Drug targets: GPCRs and NHRs Flashcards
1
Q
What are the 7 types of drug targets?
transmembrane receptors
A
- Nuclear Hormone receptor (NHR)
- Enzyme
- voltage-gated ion channels
- tyrosine kinase receptor
- transporter (transport proteins)
- ligand-gated ion channel
- G- Protein-coupled receptor (GPCR)
2
Q
How do transport proteins function?
A
By binding their substrate on one side of the membrane and then changing conformation to release the substrate on the opposite side
3
Q
What happens when a drug binds to a protein target?
A
- Drug makes specific connections (bonds) with binding domain
- Binding energy of drug → conformational effect (changes the shape of the rest of the protein)
4
Q
What is a Glucocorticoid receptor?
A
- Member of nuclear hormone receptor nuclear family
- Binds steroid hormones
- It then goes into the nucleus and controls protein transcription
- It works as a dimer - two identical copies of the same protein
- Dexamethasone binds with this protein.
5
Q
Give an example of a secondary active transport protein
A
either
Na+, H+ exchange
or
Na+ and Glucose co transport
6
Q
what is meant by ion channels being selectively permeable?
A
they select between various ions on the basis of size and charge
7
Q
How do ion channels select on the basis of charge?
A
Around the mouths of some ion channels, there are amino acid rings with the opposite charge to the ion it’s selective for, therefore will only allow the correctly charged ions in (will repel those with the same charge)
8
Q
what is ion channel opening controlled by?
A
- mechanics
- 2nd messenger
- phosphorylation
- leak
- ligand-gated
- voltage-gated
- proton- gated
- temperature- gated
9
Q
How many subunits does the Muscle nicotinic receptor have?
A
5
2 alphas, 1 beta, 1 delta and 1 epsilon
10
Q
Where are the acetylcholine binding sites in the muscle nicotinic receptor?
A
between the alphas and their neighbouring delta and epsilon subunits
11
Q
how many transmembrane domains does each muscle nicotinic receptor have?
A
4
12
Q
what are the 3 main classes of voltage-gated ion channels?
A
calcium, sodium and potassium
13
Q
What drugs don’t target proteins?
A
- Antacids – used to reduce stomach acid
- osmotic diuretics (reduce intracranial pressure)
- DNA modifying drugs (cancer therapy)
- Drugs that target membrane lipids (some antibiotics)
- Interactions tend to be non-saturable (lots of binding sites), with little specificity
14
Q
Molecular weights of drugs and receptors
A
- drugs: 100s of Daltons
- receptors: 100s of kDa
15
Q
What are the properties of the Nuclear Hormone Receptor family?
A
- All have similar structure and function
- All have same basic mechanism
○ Bind lipophilic agonist and regulate transcription of DNA = change in protein expression
16
Q
How many different types of sodium channels are there and what are the differences between them?
A
- there are 9 different voltage gated sodium channels in the human genomes
- differences between where the different subtypes are expressed
- If it has tissue specific distributions of subunits those subunits can become specialised in the tissue in which it is expressed
- the amino acid differences give the sodium channels their specialisation
17
Q
What are the advantages of having different sub-types of receptors?
Tissue specialisation
A
- if they mutate they will only cause an adverse effect in the tissue it is specialised in
- If you have a mutation in a subtype of a receptor/ channel and there is a closely related subtype which isn’t usually expressed in the tissue it is possible for the tissue to start using a different gene from which they usually do.
18
Q
What is the problem with the binding of hydrocortisone and corticosterone and how can we overcome it?
A
- they are both supposed to bind to glucocorticoid receptors but because glucocorticoid and mineralocorticoid receptors are so similar they sometimes bind to mineralocorticoid receptors. If high enough doses are given then you can overcome the enzyme protection of the mineralocorticoid receptor (that stops it becoming activated) and get mineralocorticoid side effects you don’t want.
- to overcome this you can use synthetic steroid which can select for the GR vs MR, reducing side effects
19
Q
In the context of a receptor, what is “transduction”?
A
A receptor is a protein that interacts with an information carrying stimulus and passes that information on the different form. The act of passing the information on this turned transducing the signal.
20
Q
How many transmembrane domains are there in the G protein-coupled receptor?
A
7
The N terminus is outside the cell and the C terminus inside (which means they must cross the membrane at number of times)
21
Q
What is a protein superfamily?
