Lecture 1 - General Principles and Receptors

Question 1

What must a drug do/have to produce a pharma response?

Answer 1

Non-uniform distribution and chemical influence on cell constituents (drug targets)

Question 2

What are the four types of drug target?

Answer 2

Receptors, enzymes, carrier molecules, ion channels

Question 3

What is specificity?

Answer 3

A ligand’s selectivity for a receptor. It is reciprocal. Nothing is completely specific - decreased potency –> higher dose –> sites of action other than primary assume significance.

Question 4

What are agonists/antagonists?

Answer 4

Agonists activate receptors.

Antagonists block the effect of agonists.

Question 5

What is affinity?

Answer 5

Tendency of a drug to bind to its receptor.

Kd = conc at which drug is 50% bound

Question 6

What is efficacy?

Answer 6

Ability to bind to receptor and elicit a functional response. A measure of the formation of D-R complexes.

Question 7

What is potency and what is it determined by?

Answer 7

Amount of drug needed to produce a given effect (EC50). Determined by affinity and number of receptors available.

Question 8

Way of measuring affinity? (competitive drug to receptor)

Answer 8

Incubate with radioligand are various concentrations of unlabelled competitive ligand.
% radioactivirt plotted against conc of competing ligand.
IC50 (affinity) = conc of ligand which displaces 50% of radioligand

Question 9

What is agonist concentration effect curve?

Answer 9

Measure of response measured against increasing concentrations of drug.
Semialogrithmic –> sigmoid.
Efficacy (Emax) and Potency (EC50) cam be derived from it.
Smaller EC50 = greater potency

Question 10

Receptor occupancy?

Answer 10

The magnitude of response is related to RO.
Sometimes Emax is achieved at less than 100% occupation.
Compare conc. for 50% max effect with conc. for 50% max binding. If EC50 is less than Kd, spare receptors exist.

Question 11

Partial agonists?

Answer 11

Produce submaximal response. Lower efficacy because less response at the same occupancy.

Question 12

Antagonists? Competitive and non-competitive, reversible and non-reversible

Answer 12

Drug which binds receptor but no activation.
Competitive - competes for same site, non-competitive - binds allosteric site to prevent activation.

Reversible - binds non-covalently, can be washed out, non-reversible - binds covalently so cannot be displaced.

Question 13

Reversible competitive antagonism?

Answer 13

Curve shifts to right
Slope unchanged
Emax restored by increasing agonist concentration

Question 14

Irrerversible competitive antagonism?

Answer 14

Antagonist dissociates very slowly (if at all)

No change in antagonist occupancy when agonist applied (Emax not reached)

Question 15

Inverse agonism and two-state receptor model?

Answer 15

Agonists have a higher affinty for R* than R so shift equilibrium towards R* - greater efficacy of agonist.
Inverse agonists have higher affinity for R than R* so shifts equilibrium to left.

Question 16

Four types of receptor?

Answer 16

Ligand gated ion channels, kinase linked receptors, nuclear receptors and GPCRs

Question 17

Ligand gated ion channels?

Answer 17

Rapid synpatic signalling between electrically excitable cells. Binding causes conformational changes in channel protein so ons flow to alter membrane potential.

Question 18

Example of ligand gated ion channel?

Answer 18

Nicotininc acetylcholine receptor (nAchR)

Pentameric - 2 alpha, 1 beta, 1 gamma and 1 delta subunits - membrane spanning.
2 Ach binding sites, both need to be activated to allow Na+/K+ permeability.
5 subunits cluster around central pore - contain negative amino acids making the pore cation selective.
When Ach binds the kinked alpha-helices straighten or swing out of the way, opening the channel pore.

UNWIN 1993 - reported 3D structure.

Question 19

Kinase linked receptors?

Answer 19

A large and heterogeneous group.

Similar structure to GPCRs but have different transduction mechanisms - enzymic nature of cellular domain.

Question 20

Examples of catalytic kinase linked receptors?

Answer 20

(Receptor is enzyme)
Tyrosine kinase receptors – insulin and some GFs
Serine/threonin kinase receptors - TGF-B
Guanylate cyclase (cGMP) linked receptors - ANF

Question 21

Examples of non-catalytic receptor ligands?

Answer 21

(acts through cytoplasmic tyrosine kinases)
Cytokines - interleukin, interferon
Growth hormone, prolactin

Question 22

What happens to kinase linked receptors following ligand binding?

Answer 22

Dimerization, activation. Role in activating gene trancscription (hours) - cell division, growth, differentiation, tissue repair, apoptosis, immune..

Auto phosphorylation of intracellular domain of receptor. Phosphorylated tyrosine kinase residues act as high affinity docking sites for SH-2 domain proteins.

Question 23

Examples of kinase linked receptor trandsuction pathways?

Answer 23

Ras/Raf/MAP kinase - cell division, growth, differentiation
Cytokine (JAK/STAT) pathway - synthesis and release of inflammatory mediators

GF/cytokine binds SH-2 domain protein (ENZYMES - MAPK, or PHOSPHOLIPASES - PI3K). Activate other functional proteins –> modulate gene expression.

JAK-STAT - Dimerisation of cytokine receptors attracts a cytosolic TK unit (Jak family) to phosphorylate the receptor dimer. Targets for phosphorylation by Jak are a family of transcription factors (Stats). These are SH-2 domain proteins that bind to phosphotyrosine groups on receptor-Jak complex –> get phosphorylated. ACTIVATED –> stat dimers migrate to nucleus and activate gene expression.

