Pharmacodynamics Flashcards
Drug
A drug is a chemical/substance that is used to treat a disease/condition
4 Drug Targets
Receptors, Ion Channels, Transporters, Enzymes
Exception: DNA is a target for many anti-cancer and antibiotic drugs
4 Steps of Neurotransmission
Neutrotransmitter synthesis
Neurotransmitter release
Action on receptors
Inactivation
Cholinergic Synapse
Synthesis of Acetylcholine
Choline transporter - transports choline into nerve terminal; rate limiting step in Ach synthesis
Choline Acetyl Transferase (ChAT) - enzyme involved in synthesis of Ach
Release of Acetylcholine
- Ach is packaged in synaptic vesicular transporter
- Vesicles are held in the cytoskeleton by Ca2+ sensitive vesicle membrane proteins (VAMPs)
- When an action potential reaches the terminal, voltage-dependent calcium channels open, Ca2+ rushes in triggering the fusion of vesicles with the cell membrane and release of Ach into the synapse
Receptors
Receptors are proteins that recognise a specific ligand
- Natural (endogenous) = neurotransmitter/hormone (e.g Ach, insulin)
- Synthetic (exogenous) = drug/chemical
- Binding of the ligand to the receptors alters its conformation leading to a change (either activation or inhibition depending on the ligand in cell signalling
4 Receptor Families
- Ligand gated Ion channels
- G-protein-coupled receptors (GPCR)
- Tyrosine kinase/cytokine receptors
- Nuclear/Steroid Hormone receptors
Ligand-gated ion channels receptors aka ionotropic receptors
General features
- Multi-protein subunit (oligomeric) complexes
- Conduct ions through the otherwise impermeable cell membrane
- Ion conductivity is highly selective e.g Ach, glutamate cause an increase in Na+ and K+ permeability
- Activated in response to binding by specific ligands
- Mediate fast signal transmission at synapses (fraction of a millisecond)
Nicotinic Acetylcholine Receptors - Ionotropic
- Made up of subunits from alpha, beta, delta, gamma subunit families
- Each Ach binding site is at the interface formed by the peptide loops between one of the two-alpha subunits and its neighbour
- Ach needs to bind to both sites to stimulate ion channel opening
- The five TM2 helices are sharply kinked inwards halfway through the membrane forming a constriction
- The TM2 helices are believed to snap to attention when Ach binds, opening the channel
Examples of ionotropic receptors as drug targets
- Nicotinic acetylcholien receptors - nicotine, pancuronium (antagonist) used as muscle relaxants during anaesthesia
- GABA receptors - benzodiazepines and barbiturates, muscimol
- NMDA subtype of glutamate receptors - ketamine.
G-protein coupled receptors (GPCR)
- Monomeric proteins with 7 transmembrane domains that are coupled to G-proteins
- Muscarinic acetylcholine receptors - 5 receptors subtypes that are coupled to different signalling pathways
Drug specificity and selectivity
Specificity - drug acts only at the desired drug target
Selectivetly - ?
Selective vs Non-selective
Selective - at a particular subtype eg. Acetylcholine
Non-selective - all subtypes e.g M4>M1 receptors
Pre-synaptic receptors
- Presynaptic receptors are usually Gi-lined
- Activation of them leads to inhibition of voltage sensitive Ca2+ channles
- This results in decreased neurotransmitter release (feedback loop)
- Because the pre-synpatic receptors are pharmacologically distinct from the post-synpatic receptors, specific drugs can be designed to target these receptors.
