Drugs targets 1: GPCRs and NHRs Flashcards
What do most drugs target?
Proteins
What drugs don’t target proteins?
- 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
What extracellular receptors do you usually find?
enzymes
What receptors do you find in the membrane?
- Voltage gated ion channels
- Tyrosine kinase receptor (e.g. insulin receptor)
- Transporter
- Ligand gated ion channel
- G-protein coupled receptor
What intracellular drug receptors do you usually find?
- nuclear hormone receptors
- enzymes
What is the general molecular weights of drugs and receptors?
- drugs: 100s of Daltons
- receptors: 100s of kDa
What part of the receptor does a drug interact with?
The binding domain
What in the binding domain determines whether a drug will be able to bind or not?
There will be a distinct arrangement of amino acids in the binding domain – the exact interaction of them determines whether a drug will be able to bind or not
What happens when a drug binds to a protein target?
- Drug makes specific connections (bonds) with binding domain
- Binding energy of drug -> conformational effect (changes the shape of the rest of the protein)
Give features of the glucocorticoid receptor
- Member of nuclear hormone receptor nuclear family
- Binds steroid hormone
- Once it has bound a steroid hormone it goes into nucleus and switches genes on and off – regulates protein transcription
- Works as a dimer – two identical copies of the same protein
- The drug which is bound to the protein is called dexamethasone – makes contact will a small part of the protein
What are protein super-families?
a group of more distantly related proteins that share structural and functional features – probably evolved from a common ancestor. They combine may families
How do we compare proteins?
on the basis of how similar their amino acid sequences are
What are protein families?
- proteins are very closely related
- classified on similarities between amino acids - may even bind the same drug
How do protein superfamilies arise?
By gene duplication and mutation from a common ancestor
How many different types of sodium channels are there and what are the differences between them?
- 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
What are the advantages of having different sub-types of receptors?
- 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.
What is the DRG?
dorsal route ganglia. Collection of cell bodies from sensory neurones located just outside the spinal cord. The neurones relay pain information from the periphery to the CNS
What are corticosteroids produced by?
By the adrenal cortex
What is the problem with the binding of hydrocortisone and corticosterone and how can we overcome it?
- 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
What is a receptor?
- Binds an information-carrying molecule (agonist)
Neurotransmitter
Hormone - ‘passes on the information’ in a different form
Transduction
What is it called when a receptor passes on information in a different form?
transduction
What are the three distinct classes of membrane receptors?
- receptor tyrosine kinase
- G protein-couples receptors
- ligand-gated ion channel
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.
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
Give an example of a receptor tyrosine kinase
insulin receptor
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
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)
Give an example of a GPCR
B2-adrenoceptor
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
Give features of ligand-gated ion channels
- multi-subunit transmembrane proteins
- all have at least two agonist sites
- Ion channel is part of receptor
Activation opens channel
Changes cell membrane potential
Can trigger action potential - can allow fast singalling
- lots of diversity in each family because they have multi-subunits
Give an example of a ligand-gated ion channel receptor and how many subunits it has
- nicotinic acetylcholine receptor
- 17 subunits
What happens with the ion channel in the ligand-gated ion channel receptor?
Activation opens channel
Changes cell membrane potential
Can trigger action potential
Where are nuclear hormone receptors found?
in the cytoplasm or nucleus of a cell
What do nuclear hormone receptors do?
Job is to bind lipid-soluble molecules which have crossed the membrane
Once it has binded 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.
Give features of nuclear hormone receptors
- 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
Give an example of a nuclear hormone receptor
glucocorticoid receptor
Is the nuclear hormone receptor part of a family or superfamily?
Superfamily
What does an agonist do?
bind to a site on the receptor and activate it
What do antagonists do?
Competitive antagonists bind to the agonist site and block activation
What is a ligand?
Any class of drug, hormone or neurotransmitter that binds to a receptor
What are allosteric modulators?
Bind to a different site to the natural agonist and alter receptor behaviour
How many GPCR sequences are there in the human genome
Over 800
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
What is the structure of GPCRs?
- 7 transmembrane domains (alpha helices)
- Agonist binding site is tucked down in the membrane
- The G-protein binding domain is on the inside face of receptor
Do GPCRs have diversity?
Yes they have multiple subtypes
What type of protein in the G-protein?
- trimeric protein
- it has alpha, beta and gamma subunits
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.
Which subunit of the G-protein has GDP bound to it?
The alpha subunit
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
What is the GPCR activation cycle?