Question 24

What are nuclear receptors?

Answer 24

Ligand activated transcription factors.
Sense lipid and hormonal signals (small hydrophobic molecules) which cross the plasma membrane, interact with NRs and regulate transcription of certain genes (hours).

Question 25

Class I nuclear receptors?

Answer 25

Upon binding, H-R complex translocates to nucleus, forms homodimers and bind to regions of DNA (hormone responsive elements - HREs) - located upstream of promoter region of target genes. Increase or decrease gene transcription.

Examples - glucocortocoid/mineralocorocoid receptors. Oestrogen, progesterone and androgen receptors.

Question 26

Class II nuclear receptors?

Answer 26

Present in nucleus - form homodimers with retinoid receptor (RXR). Dimers can only be activated by RXR ligand (non-permissive homodimer) or retinoic acid itself or by it's partner's ligand (permissive homodimer).
Once activated, behaves same as class I (HREs etc.)

Examples - peroxisome proliferator-activated receptor (PPAR), TH receptor and vitamin D receptor.

Question 27

GPCRs?

Answer 27

Integral membrane protein receptors - single polypeptide with 7 membrane spanning alpha-helical regions connected with alternating extracellular and intracellular loops (NH2 terminal on extracellular, COOH terminus in cytosol)

Question 28

Where does ligand binding and G protein binding happen on GPCRs?

Answer 28

Ligand - extracellular loop/transmembrane domains or both

GP - intracellular loop 3 or COOH-tail region.

Question 29

Targets and examples of GCPRs?

Answer 29

Targets - peptide hormones, neurotransmitters

Examples - muscarinic Ach receptor, adrenoreceptor, ang II receptor

Question 30

G proteins?

Answer 30

Guanosin nucleotid-binding proteins. Ga subunit contains GTPase activity - this is what differs in heterometic G proteins. Share common B and Y subunits (signalling). Anchored to membrane via FA chains - prenylation.

Question 31

Functions of G protein?

Answer 31

• Molecular switch - active when bound to GTP, inactive with GDP. Binding of ligand –> conformational change –> GP binds to receptor. GP a-subunit exchanges GDP for GTP. Ga-GTP dissociates –> interacts with target effector protein (enzyme usually).
• Upon this interaction effector protein activity is modulated –> modulates levels of 2nd messenger (cAMP, cGMP, DAG, IP3)
• Gby may also activate target 2 (ion channel or MAPK cascade). GTPase activity of Ga increases when target protein bound –> hydrolysis of GTP to GDP –> off –> reunites with subunits.

Question 32

Examples of G proteins?

Answer 32

Gas - stimulates adenylate cyclase –> increase in cAMP –> activates PKA –> phosphorylation of target proteins EG B2 adrenoreceptor activation

Gai - inhibits adenylate cyclase –> decrease in cAMP –> decreased PKA activity –> decreased phosphorylation. EG M2 Ach receptor activation.

TURNED OFF by breakdown of cAMP by PDEs to form AMP. Some are AMP selective (inhibited by milirone and rolipram). Some are cGMP selective (inhibted by sildenafil - viagra).

Question 33

How does Gaq work?

Answer 33

Stimulates PLC –> cleaves a membrane phospholipid (PIP2) –> products of enzyme cleavage are DAG and IP3 (2nd messengers)

IP3 - releases Ca2+ from intracellular stores. Affects cellular response.

DAG - directly affects activity of membrane bound PKC. PKC controls phosphorylation of Ser and Thr residues on proteins –> hormone release, change in neuroT release, inflammatory response, tumour promotion, ion transport.

Status quo is resumed by DAG being phosphorylated to form PA, while IP3 is dephosphorylated and recoupled with PA to form PIP2 again. LITHIUM BLOCKS THIS RECYCLING PATHWAY.

Question 34

What are the other three targets for drug action?

Answer 34

Enzymes, transporters, voltage gated ion channels

Question 35

How are enzymes used as drug targets?

Answer 35

• Most competitively inhibit
  Reversible - ACEi on ACE, neostigmine on acetylcholinesterase
  Irreversible - Aspirin on COX
• False substrates that undergo chemical transformation to abnormal products that subverts normal metabolic pathway (FLOURACIL)
• Prodrugs (ENALAPRIL)

Question 36

How are transporters used as drug targets?

Answer 36

Movement of ons and polar molecules across membranes. Many utilise ATP, others are coupled to the transport of ions and rely on an electrochemical gradient.

EXAMPLES
Hemicholinium - choline transporter in nerve terminal.
Omeprazole - PPI

Question 37

How are voltage gated ion channels used as drug targets?

Answer 37

Open when membrane is depolarized and underlie mechanism of membrane excitability.

• Some drugs block the ion channel - LOCAL ANAESTHETICS, TETRODOTOXIN - Na+ channels
• Some modulate the gating (GPCRs can do this by phosphorylation of PKA and B) - BENZODIAZEPINES on GABA gated Cl- channels, DIHYDROPYRIDINES on Ca2+ channels
• SULFONYLUREA drugs block ATP sensitive K+ channels by binding to associated sulfonylurea receptor (SUR), reducing K+ permeability, causing B cell depolarisation, Ca2+ entry and insulin secretion.