A
A superfamily is a group of proteins that are structurally and functionally similar. We think super families arise by duplication of ancestral genes and subsequent mutation of the redundant copies.
Not all proteins that function in the same way all members of the same super family – convergent evolution has produced several different ligand-gated ion channels superfamilies
22
Q
Define agonist
A
bind to a site on a receptor and activate it
23
Q
Define antagonist
A
competitive antagonists bind to agonist site and stop activation
24
Q
Define ligand
A
any class of drug, hormone or neuroreceptor that binds to a receptor
25
Define Allosteric Modulator
bind to a different sit to the natural agonist and alter receptor behaviour
26
What are the four main receptor types
- Receptor Tyrosine kinase
- G protein- coupled receptor
- ligand-gated ion channel
- nuclear hormone receptor
27
Define the transduction
When a receptor passes on information in a different form
28
What do tyrosine kinase receptors do?
- Extracellular facing agonist domain – bind outside the cell
- Inside the cell they have an enzyme activity – can add phosphate groups to proteins.
29
Give features of receptor tyrosine kinase
- 58 transmembrane proteins
- Bind peptide hormones, growth factors and cytokines
- Act as dimers
- Recognize specific sequences in target proteins
- Phosphorylate target protein tyronises changes behavior of target proteins = changes behavior of the cell
30
Give an example of a receptor tyrosine kinase
insulin receptor
31
What does the G protein-couples receptor do?
- Extracellular agonist binding domain
- On the inside they have a specific recognition sequence for an accessory protein – the G protein.
- When an agonist binds the receptor changes shape in a way which enables ATP to replace ADP on the g-protein which activates the G protein. The G proteins move across the membrane and interact with target proteins in the membrane and change their behaviour
32
Give features of the G protein-coupled receptor (GPCR)
- biggest receptor family (821 human genes)
- 7 transmembrane proteins
- important in nervous system, vision and olfaction
- act via accessory proteins (G proteins) - trimeric proteins
33
Give an example of a GPCR
β2-adrenoceptor
34
What do ligand-gated ion channels do?
- They have an extracellular binding site for the agonist
- When the agonist binds the built-in ion channel opens and ions can cross the membrane
- Can allow fast signaling e.g. nerve to muscle
35
Give features of ligand-gated ion channels.
- multi-subunit transmembrane proteins
- all have at least two agonist sites
- Ion channel is part of receptor
- lots of diversity in each family because they have multi-subunits
36
What are the structurally different types of the ligand-gated ion channels?
○ Cys-loop receptors
○ iGlutamate receptors
○ P2X receptors
37
Give an example of a ligand-gated ion channel receptor and how many subunits it has.
- nicotinic acetylcholine receptor
| - 17 subunits
38
What happens with the ion channel in the ligand-gated ion channel receptor?
○ Activation opens channel
○ Changes cell membrane potential
○ Can trigger action potential
39
Where are nuclear hormone receptors found?
in the cytoplasm or nucleus of a cell - Intracellular location
40
What do nuclear hormone receptors do?
- Job is to bind lipid-soluble molecules which have crossed the membrane
- Once it has bound to an agonist in binds to sequences in DNA it changes the transcription of genes that neighbour the sequences (either increases or decreases) and alters protein expression in the cell.
41
Give features of nuclear hormone receptors
- part of a superfamily
- intracellular location
- binds lipid soluble ligands such as steroid (because the ligands have to get through the lipid bilayer to get to the receptor)
- bind to DNA when activated
- effects tend to be slower than other receptor types
42
Give an example of a nuclear hormone receptor
glucocorticoid receptor
43
How many GPCR sequences are there in the human genome?
over 800
44
Give examples of common drugs acting via GPCR
- Antidepressants
- Antipsychotics: dopamine D2 receptor
- Anti-asthma: salbutamol (beta 2 AdR)
- Blood pressure: losartan (Coxaar), atenolol
- Glaucoma: pilocarpine (muscarinic receptors)
- Abuse: cannabis, heroin, LSD
45
What is the structure of GPCRs?
- all have same 7 transmembrane domains (alpha helices)
- NH2 terminus on outside of cell
- Agonist binding site is tucked down in the membrane
- The G-protein binding domain is on the inside face of receptor
46
Describe the diversity of GCPRs.
- GCPRs have multiple subtypes of diversity
47
What type of protein in the G-protein?
