellular effects. Timescale seconds. Example: muscarinic ACh receptor.
3. Kinase-linked receptors: ligand binds the receptor/enzyme complex, causing protein phosphorylation, then gene transcription, then protein synthesis, then cellular effects. Timescale hours. Example: cytokine receptors.
4. Nuclear receptors: ligand crosses the membrane and binds a receptor inside the nucleus, causing gene transcription, then protein synthesis, then cellular effects. Timescale hours. Example: oestrogen receptor.

Binding versus activation: affinity, potency, efficacy

Binding and activation are distinct processes. The scheme is: drug A (agonist) plus receptor R bind reversibly (rate constants $k_{+1}$ and $k_{-1}$) to form the complex AR, and occupation of the receptor is governed by affinity; AR then converts to the activated state AR* (rate constants $\beta$ and $\alpha$), and activation is governed by efficacy, giving the response. An antagonist B plus R form BR reversibly by the same $k_{+1}/k_{-1}$ step, but BR gives no response: binding occurs with no downstream signal.

Key terms:

• Affinity: how tightly a drug binds to the receptor. Drugs which bind at low concentration have high affinity. If a drug does not bind well its action will be shorter and the chance of binding is less. Measured by the dissociation constant $K_D$, the concentration of drug at which 50% of receptors are occupied. The higher the $K_D$, the lower the affinity (more drug is needed to occupy the receptors). Affinity is defined from concentration-binding curves: plotted against linear concentration the binding curve is hyperbolic and plateaus at $B_{max}$; plotted against log concentration it is sigmoidal, with $K$ marked at half-maximal (50%) binding.
• Potency: a measure of drug activity expressed as the amount of drug required to produce an effect of given intensity, that is how much drug is needed for a particular effect. Measured by the $E{C}_{50}$, the effective concentration of agonist producing 50% of the maximal response. The more potent the agonist, the lower the $E{C}_{50}$. On the sigmoidal curve, potency is the horizontal (x-axis) dimension.
• Efficacy: the ability of a drug to bind a receptor and cause a change in the receptor’s action, that is the ability to produce a response. Measured by $E_{max}$, the plateau of the concentration response curve. On the sigmoidal curve, efficacy is the vertical (y-axis) dimension.

Concentration response curves: biological response can be measured at a wide range of levels, both close to and distant from the initial binding event, including channel opening, signal transduction, change in blood pressure, and behavioural response. Plotted against linear drug concentration the curve is hyperbolic rising to 100% with $E{C}_{50}$ marked at 50% effect; plotted against log drug concentration it is sigmoidal, reaching $E_{max}$, with log $E{C}_{50}$ at the 50%-effect point.

Potency and efficacy vary independently: drugs can be equipotent but have very different efficacies, and a drug can be very potent but have low efficacy. On a plot of several drugs, leftward position gives potency and plateau height gives efficacy.

Classifying drugs by efficacy

• Positive efficacy: the drug activates the receptor to promote cellular response. These are agonists. Agonists have both affinity and efficacy.
• Negative efficacy: the drug binds receptors and decreases basal receptor activity. These are inverse agonists.
• No efficacy: the drug binds receptors but has no effect on activity. These are antagonists; they have affinity but no intrinsic efficacy, do not induce a conformational change in the receptor, and produce their effects by preventing agonist from binding.
• Full agonists elicit the maximum tissue response. Partial agonists produce less than the maximum response and cannot produce a maximal response even at 100% receptor occupancy, so they are less efficacious than full agonists (lower plateau, despite similar or greater potency).

Antagonism at the receptor

An antagonist is a compound that binds to but does not activate (or inactivate) the receptor, and antagonists are defined by how they bind to the receptor.

