6. Molecular pharmacology, pharmacodynamics & receptors

Receptor concept

• The molecular action of a drug/bioactive can often be explained by-, and predicted from a receptor concept

• Receptors are mostly normal regulatory structures playing a role in the physiology of the organism

• Natural ligands: neurotransmitters, hormones, or other signaling molecules

• Drugs just bind by coincidence or by design to these receptors

• Receptors: macromolecules including

  • Enzymes

  • Transporters & ion channels

  • Structures on cell membranes

  • Transcription factors

  • Nucleotides

What are the four receptor types & interactions

• Agonist/antagonist = agonist produces effect/antagonist prevents an agonist from binding = no effect.

• Ion channels = blockers (block ion channel) or modulators = transform ion channel to increase/decrease permeability

• Enzymes = inhibitor/false substrate. Inhibitor = normal reaction inhibited. False substrate = abnormal metabolite produced

• Transporters = inhibitors/false substrates. Inhibitor = transport blocked. False substrate = Abnormal compound accumulated.

Ion channels

• Multi-subunit ion channels (=ligand-gated ion channels; also: ionotropic receptors)

• Location: cell surface transmembrane

• Binding → ion flux → effect on cell excitability

• Examples of ligands: acetylcholine

  • GABA

  • Glutamate

  • Glycine

Example Ion channel

Nicotinic ACh receptor

• On the ion channel there are two binding sites for ACh.

• When ACh is bound, this causes a conformational change

GABA receptor + effect of drugs

• Also example of ion channel.

• GABA reduces the chance of response to stimulis

  • This is because when GABA binds, the ion channel opens

  • Allows Cl- to enter cell causing greater negative charger

  • During action potential you need the charge to become positive, so if resting potential is more negative than usual = harder to depolarize.

• Certain medicines (enzodiazepines) make it easier for GABA to bind. Make receptor more sensitive

• They act allosterically on the receptor (So drug doesn’t bind to active site but binds to allosteric site)

• Examples

  • Diazepam

  • Oxazepam

  • Lorazepam

Protein kinases

• Location: cell surface transmembrane

• Examples of ligands;

  • Growth factors, insulin

  • Peptide hormones

Transcription factors

• Location: cyptolasm

• One of the most important targets for today’s drugs

• Very large family of regulatory proteins

• Agonists bindings leads to a cell signalling cascade involving many chemical mediators

• Examples:

  • Steroid hormones

  • Thyroid hormone

  • Vitamin D

Example vitamin D

• Example of transcription factors

• In the DNA we have vitamin D responsive elements

• The DNA is in the nucleus but the vitamin D outside

• The vit D goes into the nucleus, forms a complex with a vitamin D receptor.

• This complex can bind to the vitamin D responsive elements.

• This leads to the expression of target genes and hence proteins

G-protein coupled receptors

• Location: cell surface, 7 transmembrane

• One of the most important targets for today’s drugs

• Very large family (~950 genes in human genome) of regulatory proteins (including olfactory, taste, light, endogenous compounds)

• Agonist bindings leads to a cell-signalling cascade involving many chemical mediators

  • Central mechanism GTP-GDP conversion

• Example of ligands:

  • Acetylcholine

  • Several a- and B-adrenergic cathecholamines

  • Eicosanoids

Molecular mechanism GPCR

1. An antagonist binds to the receptor. This causes GDP on the alpha subunit to be replaced by GTP

2. Beta gamma subunit binds to target 2 and alpha subunit binds to target 1.

3. At some point GTP is hydrolyzed into GDP so that effect stops.

cAMP system

• Intracellular ‘second messenger’ system

• Activated G-protein → activates adenylyl cyclase → ATP cAMP → activates protein kinase A

• G protein that activate cyclic AMP (cAMP) formation are known as stimulatory G-protein (Gs)

• Example: adrenalin, glucagon

Formation of cAMP explained

• Epinephrin binds to receptor protein

• This activates G-protein causing one of the three subunits to dissociate from the G-protein

• The subunit that dissociates carries GDP which is replaced by GTP when activated.

