Molecular pharmacology Flashcards
Learning objectives
- Introduce the idea of receptor families.
- Describe how gene cloning and most recently genome sequencing have expanded view of receptor families.
- Describe some of the better characterized receptor families and list aspects of their structure function activity.
- Use GPCR adrenergic receptor and nicotinic acetylcholine receptors to detail how current and emerging structures inform on protein conformations that allow receptor function.
- Describe principles of tyrosine kinases and steroid receptors.
- Introduce the key principles Pharmacogenomics.
- Use examples of how genetic determinants effect drugs by changing their Pharmacokinetics and Pharmacodynamics.
When listing the major receptor families, how can you group them by?
- Molecular characteristics
- Major drugs-indicating affinities, efficacies, and relative specificities
- Good for revising and drug related write ups
What are the two classifications of receptor families?
- Anatomical classification
- Pharmacological classification
Are adrenaline and noradrenaline shown to have a wide range of effects?
yes
Whats are the anatomical classifications of drugs?
Different vessels gave distinct response constrict or dilate
What are some pharmacological classifications of drugs?
- Ahlquist noted a rank order of potency for adrenergic agonists depending on the nature of the response.
- Constricting responses: Noradrenalin>adrenalin>isoprenaline
- *ALPHA RECEPTOR**
- Dilating responses: Isoprenaline>adrenalin> noradrenalin
BETA RECEPTOR
Differential pharmacology (structure function) and receptor associated signalling
- Known to activate the alpha- and beta-adrenergic beta responses
- Build up better idea of underlying receptor
- means no response on the drug
- Shows to distinct alpha and beta receptors
- Propranolol blocks all beta responses
- Pindolol only blocks the beta 1 responses
Tell me about molecular classification and receptor family expansion through gene cloning
- Identify and sequence the cDNA for the receptor.
- Predicts the amino acid sequence of the receptor.
- Repeat for all receptor sub-types compare amino acid to give a molecular classification.
- Often leads to identification of distinct sub-types not realized by pharmacological classification.
Methods that reveal molecular basis of receptor sub-types
Tell me about molecular methods that open up diversity in receptor families
- Hybridise under high temperatures as they are identical much like DNA
- Homologous indicate different but related proteins. Would hybridise under lower temperatures
Molecular methods that open up diversity in receptor families
What is genomic DNA made up of and what are each of these components?
- Genomic DNA is made of DNA sequence
- DNA sequence is the introns and exons
- Exons are part of genomic DNA that go onto make mRNA that encodes the protein
- Exons come together to make up coding sequence
- Exons interrupted by introns, would remove and splice introns in order to make proteins
How can a computer be used for genomic sequencing?
- Computer can remove introns to get exons. Predict by splicing on computer
- Predicted mRNA could look similar would be extinct from those already existing
What does homology mean?
the state of having the same or similar relation, relative position or structure
What is cDNA?
cDNA meaning complementary DNA (synthetic DNA in which the sequence of bases is complementary to that of DNA)
How do you build post-genomic receptor families?
How much of the human genome encodes the G-protein coupled receptors?
3-5% of genes
estimate 30,000 genes in the genome >1000 G-protein receptors
Tell me about the families in the G-protein receptor super family?
There are 5 families in the G-protein super family, they have 2 different names:
Family 1= rhodopsin family (different names for same thing)
Family 2= secretin family
Family 3= Glutamate family
Family 4= Frizzled/ TAS2 family
Family 5= adhesion family
Using family 1 of the G-protein super family as an example, tell me the sub-types of the superfamily
- Sub types of the super family using family 1 as an example
- But a subclassification of the superfamily and all the layers
- Alpha 1, alpha 2, beta 1, beta 2, beta 3 (what the starts of the sub-class represent)
Tell me the different effects that adrenaline/ noradrenaline can have an on what receptor they have to act on for this affect
Tell me about what drugs act on receptors and in what tissues for certain clinical uses…
Tell me about receptor signalling in a cDNA and a mutated cDNA
Whats some useful information for structure function studies?
Tell me about structure function studies?
- These are used to help understand receptor transduction
- 4 ways in which the receptor can be stimulated and the way it can come in via 4 different channels e.g. GPCR, ligand gated ion channels, tyrosine kinases, steroid receptors
What do the G-proteins that associate with GPCRs have?
They are heterotrimeric meaning they have 3 different subunits: alpha, beta and gamma
alpha and gamma are attached to the plasma membrane by lipid anchors
How does the alpha subunit exchange on G proteins exchange GDP for GTP?
- When GDP is bound to the alpha subunit, the alpha subunit remains bound to the beta-gamma subunit to form an inactive trimeric protein
- This conformational change causes the alpha subunit to give up its GDP in exchange for GTP
- Once bound to GTP, the alpha subunit dissociates from the beta-gamma complex.
