Principles of Pharmacology Flashcards
To Memorise Key Info From Block 1
What is a drug?
A drug is a chemical substsance or natural product that affects ther function of cells, organs, systems or the whole body (i.e. is bioactive).
What is Pharmacokinetics?
Pharamacokinetics - essentially what the body does to the drug. Typically a generic term to descrive the fate of a drug molecule following administration to a liviong organism or how a drug molecule is affected by exposure to living cells.
What is Pharmacodynamics?
Pharmacodynamics - Typically used as a generic term to describe the mechanism of drug action or or what happens to cells, organs, systems, etc., as a result of drug exposure.
How did Pharmascology develop anyway?
Well, I’ll tell ya fella!
It emerged as a scientific discipline in the late 19th century. Principles of experimentation rather than dogma. Herbal and other remedies had been used for millenia, pharmacopoeias written, apothecaries flourished!
…However there was little to no evidence tht these treatments were actually succesful.
Where do drugs come from?
Drugs come from:
- Natural products (e.g. plants and animals)
- Serediptity (by accident)
- Changing the structure of an existing molecule (structure-activity relationships)
- Using an existing drug in a new disease (re-purposing)
- Computer-aided design
- Studying disease processes
Give six examples of drugs we derive from plants?
Six examples of drugs derived from plants include:
- Willow Tree - Aspirin (painkiller)
- Cocoa Plant - Cocaine (local anaesthtic)
- Cinchona Tree - Quinine (anti-malarial)
- Poppy - Morphine (painkiller)
- Foxglove - Digoxine (heart failure treatment)
- Guggul Tree - Statins (cholesterol lowering)
Give two examples of drugs derived from animals
Two examples of drugs derived from animals include:
- Leech - Hirudin (anticoagulent)
- Cone Snail - Zinconotide (powerful painkiller)
Give an example of Seredipity in drug discovery
In 1928, alexander Fleming first noted the antibacterial properties of Penicillium mould.
In 1938, Howard Florey and Emst Chain isolated penicillin from the mould and tested it in human volunteers
100 litres of broth was required for the production of one day’s dose. So they re-extracted from the urine of the volunteers as the drug is not metabolised by the body
During WWII, the USA developed large scale production technology
Give an example of Drug re-purposing.
Sildenafil
Was discovered in 1989 by Pfizer pharmaceuticals, Sandwich, Kent.
They were looking for a drug thatlowers blood pressure (antihypertensive).
The clinical trials showed an unusal side effect (made PP hard).
ViagraTM sold around 10 million prescriptions in its first year.
Experimental vs. Therapeutic drug?
Experimental drugs are used in labs to explore biological processes or are in development for clinical use
Therapeutic drugs are those that are approved for the treatment of disease in (humans or animals).
What are the three types of drug names?
The three types of names for drugs are:
- Chemical - IUPAC name that describes the chemical sturcture of the drug. E.g. N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine.
- Generic - International non-proprietary name given to a molecule. E.g. Fluoxetine.
- Proprietary - ‘trade’ name(s) given to an approved drug by the manufacturer. E.g. Prozac.
- Drugs in development are also typically given a ‘code-name’ to disguise their identity.
- When referring to a drug always use the generic name and do not use capital letters.
Give 4 Examples of Drug Targets:
4 Drug Targets:
- Receptors
- Ion Channels
- Enzymes
- Transporters
- Drugs can also target circulating proteins like bacterial cell walls.
Explain Pharmacological variability
Pharmacological Variability
- Drug responses are not always the same.
- Responses vary between people (i.e. inter-individual variability)
- Responses can also vary within the same person (i.e. intra-individual variability)
What are the main causes of Pharmacological variability?
The main causes of Pharmacological Variability are:
- Age
- Genetics
- Disease state
- Drug Interactions
- Environment
Memorise this diagram of the absortion, metabolism and excretion of drugs.
List routes of drug penetration into cells.