• The subunit then binds to adenylyl cyclase.

• Adenylyl cyclase, when activating, catalyzes the formation of cAMP from ATP.

• The cAMP then binds to a protein kinase A which then activates phosphorylase that turns glycogen into glucose

Phospholipase C pathway

1. Starts with hormone binding to receptor. This complex binds to G-protein

2. As a result GDP is replaced with GTP

3. alpha subunit of G dissociates from beta and gamma subunits and activates phospholipase C

4. Active phospholipase C cleaves phosphatidyl-inositol 4,5-bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol

5. IP3 binds to a specific receptor on the endoplasmic reticulum, causing release of sequesteral Ca2+

6. Calcium and diacylglycerol activate protein kinase C

7. Protein kinase C catalyzes phophorylation of cellular proteins that mediate cellular responses.

How is the GPCR signal regulated?

• GTP activated alpha-subunit has intrinsic GTPase activity

  • GTP → GDP + P

  • Inactivates alpha subunit

  • → re-association with bg-subunits

• Phosphodiesterase (PDE) hydrolyzes cAMP → AMP

• Without these mechanisms, we would not be able to turn off receptor signals

Receptor desensitization

• Happens in drug addicts

• Some receptors are phosphorylated via specific receptor kinases

• The phosphorylated receptor may then bind to a protein b-arrestin that promotes removal of the receptor from the membrane by clathrin-mediated endocytosis

Gs and Gi proteins

• An inhibitory Gia, when it binds GTP, inhibits Adenylate cyclase

• The stimulatory Gsa, when it binds GTP, acivates Adenylate cyclalse

• Different effectors & their receptors induce Gia to exchange GDP for GTP than those that activate Gsa

• They may act simultaneously

What is asthma and what are its pharmacological targets?

• Asthma in brief: recurrent and reversible airway obstruction

  • Inflammatory changes in the airways

  • Bronchial hyper-responsiveness

  • Attack in two phases

• Pharmacological targets

  • Reduction of inflammatory response

  • Bronchodilation

Pharmacological treatment asthma

• B2 - adrenergic agonists relax smooth muscle

• Short acting (salbutamol, terbutaline) or long acting (salmeterol, formoterol etc)

• Usually given as local formulation (inhaler)

• Use depends on type of asthma, often combined with glucorticoids

• Sometimes: muscaranic receptor antagonists (ipratropium)

• Not that common anymore: PDE inhibitors (theophylline)

  • PDE breaks down cAMP which normally gives bronchodilation. Therefore PDE inhibitors will prevent bronchodilation

cannabinoid receptors

• Important endogenous regulation system

• Two G-protein coupled receptors described so far (CB1 and CB2)

• Many endogenous and exogenous cannabinoids

• Specific transporters

Range of effects of cannabinoids

• Appetite stimulation

• Anti-inflammatory

• Hallucinogenic euforic

• Anti-tumor

• Psychosis risk

Endogenous CB ligands: endocannabinoids

• We have our own endocannabinoid system (ECS)

• The ECS is a highly important regulation system in our body (metabolism, immune defense, pain)

• Endocannabinoids are formed from fatty acids

• This is an important system for food intake

Initial hypothesized role of ECS in obesity

• Stimulation with EC → increased food intake

• CB1 stimulation increases differentiation and lipogenesis in pre-adipocytes

• Plasma levels of EC’s are increased in obese human subjects and in animal models of diet-induced obesity

• CB1 mouse is protected from diet-induced obesity

• Can we treat/prevent obesity if the ECS is inhibited?

Effects and side effects of CB1 blockers

• Good effects:

  • Weight loss

  • Decrease HbA1C

  • Increase Adiponectin

  • etc.