- trimeric protein
| - it has alpha, beta and gamma subunits
48
Give a quick summary of how G-protein coupled receptors work
Our agonist interacts with G-protein coupled receptor. Activated protein interacts with G-protein. G-protein subunits will split – alpha subunits and beta and gamma subunits move off on their own. These components of the G protein then activate the target protein/ effector (e.g. enzyme or ion gated channel), then this will enable the (intracellular) second messenger to change the behaviour of the cell.
49
Describe the amplification process of the G protein
1 agonist activates 1 GCPR → activated GCPR interacts with multiple G-proteins → target proteins are switched on → many molecules of send messengers are created. = cascade
- Cyclic System
50
Which subunit of the G-protein has GDP bound to it?
The alpha subunit
51
Why can the activation of one GPCR lead to many secondary messengers?
Once a GPCR has been activated in can interact with multiple G proteins which can all act on many different effector/target proteins
52
What is the GPCR activation cycle?
1) G-protein has GDP bound to it.
2) Agonist binds to GPCR. This allows the G-protein to interact with the GPCR = the G-protein’s alpha subunit bound GDP is replaced by GTP
3) The binding of GTP triggers the G-protein to split into two parts (structure changes)
a) the alpha subunit with the GTP attached
b) the beta and gamma subunits. Originally it was thought that only the alpha subunit was active as a regulator of target proteins, but now it is clear that alpha-GTP and the Beta-gamma parts are active.
- The effector proteins generate the second message (beta and gamma can also signal in the membrane)
4) The alpha subunit has a built in GTPase activity and eventually will hydrolyse the GTP to GDP + Pi. = terminates the signalling process.
5. To complete the cycle the agonist also must dissociate from the receptor
53
What is the G protein structure?
- Heterotrimeric G protein
- Different types of alpha subunits
- Four families of G proteins: Gi, Gs, Gq, G12/13
- Main difference are alpha subunits present
- beta-gamma subunit complex
- three subunits tightly pressed against inner face of membrane
54
Name the 4 components of the G protein signalling process.
1. agonist ligand
2. GPCR (membrane bound)
3. G-protein (guanine nucleotide binding protein). This is a trimeric (3 subunit) membrane protein that binds GDP and GTP.
4. a protein (usually an enzyme) that generates the second message. The activity of this protein is regulated by the G-protein.
55
What are the different subunits in the Gi family and what do they do?
- Gi/0L ai, a0 – inhibits adenylyl cyclase (AC)
- Gt at (transducing) – activation of PDE-6 (vision)
- Gg agust (gustducin) activation of PDE-6 (taste)
56
What are the different subunits in the Gs family and what do they do?
- Gs – as – activation of AC
| - Golf – aolf – activation of AC (olfaction)
57
What are the different subunits in the Gq family and what do they do?
Gq – aq – activation of phospholipase C
58
What does the beta-gamma subunit complex do of the G protein?
- Modulates a wide range of ion channels, enzymes
- Activates: cardiac potassium channels, B-adrenoceptor kinase
- Inhibits: N, P and Q type calcium channels
59
What does the beta-gamma subunit complex do of the G protein?
anchors subunits in the membrane
60
What is adenylate cyclase a key component of?
The cAMP pathway: Gs protein
61
Describe what happens in GPCR signaling with adenylyl cyclase?
cAMP pathway: Gs
1. G-protein coupled receptor activated by agonist + interacts with Gs.
2. Interaction = conformational changes in G-protein. GTP replaces GDP.
3. αs-subunit separates from β & γ.
4. αs-subunit is anchored in the membrane and can diffuse through the membrane and interact with adenylyl cyclase. = activates enzyme
○ adenylyl cyclase turns ATP → cyclic AMP
5. cyclic AMP can have direct signalling effects e.g. modulates membrane channels.
6. Protein kinase A (tetrameric protein)
○ In its inactive form it consists of 2 catalytic subunits with regulatory subunits that block the catalytic site.
7. When 2 cAMP molecules bind to catalytic subunits the catalytic sites are freed → activated protein kinase A.
8. Protein kinase A phosphorylates other proteins (turns proteins on/off)
62
What are the Actions of Protein Kinase A?
- Promote lipolysis in fat cells
- Mobilises glucose in skeletal muscle and hepatocytes, by the break down of glycogen.