- Diffusion through lipid membrane
- Major route for lipophilic drugs
- Diffusion through aqeous channels
- Most drugs too large!
- Carrier-mediated transport
- Major route for hydrophilic drugs
- Pinocytosis
- Transport of insulin into brain
What does the oral absorption of drugs require?
The oral absorbtion of drugs require permeation of epithelial cell membrane so the physiochemical properties of the drug are important such as the mw, pKa, log P (provides an indication on whethter the drug will be absorbed by the plant/animal) etc.
What is Lipinski’s rule of 5 (1997)?
Lipinski’s rule of 5 is based on observation that most orally administrated drugs are relatively small and moderately lipophilic molecules.
- A Mr less than 500 daltons.
- No more than 5 H-bond donors (total number of N-H and O-H bonds).
- no more than 10 H-bond acceptors (all nitrogen/oxygen atoms).
- an octanol-water partition coefficient log P not greater than 5.
Diagram of Phase I of Drug Metabolism.
In Phase I the drug is actually made slightly more reactive. Seemingly counterintuitive as metabolism is supposed to make the
Where does Phase I metabolism occur?
Phase I metabolism occurs in the hepatocytes within the liver.
Diagram of Phase II metabolism.
Take a look at this Concentration-Time graph detailing some basic pharmacokinetic parameters
Give some examples of sites of drug action
The human body has 100 trillion cells with 200 different cell types. Some sites of drug action include:
- Primary tissues: muscles, nerves, epiuthelial, bone, connective etc.
- Tissues controlled by: innervation, EC fluids, blood supply, exocrine and endocrine secretions.
- Many drugs mimic (or block) the action of endogenous molecules (e.g. neurotransmitters, hormones)
- they act at specific sites such as ion channels, receptors, enzymes, transporters.
How do drugs act?
Drug molecules exert a chemical influence on constituents of cells to produce a pharmacological response. In order to this, the drug molecule must get close enought to the cellular constituents so they can interact chemically.
The interaction leads to an alteration in the molecular/cellular function.
The drug molecules must bind to the specific constituents of cells (aka Drug Targets) in order to produce an effect.
Why do we have receptors?
We primarily have receptors for the purpose of cell-cell comms
See, this is important in the contect of:
- Neurotransmission (e.g. nerve-nerve; nerve-muscle)
- Effects of chemical mediators in bloodstream (e.g. adrenaline on heart)
- Hormone and growth factor signalling (e.g. action of insulin on muscle)
What is a receptor?
A receptor is a recognition molecule for a chemical mediator through which a response is transduced. they are usaually a protein or complex of two or more proteins and often expressed on the surface of cells (with some exceptions).
Many drugs act to mimic or block rhe effects of endogenous molecules at their receptors.
Describe the basic Lock and key concept.
Well, it’s a very simple analogy. the receptor is the lock and the ligand is the key. Some ‘keys’ fit in the lock and others do not. What determines if a lock will fit in the key is the chemical structure of the lock and key. Are they complementary to each other?
Study this diagram of the basic receptor structure broooo.
Study this very simple flow chart of Signal transduction.
Study this diagram of four different types of receptors.
Study this diagram of effector mechanisms
Describe what: Ligands, Agonists and Antagonists are.
A ligand is any chemical that binds to a receptor.
An agonist is a drug that binds to a specific site on a receptor and mimics the effect of the endogenous ligand for that site.
An agonist is a drug that binds to a specific site on a receptor, blocks the effect of the endogenous ligand (same/different binding site as ligand).
Go on, explain the more advanced lock and key concept.
Okay, so the receptor is basically the lock, the endogenous ligand is the key. Now, an agonist drug fits into the lock; mimics the action of the key (like picking a lock) and activating the receptor (opening the door). However, an antagonist drug also fits into the lock; gets stuck and prevents the opening of the door (activation of protein) by agonists or endogneous ligands.
What is affinity and efficacy?