- Drugs which block presynpatic receptors can result in a 10-fold increase in neurotransmitter release
- Eg. M2 receptor on pre-synaptic membrane
Examples of drugs that act through GPCRs
B-adrenoreceptors - isoprenaline
Adenosine receptors - caffeine, theophylline
Dopamine receptors - L-dopa, haloperiodol
Opioid receptors - morphine, codeine
Serotonin receptors - buspirone, ondansetron
Muscarinic receptors - atropine
Cannabinoid receptors - cannabis, rimonabant, Sativex
Receptor Tyrosine Kinases
- RTK mediate the actions of growth factors, cytokines and certain hormones (e.g insulin)
- Have an extracellular part that the ligand binds to, and an intracellular part that functions as a kinase
- Kinases are enzymes that transfer phosphate groups from ATP to a substrate (ie.phoshphorylation)
- For RTK, the phosphate groups are transferred to tyrosine amino acid residues on intracellular target proteins
- Phophorylation can control protein function by changing the activity of an enzyme to an ‘on’ or ‘off’ state, altering its subcellular location or interaction with other proteins
Phosphorylation of the cytoplasmic domain creates effector protein binding sites
- Adaptor proteins
- Kinases
- Phosphatases
- Lipases
Vascular Endothelial Growth Factor Receptors
- Essential for angiogenesis (i.e blood vessel formation) during development, pregnancy, wound healing
- Also associated with pathophysiological conditions e.g cancer, rheumatoid arthritis, cardiovascular disease
- Multiple receptors/multiple ligands - we will look briefly at VEGFR2
VEGFR2
- Ligand stimulated receptor dimerisation
- Autophosphorylation of tyrosine residues in cytoplasmic domain
- Associates with SH2 domain proteins
- Activation regulates a multiple of biological functions
- Endothelial cell survival
- Endothelial cell proliferation
- Endothelial cell migration
- Nitric oxide and prostaglandin I2 production
- Increase vascular permeability
VEGFR-2 Diagram
A signal transduction pathway that drives proliferation
- Receptor activation leads to activation of PLCy by phosphorylation
- PLCy-hydrolyses PIP2 to DAG + IP3
- DAG activates PKC
- PKC activation leads to activation of ERK via Raf and MEK
- ERK activation leads to increases gene transcription
- Potential therapeutic use:
- Angiogenesis inhibitors - block endothelial cell growth in tumours
- Angiogenesis stimulators - promote blood vessel growth following ischaemic conditions e.g heart diseaes, limb ischemia
Nuclear/steroid hormone receptors
- Located intracellularly in the cytosol and nucleus
- Function as transcription factors - the ligand binds to the receptor, translocates to the nucleus and stimulates transcription of specific target genes
Summary diagram of receptors
How do drugs bind to receptors?
- Van der Waals forces - weak forces
- Hydrogen binding - stronger
- Ionic interactions - between atoms with opposite charges, stronger than hydrogen, weaker than covalent
- Covalent binding - essentially irreversible
Affinity Constants
Kd = concentration when fractional occupancy = 0.5
Affinity
Attraction of drug for a receptor. Defined as the extent or fraction to which a drug binds to receptors at any given drug concentration. The higher the affinity of the drug for the receptor, the lower the concentration at which it produces a given level of receptor occupancy
Kd (dissociation/affinity constant)
- Ligand concentration at which half of the receptor population has ligand bound. Lower Kd = higher affinity
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Biological response to a drug, diagrams
Efficacy
- The ability of a drug to bind to a receptor and cause a change in the receptor’s action is termed efficacy and measured by Emax
- A drug with positive efficacy will activate a receptor to promote cellular response - agonist
- A drug with negative efficacy will bind to receptors to decrease basal receptor activity - inverse agonist
- A drug with no efficacy will bind to the receptors but have no effect on activity - antagonist
Potency
- EC50 is used to measure the potency of an agonist
- EC50 is the effective concentration of an agonist that produces 50% of its maximal response
- The more potent the agonist, the lower the EC50
- Antagonist potencies are more complicated to determine but a similar principle holds
Agonism
- Drugs that elicit the maximum tissue response are referred to as full agonists, drugs that produce less than maximum response are partial agonists
- Partial agonists can not produce maximal response even at 100% receptor occupancy
- While affinity is thought to be a function of only the drug and the receptor, the maximum response to a tissue to a drug is determined by efficacy and tissue properties. A drug may appear to be a partial agonist in one tissue and a full agonist in another
Affinity vs biological response
- Concentration-response curves are not a good measure of the affinity of agonist drug for their receptor
- The relationship between receptor occupancy and response is not strictly proportional:
- considerable amplification may exist - it may only take a low level of receptor occupancy to cause a maximal response in some tissues
- many factors downstream from the receptor binding may interact to produce the final response
Inverse agonism
- Some receptors have a constitutive level of activity (i.e they are active) even when no ligand is present (e.g serotonin receptors)
- Inverse agonists decrease the activity of these receptors to below basal levels (negative efficacy)
Antagonists
- A compound that binds to but does not actiavte (or inactivate) the receptor
- Antagonists have affinity but NO efficacy
- The type of antagonist is defined by how much they bind to the receptor
Reversible competitive antagonism
- This type of antagonism binds to the receptor in a reversible manner to compete directly with agonist binding
Irreversible antagonism
- These types of antagonists bind covalently to the receptor. It is not reversible
- Therefore, it reduces the number of receptors available to the agonist.