Reversible competitive antagonism

• Reversible: the drug binds non-covalently to the receptor.
• Competitive: the drug binds the same site as the endogenous agonist (the orthosteric site), competing directly with agonist binding.
• Antagonist: affinity but no efficacy.
• Effect is surmountable: the action of the agonist can be restored by increasing agonist concentration.
• On the curve: a progressive rightward shift of the concentration response curve with the maximum response unchanged. Example: isoprenaline (agonist) with increasing concentrations of propranolol (0, $10^{-8}$, $10^{-7}$, $10^{-6}$ mol/l) shifts the curves progressively rightward.

Irreversible competitive antagonism

• The drug binds covalently to the receptor’s orthosteric site.
• Not surmountable.
• Reduces the number of receptors available to the agonist.
• On the curve: as antagonist concentration rises (0, 1, 10, 100) the maximum achievable response falls progressively (curves flatten, plateau drops), unlike the reversible case where only potency changes.

Direct comparison (change in level of receptor activation vs ligand concentration): agonist alone is leftmost and reaches the highest plateau near 100%; agonist with a reversible competitive antagonist is shifted right but still reaches the same maximum; agonist with an irreversible antagonist is shifted right and capped at a lower maximum. Antagonist alone sits flat at the constitutive level of receptor activation, that is it does not raise activation above baseline.

Allosteric modulators (non-competitive drugs)

• All mechanisms above involve binding at the orthosteric site (the site of endogenous ligand binding). Receptor proteins may contain other, allosteric binding sites.
• Binding to an allosteric site might: increase or decrease the affinity of the orthosteric ligand; increase or decrease the efficacy of the orthosteric ligand; activate or inactivate the receptor in the absence of an orthosteric ligand.
• These drugs are termed positive allosteric modulators or negative allosteric modulators.
• The slide poses the question of why a positive allosteric modulator might be preferable to an orthosteric agonist [slide does not elaborate].

Non-receptor mechanisms of drug “antagonism”

• Chemical antagonism: two substances combine in solution, for example chelating agents binding heavy metals to reduce their toxicity, or infliximab, a neutralising antibody that sequesters the inflammatory cytokine tumour necrosis factor (TNF).
• Pharmacokinetic antagonism: reducing the concentration of the drug at the active site, for example reduced absorption of the drug from the GI tract.
• Interrupting receptor-response: blocking downstream of the receptor, or non-competitive blockage, for example nifedipine blocking Ca2+ channels and so preventing smooth muscle contraction produced by any receptor.
• Physiological antagonism: two drugs whose opposing actions in the body tend to cancel each other out, for example histamine-induced vasodilation versus adrenaline-induced vasoconstriction.

Ion channels as drug targets

• An ion channel is a membrane protein in the lipid bilayer whose gating state controls ion permeation: when open, ions pass straight through into the cell; when closed, ions are blocked.
• Two key types: ligand-gated channels, a type of receptor in which channel opening follows drug/ligand binding; and voltage-gated channels, which open in response to changes in membrane potential.
• Two modes of drug action on channels: blockers physically obstruct the pore so permeation is blocked; modulators alter channel structure, increasing or decreasing the opening probability.
• Voltage-gated channels: drugs can block Na+ channels. Examples are local anaesthetics such as lignocaine, and anti-epileptics (phenytoin, carbamazepine). Blocking these Na+ and related channels at the sensory nerve ending interrupts the pain-signalling pathway from the periphery (nerve ending, dorsal root ganglion, spinal cord) to the brain. Channel and receptor subtypes shown at the nerve ending: Kv4.2, Nav1.6, Nav1.3, Nav1.8, Nav1.7, Nav1.1, ASIC, TRPV1, TRPA1, P2X3.