The affinity of a drug describes how well it binds to the active site of the target protein.
The efficacy of a drug describes how much the drug evokes a cellular response from the target protein as a result of this binding.
Endogenous ligands and agonists possess both affinity and efficacy. Antagonists only possess affinity and lack efficacy.
What is Neurotransmission?
Neurotransmission is a chemically-mediated form of the cell to cell comms. it is seen in Peripheral Nervous System (Neuromuscular Junction, Autonomic Nervous System, and Enteric Nervous System). It is also seen in the Central Nervous System.
What are the 13 Key processes in Neurotransmission?
- Uptake of precursor molecules (via transporter)
- Synthesis of the neurotransmitter from the precursor molecule. (via enzyme action)
- The molecule is then packaged into a vesicle. (involves a transporter)
- An enzyme then breaks down the excess transmitter. (enzyme action)
- An AP is carried down the nerve terminal leading to depolarization. (via an ion channel)
- The AP reaches the nerve terminal and causes the Ca2+ channel to open and allows for the pre-synaptic entry of Ca2+. (ion channel)
- A Neurotransmitter is then released (not sure where the drug target here is, however)
- The neurotransmitter crosses the synaptic cleft.
- It then binds to the postsynaptic cell. (receptor)
- An enzyme in the synapse of the postsynaptic cell then breaks down the NT.
- The broken-down NT id the re-uptaken into the synaptic terminal. (via transporter)
- The broken-down NT can also be taken into non-neuronal cells via (transporter too)
- The broken-down Nt may even bind to the pre-synaptic cell membrane (receptor)
All the bracketed proteins are target sites for drugs to interact within neurotransmission.
What are the receptor types involved in neurotransmission?
Type 1 (Ligand-gated ion channels) and Type 2 (G protein-coupled receptors). Both of these receptors have a relatively fast signal transmission (milliseconds and seconds, respectively). type 3 and type 4 receptors take hours to days respectively and are just not viable.
Describe Drug-receptor selectivity.
- There is reciprocal selectivity between drugs and their target receptors.
- Individual drug classes only bind to certain receptors and individual receptors only recognize certain drugs.
- No drugs are 100% selective in their binding “they are dirty in that sense”.
- Most are reasonably selective at regular conc. but all have non-selective (or ‘off-target’) effects at high conc.
What is Receptor Heterogeneity?
Most endogenous chemical mediators have more than one receptor; often whole families and sub-families. For example, take ACh, it’s an NT that acts within the CNS, autonomic, somatic, and enteric nervous systems.
- Acts on nicotinic ACh receptors (ligand ion channels)
- Acts on muscarinic ACh receptors (G protein-coupled receptors)
Receptor heterogeneity is conferred by receptor type, subunit composition, and the amino acid sequence of proteins.
How are Receptors classed?
- Understanding of different receptor types and their classification has come from the use of drugs.
- Traditional anti-histamines blocked the effects of histamine on blood vessels and smooth muscle but not the effects of histamine on gastric acid secretion.
- Is there a different type of histamine receptor that is expressed in the stomach?
- Testing a range of drugs with different specificities allowed ID of H1-receptors and H2-receptors.
- Sir James Black discovered H2 receptors and H2 blockers.
Study the list of Major NTs and Receptors
Describe the relationship between Receptor Occupancy (%) against Agonist Concentration.
Receptor occupancy increases with increasing agonist concentration. As seen in the graph, this is not a linear relationship.
Describe the relationship between the concentration of the agonist against the response.
The relationship between the response (% maximum) against the agonist concentration is very exaggerated. Here is a graph to illustrate what I mean.
Use the graph on this flashcard for reference, what is an EC50?
The EC50 is the concentration of an agonist that elicits a half-maximal response. It’s an important efficacy measure that allows for comparison between drugs.
Study how a concentration-response curve looks on a semi-log scale.