- Relaxation in smooth muscle
63
Name the receptors coupled to Gs
- β-adrenoceptors
- Dopamine receptors D1 and D5
- Glucagon receptor
- Cannabinoid receptor CB2
- Histamine H2 receptor
- Luteinizing hormone receptor
- Follicle stimulating hormone receptor
64
What happens when Gi acts on the adenylyl cyclase pathway?
| cAMP pathway: Gi
1. Activated G-protein interacts with Gi.
2. Replacement of GDP with GTP.
3. G-protein dissociates (αi-subunit separates from β & γ)
4. αi inhibits adenylyl cyclase = reduce cAMP in cell = opposite effects to Gs pathway = less activation of protein kinase A
65
Which receptors are coupled to Gi?
- Muscarinic acetylcholine receptors M2 and M4
- Cannabinoid receptors CB1 and CB2
- Dopamine D2, D3 and D4
- As adrenoceptors
- GABAB receptors
66
What happens in the GPCR signaling phospholipase C (IP3) pathway?
cAMP pathway: Gq
1. G-protein activated by agonist and interacts with Gs.
2. Replacement of GDP with GTP.
3. G-protein dissociates (αq-subunit separates from β & γ)
4. αq-subunit interacts with phospholipase C enzyme
5. PLC cleaves membrane lipids specifically phosphatidylinositol 4-5-bis (PIP2) → IP3 (phospho-sugar head group)+ DAG (lipid + signalling molecule)
6. IP3 is water soluble → diffuse through cytoplasm to intracellular calcium stores (e.g. endoplasmic reticulum & sarcoplasmic reticulum)
7. IP3 receptor (ligand-gated calcium channel) on calcium store binds 4 IP3 molecules → ion channel opens → calcium leaves
○ Calcium is a powerful intracellular signalling entity. Acts via calmodulin to modulate the function of proteins. (involved in smooth muscle contraction)
8. Protein Kinase C (PKC) is activated when calcium &/or DAG (diacylglycerol) bind.
9. PKC phosphorylates other proteins (turns proteins on/off)
67
Which receptors are coupled to Gq?
- Muscarinic acetylcholine receptors M1, M3 and M5
- Histamine H1 receptor
- Angiotensin II type 1 receptor
- α1 adrenoceptors
68
How many nuclear hormone receptors (NHRs) are there in man?
48
69
Give examples of NHRs
○ Progesterone, oestrogen, androgen receptors
○ Thyroid hormone receptor
○ Glucocorticoid, mineralocorticoid receptors
○ Vitamin D receptor
○ Retinoic acid receptors
70
What are NHRs connected to?
heat shock protein which helps keep it stable in the cytoplasm
71
Nuclear hormone receptors are a superfamily of receptors for lipophilic substances. Give some examples of these
Steroid hormones
- Corticosteroids (adrenal steroids)
- Sex hormones
72
Describe the basic mechanisms of NHRs.
- When activated, bind to DNA
○ Increase transcription of specific genes
○ Decrease transcription of specific genes
- Effects (protein transcription) tend to be slower than other receptor types
73
What is the NHR structure from left to right?
- At the N-terminal end of the protein is a loosely folded region.
- This is followed by the DNA binding domain (DBD)
- hinge region
- the ligand binding domain (LBD
- C terminal domain tail.
74
What is the NHR transactivation mechanism?
1. After translocation to the nucleus, the NHR dimer interacts with transcription factors → binds to a section of DNA (hormone responsive element (HRE))
2. RNA leaves nucleus → translated to protein in cytoplasm
3. It then recruits coactivator proteins and RNA polymerase & promotes transcription of mRNA = expression of proteins.
4. The gene next to this will then be transcribed at an increased rate
75
Describe the basic mechanism of NHR?
1. Steroid hormone outside the target cell is lipid soluble so easily slip through the membrane
2. Interacts with glucocorticoid receptors (GCR) which are normally bound to Heat Shock proteins (HSP)
3. This interaction with the steroid causes the HSPs to dissociate.
4. Receptors dimerize and enter the nucleus via a nuclear pore.
5. Monomeric receptors can also enter the nucleus.
76
Define Transactivation
Increasing transcription of a gene = increase protein levels.
77
What is the NHR transrepression mechanism?
- NHR monomer interacts with transcription factors and forms a dead end complex on DNA blocking the transcription
- Leads to reduced expression of gene
78
Define Transrepression
Decreasing transcription of a gene = decrease protein levels.
79
Do most NHRs have transactivating or transrepressing effects or both?
Both
80
Define Endogenous Ligand:
a natural ligand that binds to the receptor as part of a signalling process