- Lowers plateu on response curve
Receptor reserve
A proportion of receptors needed to be activated in order to produce maximal response
Different agonisms, summary diagram
Allosteric modulators
- The primary binding site for the endogenous ligand is called the orthosteric binding site
- Many proteins possess more than one binding site
- An allosteric binding site is a non-overlapping and spatially distinct site that is conformationally linked to the orthosteric binding site on a protein
- Allosteric modulators are ligands that can bind to the allosteric site to modulate (i.e influence) the binding of the endogenous ligand and/or the signalling properties at the orthosteric site
Name of allosteric modulators that can alter affinity
- Positive allosteric modulators potentiate the effects of the orthosteric ligand by increasing the ligand association rate and/or a decrease in ligand dissociation rate (the latter being more common)
- Negative allosteric inhibition can arise from opposite changes
- Neutral allosteric modulators can also occupy the allosteric site but have no effect (functionally silent)
Difference between allosteric modulation and competitive antagonism
A competitive interaction results in a thereotically limitless rightward shift of the concentration-occupancy curve for orthosteric ligand, A.
An allosteric enhancer or allosteric inhibitor exhibits progressive inability to maximally shift the orthosteric ligand occupancy curve at maximal modulator concentration.
Advantages of allosteric modulators
- Ceiling effect (saturability): The extent of modulation is limited by the cooperativity between the orthosteric and allosteric ligand; decreaed risk of overdose.
- Increase selectivity: Allosteric site are evolutionarily less conserved making it easier to develop a drug that selectively targets a specific member in a family of receptors
- Maintenance of spatial and temporal signalling by the endogenous ligand: Allosteric ligands act to fine-tune the actions of the endogenous ligand when it is bound at the orthosteric site. The spatial and temporal pattern of activity of the endogenous ligand is still maintained.