Enzymes as drug targets

• An enzyme is a biological catalyst; it speeds up the rate of a specific chemical reaction. A cell contains thousands of different types of enzymes, each specific to a particular chemical reaction. The catalytic cycle is: substrate approaches the enzyme, binds to form an enzyme-substrate complex, then products are released and free enzyme is regenerated.
• Three modes of enzyme-targeted drug action: an inhibitor binds the enzyme so the normal reaction is inhibited; a false substrate is processed to produce an abnormal metabolite; a prodrug is processed to produce the active drug.
• Worked pathway (catecholamine synthesis): TYROSINE, via tyrosine hydroxylase, to DOPA; DOPA, via DOPA decarboxylase, to methyl DOPAMINE; methyl DOPAMINE, via dopamine beta hydroxylase, to methyl NORADRENALINE. Drug interactions on this pathway: L-dopa feeds in at DOPA (Parkinson’s disease; a prodrug); methyl dopa feeds in at DOPA (hypertension; a false substrate); carbidopa acts at DOPA decarboxylase (peripherally restricted, inhibits L-dopa metabolism). The end product methyl noradrenaline is linked to alpha 2 receptor agonism.
• Two types of enzyme inhibition:
  • Non-competitive inhibition: the inhibitor binds the enzyme at a site distinct from the substrate, changing the enzyme’s shape so the substrate can no longer bind. Example: aspirin binding to COX. Enzyme cyclooxygenase, substrate arachidonic acid, product prostaglandin H2, inhibitor acetylsalicylic acid.
  • Competitive inhibition: the inhibitor competes directly for the enzyme’s active site so the substrate cannot bind. Example: captopril binding to ACE. Enzyme angiotensin converting enzyme, substrate angiotensin 1, product angiotensin 2, ACE inhibitor captopril.

Transporters as drug targets

• Transporters carry ions and small molecules across membranes. Pharmacologically important roles: transport of ions or organic molecules across the renal tubule, intestinal epithelium and blood brain barrier; uptake of neurotransmitter or neurotransmitter precursors by nerve terminals; transport of drug molecules or metabolites across cell membranes.
• Drugs usually act to inhibit transporters.
• Monoamine reuptake transporters: serotonin (SERT), norepinephrine (NET) and dopamine (DAT). Drugs acting here include antidepressants (tricyclic antidepressants, SSRIs, serotonin-norepinephrine reuptake inhibitors) and psychostimulants such as methylphenidate (NET/DAT). SERT diagram: serotonin is transported through a central binding cavity of SERT; escitalopram occupies an allosteric binding cavity distinct from the central cavity, an SSRI binding at an allosteric site to block serotonin reuptake.
• ABC transporters and p-glycoprotein (P-gp, multidrug resistance protein): export drugs from the cytosol to outside the cell; often highly expressed in cancer cells, exporting drugs from the cell and reducing their activity/effectiveness. Mechanism: the transporter cycles from an inward-facing conformation (binding drug via the TMD1/TMD2 domains from inside the cell, with NBD1/NBD2 nucleotide-binding domains) to, after binding 2 ATP at NBD1/NBD2, an outward-facing conformation that releases the drug to the outside, that is ATP-driven efflux.
• Where P-gp acts:
  • GI epithelium: pumps xenobiotics (toxins or drugs) back into the lumen, decreasing absorption.
  • Liver: pumps xenobiotics into the bile ducts, cleared into faeces.
  • Proximal tubule of the kidney: pumps xenobiotics into the urine collecting ducts.
  • Blood brain barrier: pumps xenobiotics back into the capillaries, out of the brain.
  • Some cancer cells express large amounts of P-gp, rendering them multi-drug resistant.
• P-gp is an efflux pump, so drugs that inhibit it, such as verapamil, increase the bioavailability of susceptible drugs, while drugs that induce it, such as rifampicin, can reduce bioavailability of some drugs. Blocking the pump in a resistant cell lets the medicine accumulate inside the cell instead of being pumped out, restoring sensitivity.

Selectivity and adverse effects

Important

To be useful, a drug must act selectively on a particular target, but no drug acts with complete specificity. In general the lower the potency of the drug, the higher the dose needed, and the more likely it is to have more than one target and a wider range of off target adverse effects.