Tell me the primary, secondary, tertiary and quaternary structure of the GPCR?
- Primary structure: Multifaceted family made up related sub-families (see previous lecture)
- Secondary structure: all receptors predicted to be seven transmembrane spanning
- Tertiary structure: Rhodopsin and many structures support the 7 transmembrane model.
- Quaternary structure: growing evidence that some GPCR may operate as dimers of individual subunits (e.g., Family 3).
Tell me about transduction of GPCRs?
- Bind agonist supplied from outside
- Alter receptor conformation
- Make contact to activate G-protein
Tell me about the secondary structure of an adrenergic GPCR?
Tell me about adrenergic agonist binding sites in transmembrane domains
Tell me about the important aspects of the agonist binding site and the conformational changes to fully activate?
- Agonist binds moves helices (5 and 6) creates a G-protein binding site.
- Poor activation of the receptor if G-protein does not bind.
- Agonist bind moves helices (5 and 6) G-protein binds stabilizes change in helices conformation.
- Suggests interdependence of the agonist, binding transmembrane conformation and
- G-protein binding.
Give an example of a ligand-gated ion channel
Nicotinic Acetyl Receptor super-family
Tell me about the primary structure of ligand gated ion channels
Primary structures
- A number of related genes encoding sub-units that make up distinct members of this family.
- Acetylcholine (Nicotinic) and 5-HT (5-HT3) excite allowing Na+ to flow in
- Glycine and GABA inhibit by allowing Cl- to flow in
Tell me about the secondary structure of the ligand-gated ion channels?
Secondary structure
Each subunit exhibit a common and distinct shared trans-membrane topology.
Tell me about the Tertiary structure of the ligand-gated ion channels?
Tertiary structure
good models of tertiary structure are now supported by structural information for whole receptor or domains of the receptor.
Tell me about the quaternary structure of the ligand gated ion channels
Quaternary structure
exist as oligomeric receptors made up of a mix and match of individual subunits. 5 subunits associate to generate a PENTAMERIC functional receptor
For fast and self-contained ligand gated ion channels whats the transduction mechanism?
- Bind agonist from the outside
- Conformational change or gate of ion channel
- Select specific ions to flux through ion channel
- pass through membrane.
- Operate on a millisecond time scale
Tell me the key features of the nicotinic acetylcholine ligand gated ion channel superfamily
What do protein alignments reveal and what does this mean?
- The protein alignments reveal ancient nicotinic receptor superfamily like proteins in bacteria
- This means it is easier to overexpress the protein and produce large amounts for structural studies
Where is the acetylcholine binding site?
Acetylcholine binding site is at the alpha subunit and on neighbouring non-alpha subunit
Tell me about the organisation and key features of the M2 ion channel domain?
- Twisting the hydrophobic residues in M2 helix could open the ion channel but would twist the aligned hydroxyl residues (so nice idea that probably wrong)!
- Amino acid alignment that is generally conserved in M2 of this receptor family. Charged residues (-2), hydroxyl residues 2 and 4 and hydrophobic residues (9 13 16) co-ordination, selectivity and flux.
What moves the M2 ion channel lining helix so it opens receptor ?
- Twisting the agonist binding site and levering the underlying ion channel moves the M2 ion channel lining helix so it opens receptor
- Rotation in an individual subunit co-ordinated across subunits of oligomeric protein increases the size of the ion channel thus it opens.
Tell me about the 1˚, 2˚, 3˚ and 4˚ structure of the tyrosine-kinase linked receptors
- Primary structure A number of different growth factor receptor gene families
- (e.g., NGF, cytokines and Insulin receptors same basic transduction pathway)
- Secondary structure Single transmembrane spanning or membrane associated domain.
- Tertiary structure. Isolated ligand binding domain or kinase domains confirm likely structures.
- Quaternary Clear dimerization required during signal transduction and some exist as oligomers before ligand
Tell me the transduction mechanism of the tyrosine-kinase linked receptors
- Activate by extracellular ligand
- Conformational change drives receptor dimerization
- Dimerization activates intrinsic tyrosine kinase or recruits
- non membrane associated kinase
- Creates site for phosphotyrosine or SH2 binding sites
Tell me about steroid receptors
- Cholesterol starting point for synthesis of mineralocorticoids (aldosterone), glucocorticoids (corticosterone) and sex steroids (e.g., oestrogens).
- Lipid soluble, diffuse into the cell and act on intracellular receptors.
Tell me the transduction mechanism of the steroid receptors
- Diffuse into cell and nucleus and bind Hsp-90 inactivated receptor.
- Dissociate HSP-90 and dimerize to give active form of the receptor.
- Activate transcription