Example of inactivation of an enzyme
- Once in the synapse, neurotransmitter must be quickly removed or chemically inactivated in order to prevent constant stimulation of the post-synpatic cell
- There are two key mechanisms of inactivation
- enzymic degradation (e.g acetylcholine)
- transport back into the pre-synaptic terminal (e.g serotonin)
Inhibitors of acetylcholine esterase
- The effect that a specific AChE inhibitor can have on the body depends largely on the chemical properties of the molecule and the strength of the bond it forms with AChE
- Irreversible AchE inhibitors are highly toxic
- organophosphorus compounds or nerve gases
- form incredibly stable phosphorus bonds with AchE which resists hydrolytic cleavage = AChe activity is inhibited leading to an excessive build-up of ACh at synapses
- Lead to profuse sweating, dimmed vision, uncontrollable vomiting, convulsions, bronchial contriction, and at last, paralysis and asphyxiation from respiratory failure
Reversible AchE inhibitors
- Bind to AChe for a short time
- Used in disorders characterized by a decrease in cholinergic function - AChE inhibitors increase the duration that acetylcholine is in the synpase so one molecule can activate more receptors
- Used a treatment for mysthenia gravis
- an autoimmune disorder characterized by debilitating muscle weakness caused by a a progressive breakdown of ACh receptor sites on the muscular endplate
- Pyridostigmine bromide, (Mestinon or Regonol), is the most widely used. Cannot pass the blood-brain barrier, so their action is limited to the inactivation of AChE at the neuromuscular junction. Can’t get into brain = no psychotopic effects
- Used in the treatment of Alzheimer’s disease
- brain cell loss results in a progressive and significant loss of cognitive function
- drugs must readily cross the blood-brain barrier
- Tetrohydroaminoacridine (Tacrine or Cognez), and Donepezil (Aricept)
- Can help to ease some of the memory and language deficits
Diagram of acetylcholinesterase inhibitors
Inactivation by transport back into the pre-synaptic terminal (e.g serotonin) - Serotonin (5HT) transporter
- 5HT is involved in sleep,appetite, memory, sexual behaviour, neuroendocrine function and mood
- It is synthesized from the amino acid precursor tryptophan, packaged in vesucles, and released into synpases following an action potential
- Reuptake, determines the extent and duration of receptor activation
- SSRI - selective serotonin reuptake inhibitors
Serotonin Transporter
- Na+ binds first to the carrier, followed by 5-HT. Cl- is needed for transport by not for transmitter binding
- The molecules translocate across the membrane and dissociate. K+ then binds and translocates the outside of the membrane and dissociates to complete the cycle
What don’t we learn from assays?
- No information about efficacy
- activation/inhibition ; agonism / antagonism / inverse agonism
- BUT, helps in designing efficacy assays to know drug affinity for target
- A drug with high affinity has the potential to have high potency (ie. produce an effect at a low concetration) but we CANNOT determine this from a binding assay alone.
- NO information about binding to other targets
Binding assay: Basic Procedure
- Incubate labelled compound (drug/ligand) with tissue/cell sample
- Parameter measurements rely on assay to be at “equillibrium” when measurements are taken
- Rate ligand associates = Rate ligand dissociates
- Separate bound ligand from free ie. rapid wash to remove free ligand
- Measure signal - scintillation counting, radioisotope sensitive film
What information does binding assay give?
- Gives information about a labelled ligand
- Parameters: Kd and Bmax
- Apply labelled ligand at various concentrations
Binding assay: Non-specific binding estimation
- Non-specific binding can be estimated:
- Incubate labelled ligand with high concentration of an equivalent non-labelled ligand
- The non-labelled ligand will bind to all the “specific” (receptor) binding sites but have little effect on non-specific binding (non-saturable component)
- NB: the “competitor” (non-labelled ligand) should bind selectively to the receptor of interest
- Therefore:
- Only labelled ligand present => “total binding”
- Labelled ligand + high concentration unlabelled ligand => “non-specific binding”
- Difference between total and non-specific binding = “specific receptor binding”
Saturation binding data graph
Competition binding: Why used?
- Used when interested in an unlabelled compound (can’t run a saturation experiment)
- Can the unlabelled compound compete with a radioligand for the same binding site?
- Labelled ligand with known properties ie Kd still required; acts as an indicator of binding of the unlabelled ligand
- NB: only get information about binding site where the ligand binds
- Measured affinity of unlabelled ligand is called Ki, inhibition constant
- Dissociation consant of the inhibitor of labelled ligand binding
- Inhibitor = competitor / unlabelled ligand of interest
Competition binding: Procedure
- Labelled ligand applied at approximate Kd concentration
- ie without any competitor, and at equilibrium, approximately 50% of the total receptor binding sites will have ligand bound (be “occupied”)
- Constant concentration for all assay points
- Unlabelled ligand of interest applied at a range of concentration in the presence of the labelled ligand
- Aka “cold” ligand
Competition binding assay graph
IC50 to affinity
Summary: Saturation vs Competition Assays