• Not all adverse effects are off target: opioid analgesics such as morphine produce analgesia as well as respiratory depression and constipation, all through interactions with the mu-opioid receptor.
• Affinity/potency of FDA approved drugs: a frequency distribution of small-molecule drug potencies (frequency vs $-{\log }_{10}$ affinity, x-axis 1.8 to 11.8) is roughly bell-shaped and unimodal, peaking around $-{\log }_{10}$ affinity 7.3 to 7.8, with bands labelled mM (weakest affinity), µM (mid-range) and nM (strongest). Most approved drugs therefore have nanomolar affinity for their targets, with frequencies tapering off towards both the mM and the higher nM/sub-nM extremes.

Desensitisation, tolerance and resistance

Drug effect can diminish following continuous or repeated administration:

• Desensitisation: drug effect reduces in a few minutes.
• Tolerance: drug effect reduces over hours to days.
• Resistance: loss of sensitivity to cytotoxic or antimicrobial drugs.

Mechanisms: receptor phosphorylation; receptor internalisation; intracellular feedback loops; drug metabolism; physiological adaptation.

Revision summary of drug actions by target class

• Receptors: an agonist or inverse agonist acts by direct action or transduction mechanisms, giving ion channel opening/closing, enzyme activation/inhibition, ion channel modulation, or DNA transcription; an antagonist gives no effect, with endogenous mediators blocked.
• Ion channels: blockers block permeation; modulators increase or decrease opening probability.
• Enzymes: inhibitor inhibits the normal reaction; false substrate produces an abnormal metabolite; prodrug produces active drug.
• Transporters: normal transport moves agonist/substrate across the membrane; an inhibitor blocks transport; a false substrate leads to an abnormal compound accumulating.

An ambiguous revision graph shows two curves, A reaching a higher plateau and B a lower plateau at a similar EC50, and asks for two possible interpretations of what each curve could illustrate, for example full versus partial agonist, or agonist alone versus agonist plus irreversible antagonist.

Self-test

1. Define pharmacodynamics.
2. List the four main protein classes that are drug targets, with the approximate proportion of current drugs acting on each.
3. Describe the four receptor superfamilies, giving for each the signalling route, the timescale and an example receptor.
4. Define affinity and explain how $K_D$ measures it, including what a higher $K_D$ means.
5. Distinguish potency from efficacy, naming the parameter that measures each.
6. Explain why two drugs can be equipotent yet differ greatly in efficacy.
7. Describe the steps from agonist plus free receptor to response, stating which step affinity governs and which efficacy governs.
8. Distinguish an agonist, an inverse agonist and an antagonist using the terms affinity and efficacy.
9. Explain why a partial agonist cannot produce a maximal response even at 100% receptor occupancy.
10. Describe the four defining features of reversible competitive antagonism.
11. Predict what happens to the agonist concentration response curve when an irreversible competitive antagonist is added, and explain why it differs from the reversible case.
12. A patient’s response to an agonist is not restored by increasing the agonist dose in the presence of an antagonist. Explain which type of antagonism this indicates and why.
13. Explain what an allosteric site is and list the three things binding to one might do.
14. List the four non-receptor mechanisms of drug antagonism, with an example of each.
15. Distinguish ligand-gated from voltage-gated ion channels, and distinguish channel blockers from channel modulators.
16. Explain how local anaesthetics such as lignocaine produce their effect, and name two anti-epileptics acting by the same channel mechanism.
17. List the three modes by which drugs act on enzymes, with an example drug for each from the catecholamine pathway shown.
18. Distinguish competitive from non-competitive enzyme inhibition, giving the example enzyme, substrate, product and inhibitor for each.
19. Describe the steps by which P-glycoprotein exports a drug from a cell.
20. List the four normal body sites where P-gp pumps xenobiotics, stating where it moves them in each case.
21. Predict the effect on the bioavailability of a susceptible drug of co-administering verapamil, and of co-administering rifampicin, and explain why.
22. Explain the relationship between a drug’s potency and its likelihood of off target adverse effects.
23. Explain, using morphine as the example, why not all adverse effects are off target effects.
24. Distinguish desensitisation, tolerance and resistance, and list the mechanisms that can underlie loss of drug effect.
25. Integrative: two sigmoidal curves are plotted, A with a higher plateau and B with a lower plateau at a similar EC50. Suggest two different pharmacological explanations for this pattern and state how you would tell them apart.