• Side effects: joy of eating removed = more depression and anxiety = more suicide

Two-state receptor model

• Every now and then spontaneously, the system goes from resting state to activated state

• This spontaneous activation is what we call intrinsic activity

• When in activated state, response is created.

• Agonists shifts equilibrium to active state R*

Agonist/antagonist working shown in two-state receptor model

Agonist

• Drug binds to receptor = occupation, governed by affinity (higher affinity = stronger binding)

• Activation governed by efficacy

Antagonist

• Binds to receptor and forms a complex

• Does not make equilibrium shift to activated state

• = no response

• But by binding to receptor prevents agonist from binding

What is efficacy?

What is the maximum response?

What is potency?

At which concentration do you achieve EC50?

Full versus partial agonists

• Full agonist: a ligand that binds to a receptor and shifts the equilibrium toward thea ctivate state, producing the maximum possible biological response

• Partial agonist = A ligand that binds to the exact same active site on the receptor but elicits only a sub-maximal biological response, even when given at high concentrations that occupy 100% of the receptors.

• Ligand b is partial agonist with the same affinity as the full agonist

What happens with the repons when you mix a full and partial agonist?

The response will be somewhere between a and b (can have inhibitory effect)

• Competition for Receptors: Both the full and partial agonist compete to occupy the same active binding sites on the receptors.

• Sub-Maximal Activation: Receptors bound by the partial agonist only produce a fraction of the maximum signal (e.g., around 50% response).

• Competitive Inhibition: While a receptor is occupied by the partial agonist, the full agonist is physically blocked from binding to that site. Because the full agonist cannot occupy 100% of the available receptors, it can no longer produce its 100% maximal effect Emax

Competitive vs non-competitive antagonist

• Competitive: binds at the same binding site as agonist

• Non-competitive: binds at different binding site as antagonist

Graphs for reversible vs irreversible antagonism

What are reversible and irreversible antagonism?

• Reversible: The antagonist binds to the receptor, but the interaction is non-covalent and dynamic—meaning the antagonist can associate and dissociate from the binding site

• Is surmountable = if you add a sufficiently high concentration of the agonist, the agonist statistically outcompetes and displaces the antagonist from the receptor active sites

  • Graph: the concentration-response curve shifts to the right. A higher concentration of agonist is required to produce the same biological response

• Irreversible: The antagonist forms a covalent bond to the receptor, preventing it from dissociating

• Unsurmountable: Because the antagonist does not let go of the receptor, increasing the agonist concentration cannot push the antagonist off the obund sites.

  • Graph: The curve flattens, maximal achievable response is permanently reduced because a fraction of the total receptor population is rendered permanently inactive

Reversible vs irreversible agonist

1. Reversible Agonists (Typical Agonists)

• Binding Nature: Almost all natural neurotransmitters and conventional drugs (e.g., adrenaline, acetylcholine) bind reversibly through non-covalent interactions^[3].

• Dynamic Equilibrium: Reversible agonists undergo continuous association and dissociation with the receptor^[2][5].

• Surmountability: Because binding is dynamic, a reversible agonist can compete with and displace a reversible competitive antagonist if the agonist concentration is sufficiently increased^[1][2].

• Signal Termination: The biological response turns off naturally as the drug dissociates from the receptor and is metabolized or excreted^[6][7].

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2. Irreversible Agonists

• Binding Nature: An irreversible agonist forms a covalent bond (or has extremely slow dissociation) with the active site of the receptor^[1].

• Persistent Activation: Once attached, it continuously drives the receptor into the active state without letting go^[1][5].

• Receptor Desensitization: Because the receptor is continuously stimulated without a break, the cell rapidly initiates receptor desensitization^[8]. The receptor becomes phosphorylated and internalized (broken down via endocytosis), effectively removing it from the cell membrane^[7][8]. Paradoxically, prolonged exposure to an irreversible or long-acting agonist often leads to a loss of responsiveness (tolerance) due to receptor downregulation^[8].