The relationship in this graph is sigmoidal. So it is easier to see where EC50 is.
Use the graph to see the difference between potency and efficacy.
In the example graph, Drug A and Drug B are full agonists i.e. they produce ~100% maximum response from the activated proteins.
However, Drug A is more potent than Drug B. This is because Drug A has a lower EC50 compared to Drug B.
Drug A and Drug C have the same potency. because they have equal EC50 values.
Drug C is a partial agonist as it has lower efficacy than Drug A and Drug B.
Describe Competitive vs Non-competitive Antagonism.
Competitive Antagonism
- Binds to the same site as the agonist (or endogenous ligand)
- Competes with the agonist/endogenous ligand for binding.
- Conc. and affinities of agonist/antagonist determine the overall ‘winner’.
- The effect of the antagonist can be overcome by increasing agonist concentration.
- Some competitive antagonists bind covalently (irreversibly) to the agonist binding site. They cannot be displaced by the agonist but the extent of antagonism depends on the agonist concentration.
Non-Competitive Antagonism
- Binds to a site that is distinct from the agonist binding site.
- Causes change in conformation of receptor that restricts agonist binding.
- Effect of antagonist cannot be overcome by increasing agonist concentration.
Study this diagram detailing the % response with an agonist alone, an agonist + NC antagonist, and the same agonist + competitive antagonist.
Explain what the Two-State Model is.
Two State Model
Most receptors exist in two conformational states, resting (R) and activated (R*). However, in the absence of an agonist, the equilibrium lies to the left (i.e. most receptors are resting). Full agonists bind preferentially to R* and shift the equilibrium to the right, invoking a response. In this sense, the higher the affinity the agonist has for R*, the higher the efficacy.
Antagonists have equal affinity for R and R*. They effectively do not shift the equilibrium. This is because they have lack efficacy and prevent drugs to bind and shift the equilibrium.
Explain how the Two-State model works when there is a constitutively active receptor involved.
- Some receptors are ‘constitutively active’ like the cannabinoid receptor.
- In the absence of agonist, appreciable proportion of these receptors are in the R* state.
- These receptors have inverse agonists; they bind preferentially to R and shift the equilibrium to the left.
- This switches receptor activation off and reduces response.
- The higher the inverse agonist affinity for R = the greater the efficacy.
- Antagonists block agonists and inverse agonists equally.
Study the graph displaying how agonists and inverse agonists are affected by antagonism.
When can the true effects of partial agonists and inverse agonists be observed?
Partial Agonists
- True effects of partial agonists are only seen in the absence of full agonists.
- Partial agonists look like competitive antagonists in the presence of full agonists.
Inverse Agonists
- True effects of inverse agonists are only seen when the receptor is constitutively active.
- In the absence of constitutive activity, inverse agonists look like weak competitive antagonists.
Here’s a fun analogy for agonists, inverse agonists, partial inverse agonists, partial agonists, competitive and non-competitive antagonists.
What does an Allosteric Modulator do?
Allosteric Modulators
- Allosteric modulators bind at a distinct site to affect affinity and efficacy of agonists.
- They can be positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs).
- NAMs can look like competitive antagonists (affinity) or non-competitive antagonists (efficacy)
- PAMs make the “volume knob” easier to turn (affinity) or increase the efficacy.
What is a biased Agonist?
Biased Agonists
- Receptors are typically coupled to effector mechanisms that elicit the response following against binding.
- Some receptors are coupled to more than one effector mechanism.
- Conventional Agonism - Different agonists bind to the same receptor and activate effector mechanisms to the same extent.
- Biased Agonism - Different agonists bind to the same receptor and preferentially activate one effector mechanism.
Define an Agonist.
An Agonist is a compound that binds to and activates a receptor.
Define an Antagonist.
An Antagonist is a compound that reduces the effect of an agonist at a receptor.
Define an Inverse agonist.
An inverse agonist is a •compound that binds to the same receptor site as an agonist but produces the opposite effect (constitutively active receptors only).