Answers

Reveal answers

1. The relationship between drug concentration at the site of action and the resulting effect.
2. Receptors 53%, enzymes 22%, transporters/carrier molecules 9%, ion channels 8% (other 8%).
3. Ligand-gated ion channels (ionotropic): ligand binding directly opens or closes the channel, causing hyperpolarisation or depolarisation then cellular effects; milliseconds; nicotinic ACh receptor. G-protein-coupled (metabotropic): ligand binds receptor coupled via an activating or inhibiting G-protein, changing excitability and/or generating second messengers, leading to Ca2+ release or protein phosphorylation then cellular effects; seconds; muscarinic ACh receptor. Kinase-linked: ligand binds the receptor/enzyme complex, causing protein phosphorylation then gene transcription then protein synthesis; hours; cytokine receptors. Nuclear receptors: ligand crosses the membrane and binds a receptor in the nucleus, causing gene transcription then protein synthesis; hours; oestrogen receptor.
4. Affinity is how tightly a drug binds the receptor; drugs binding at low concentration have high affinity. $K_D$, the dissociation constant, is the concentration of drug at which 50% of receptors are occupied. A higher $K_D$ means lower affinity, because more drug is required to occupy the receptors.
5. Potency is the amount of drug required to produce an effect of given intensity, measured by $E{C}_{50}$ (concentration giving 50% of maximal response); the more potent the drug the lower the $E{C}_{50}$. Efficacy is the ability to bind and change the receptor’s action, that is to produce a response, measured by $E_{max}$, the plateau of the curve.
6. Potency is the leftward position of the curve on the log concentration axis and efficacy is the height of its plateau; these are independent, so curves can share an $E{C}_{50}$ while plateauing at different maxima. A drug can also be very potent but of low efficacy.
7. Drug A plus receptor R bind reversibly (rate constants $k_{+1}$, $k_{-1}$) to form AR; this occupation step is governed by affinity. AR then converts to the activated state AR* (rate constants $\beta$, $\alpha$); this activation step is governed by efficacy and produces the response.
8. An agonist has affinity and positive efficacy, activating the receptor to promote cellular response. An inverse agonist has affinity and negative efficacy, binding to decrease basal receptor activity. An antagonist has affinity but no efficacy: it binds without inducing a conformational change and acts by preventing agonist binding.
9. Because a partial agonist has lower efficacy than a full agonist; it binds with affinity but does not produce a maximal response, so its plateau is lower even when all receptors are occupied.
10. Reversible: binds non-covalently. Competitive: binds the same (orthosteric) site as the endogenous agonist. Antagonist: has affinity but no efficacy. Surmountable: the agonist’s action can be restored by increasing agonist concentration, seen as a rightward shift of the curve with maximum response unchanged (isoprenaline with propranolol 0 to $10^{-6}$ mol/l).
11. The maximum achievable response falls progressively as antagonist concentration rises (curves flatten, plateau drops) rather than simply shifting rightward. The antagonist binds covalently to the orthosteric site, so it is not surmountable and it reduces the number of receptors available to the agonist.
12. Irreversible competitive antagonism, because covalent binding permanently removes receptors from the pool available to the agonist, so the effect is not surmountable; in reversible competitive antagonism increasing agonist would restore the response.
13. An allosteric site is a binding site on the receptor protein distinct from the orthosteric site (the site of endogenous ligand binding). Binding there might increase or decrease the affinity of the orthosteric ligand, increase or decrease its efficacy, or activate/inactivate the receptor in the absence of an orthosteric ligand; such drugs are positive or negative allosteric modulators.