- Agonist binds to receptor changing receptor conformation and allowing it to interact with the G protein.
- Receptor interacting with G protein changes the structure of the G protein and allows the alpha subunit to release it’s GDP and bind instead a GTP molecule (replacement, not phosphorylation)
- Alpha subunit separates from beta and gamma subunit, travels through the membrane and interact with enzymes or other effector proteins. The effector proteins generate the second message (beta and gamma can also signal in the membrane)
- To stop signalling alpha subunit has built in GPAse activity, which breaks down GTP to GDP and phosphate, means the alpha subunit can reassociate with alpha and gamma and is back to the start of the cycle
- To complete the cycle the agonist also must dissociate from the receptor
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
- incorporates lipid tails which come off the protein components of the subunits- Has a C terminus and N terminus ends of the tail on each G protein receptor subunit
- TM domain near the N terminus end of the tail
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)
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)
What are the different subunits in the Gq family and what do they do?
Gq – aq – activation of phospholipase C
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
What does the lipid tail of the G protein do?
anchors subunits in the membrane
What is adenylate cyclase a key component of?
The cAMP pathway: Gs protein
What happens in GPCR signalling with adenylyl cyclase?
- agonist binds to Gs GPCR
• The alpha subunit diffuses through the membrane and activates the enzyme adenylyl cyclase which turns ATP into cyclic AMP (cAMP)
• cAMP has a direct effect on CNG and HCN channels
• cAMP has a main effect through protein kinase A (PkA)
• Protein kinase A has two catalytic subunits with regulatory subunits
• When two cAMP molecules bind to each of the regulatory subunits the catalytic sites are freed and PkA becomes activated
• Protein kinase phosphorylates other proteins which can either switch them on or off. There are a lot of targets for PkA and it depends on the cell type. E.g. in adipocytes actions of PkA promotes lipolysis, in smooth muscle PkA effects can cause relaxation
What receptors are coupled to protein Gs?
- B-adrenoceptors
- Dopamine receptors D1 and D5
- Glucagon receptors
- Cannabinoid receptor CB2
- Histamine H2 receptor
- Luteinizing hormone receptor
- Follicle stimulating hormone receptor
What happens when Gi acts on the adenylyl cyclase pathway?
- GTP replaces GDP
- However alpha i inhibits adenylyl cyclase which inhibits the production of cAMP
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
What happens in the GPCR signalling phospholipase C (IP3) pathway?
- G protein interacts with Gq which has an alpha q subunit
- GTP -> GDP
- Alpha subunit will be released
- Alpha q activates phospholipase C (PLC)
- PLC cleaves phospholipids
- PLC cleaves PIP2 to release diacylglycerol and IP3
- IP3 is the phospholipid head group from the phospholipid
- Diacylglycerol (DAG) is a lipid and stays in the membrane
- IP3 is water soluble so diffuses into the cytoplasm and makes it’s way to intracellular calcium stores
- On the calcium stores there is a receptor for IP3 – ligand gated ion channel. Binds 4 molecules of IP3 and ion channel opens to let out calcium
- When protein kinase C (PKC) has bound diacylglycerol or calcium it becomes activated and phosphorylates proteins
What receptors are coupled to Gq?
- Muscarinic acetylcholine receptors M1, M3 and M5
- Histamine H1, receptor
- Angiotensin II type 1 receptor
- A1 adrenoceptors
How many nuclear hormone receptors (NHRs) are there in man?
48
Give examples of NHRs
- Progesterone, oestrogen, androgen receptors
- Thyroid hormone receptor
- Glucocorticoid, mineralocorticoid receptors
What are NHRs connected to?
heat shock protein which helps keep it stable in cytoplasm
What is the NHR mechanism?
- Lipid soluble agonist crosses membrane
- Interacts with nuclear hormone receptor in cytoplasm
- When agonist binds the heat shock proteins leave and the receptor dimerises (two monomers come together)
- dimer NHR enters the nucleus
Nuclear hormone receptors are a superfamily of receptors for lipophilic substances. Give some examples of these
- Steroid hormones
Corticosteroids (adrenal steroids)
Sex hormones
What is the NHR structure from left to right?
N-terminal domain, DNA binding domain, hinge region, agonist binding domain, c-terminal domain
What is the NHR transactivation mechanism?
- Nuclear hormone receptor dimer interacts with transcription factors then binds to a specific recognition sequence in the DNA
- The gene next to this will then be transcribed at an increased rate
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
Do most NHRs have transactivating or transrepressing effects or both?
Both