14. Chemical antagonism, two substances combining in solution, for example chelating agents binding heavy metals, or infliximab sequestering TNF. Pharmacokinetic antagonism, reducing drug concentration at the active site, for example reduced GI absorption. Interrupting receptor-response, blocking downstream of the receptor or non-competitive blockage, for example nifedipine blocking Ca2+ channels to prevent smooth muscle contraction produced by any receptor. Physiological antagonism, two drugs with opposing actions cancelling out, for example histamine-induced vasodilation versus adrenaline-induced vasoconstriction.
15. Ligand-gated channels are a type of receptor in which opening follows drug or ligand binding; voltage-gated channels open in response to changes in membrane potential. Blockers physically obstruct the pore so permeation is blocked; modulators alter the channel so opening probability increases or decreases.
16. They block Na+ channels at the sensory nerve ending, interrupting the pain-signalling pathway running from the nerve ending through the dorsal root ganglion and spinal cord to the brain. Phenytoin and carbamazepine are anti-epileptics acting by Na+ channel block.
17. Inhibitor, the normal reaction is inhibited, for example carbidopa inhibiting DOPA decarboxylase. False substrate, an abnormal metabolite is produced, for example methyl dopa (hypertension). Prodrug, active drug is produced, for example L-dopa (Parkinson’s disease).
18. Competitive inhibition: the inhibitor competes for the enzyme’s active site so substrate cannot bind; captopril on angiotensin converting enzyme, substrate angiotensin 1, product angiotensin 2. Non-competitive inhibition: the inhibitor binds at a site distinct from the substrate and changes the enzyme’s shape so substrate can no longer bind; aspirin (acetylsalicylic acid) on cyclooxygenase, substrate arachidonic acid, product prostaglandin H2.
19. P-gp starts inward facing and binds the drug from inside the cell via its TMD1/TMD2 domains; binding of 2 ATP at the NBD1/NBD2 nucleotide-binding domains switches it to an outward-facing conformation, which releases the drug to the outside of the cell. It is ATP-driven efflux.
20. GI epithelium, back into the lumen (decreasing absorption); liver, into the bile ducts (cleared into faeces); proximal tubule of the kidney, into the urine collecting ducts; blood brain barrier, back into the capillaries, out of the brain.
21. Verapamil inhibits P-gp, so less drug is pumped back out and bioavailability of susceptible drugs increases. Rifampicin induces P-gp, so more drug is effluxed and bioavailability of some drugs is reduced.
22. No drug acts with complete specificity. In general the lower the potency, the higher the dose needed, and the more likely the drug is to hit more than one target and to produce a wider range of off target adverse effects.
23. Morphine produces analgesia and also respiratory depression and constipation, all through interactions with the same mu-opioid receptor, so these adverse effects arise at the intended target rather than off target.
24. Desensitisation is a reduction in drug effect within a few minutes; tolerance is reduction over hours to days; resistance is loss of sensitivity to cytotoxic or antimicrobial drugs. Mechanisms: receptor phosphorylation, receptor internalisation, intracellular feedback loops, drug metabolism, physiological adaptation.
25. Possibility one: A is a full agonist and B a partial agonist, B having lower efficacy so a lower $E_{max}$ even at full occupancy. Possibility two: A is the agonist alone and B the same agonist in the presence of an irreversible antagonist, which reduces the number of available receptors and so caps the maximum response. They could be distinguished by whether the lower plateau is intrinsic to drug B or appears only when a second drug is present, and by whether increasing agonist concentration restores the maximum (it would not in either case, unlike reversible competitive antagonism, which shifts the curve rightward with $E_{max}$ unchanged).