Define an Allosteric Modulator.
An Allosteric Modulator is a compound that binds to a receptor site distinct from the agonist binding site, inducing a conformational change that alters the affinity or efficacy of agonist binding (at the orthosteric site)
Define Specificity
a measure of the number of receptor sites that a given drug may bind to or the range of effects it may produce
Define Selectivity.
: the degree to which a drug binds to a given receptor site relative to other receptor sites (related to affinity)
What are the two types of ion channels directly involved in drug action?
Two major types of ion channels directly involved in drug action:
- Voltage-gated ion channels (VGICs)
- Ligand-gated ion channels (LGICs); considered as receptors
Other ion channels (cell surface and intracellular) may be activated indirectly via GPCRs (not considered here).
What is the passage of specific ions detemined by?
Passage of specific ions is determined by selectivity of channel pore.
What is ion flux driven by?
Ion flux is driven by the electrochemical gradient; direction of ion travel (influx or efflux) is determined by:
- Concentration gradient (many cell types)
- Electrical (or charge) gradient (excitable cells only)
Here is a diagram depicting the elctrochemical gradient.
What are some of the basics of voltage-gated ion channels?
- VGICs expressed in electrically excitable cells (e.g. muscle, nerve)
- Permeable to sodium (Na+), potassium (K+), calcium (Ca2+), chloride (Cl-)
- Comprise one or more a-subunit proteins that associate with ancillary subunits; modify function but not necessary for basic channel activity
- Gated (i.e. activated) by changes in membrane potential:
- Responsible for action potentials (Na+, K+)
- Pre-synaptic regulation of neurotransmitter release (Ca2+)
- •Key targets of several drug classes; antiepileptics, antihypertensives, local anaesthetics etc.
How do Voltage Gated Ion Channels and Ligand Gated Ion Channels compare?
Here is a diagram of the structural topology of key VGICs.
Give a brief overview on how a VGIC works.
- They are initially closed at resting membrane potential (-70mV)
- They hen rapidly open in response to changes in membrane potential
- Involved in depolarisation and repolarisation and neurotransmitter release
- Channel opening is mostly transient and rapidly inactivates
- Cycle through 3 conformational states; resting (closed), activated (open) and inacivated.
- Ball and chain mechanism and changes to conformational shape of transmembrane protein.
How does Phenytoin ineract with the Voltage Gated Sodium Channel?
- Phenytoin is a classical sodium channel blocking antiepileptic drug
- Binds preferentially to the inactivated state of the channel
- Slows conformational recycling back to resting state (does not block pore)
- Extends the refractory period between individual action potentials; reduces ability of neurons to fire at high frequency
- Local anaesthetics work in similar way but block nerve conduction completely
How do Gabapentin (GBP) and pregabalin (PGB) interact with Voltage Gated Calcium Channels?
- Gabapentin (GBP) and pregabalin (PGB) are newer antiepileptic drugs
- Bind to ancillary a2-d1 subunit of voltage-gated calcium channel
- The a2-d1 subunit associates with Cav2.1 a-subunit to form P/Q-type channel
- GBP and PGB indirectly block the P/Q-type channel
- Involved in neurotransmitter release at synapse; glutamate?
How does Retigabine (RTG) interact with the Voltage-Gated Potassium Channel?
- Retigabine (RTG) is a newer antiepileptic drug; now withdrawn due to serious adverse effects
- Potassium channel activator; selective for Kv7 channels; responsible for M-current
- M-current is non-inactivating potassium current that regulates neuronal excitability
- Contributes to refractory period between action potentials; limits repeated activation
- RTG enhances M-current, holds membrane potential below threshold, and reduces firing frequency
Describe the subunuit composition of LGICs
- LGICs consist of complexes of multiple independent protein subunits assembled around a central ion pore; heteromultimers.
- Heterogeneous assembly of multiple subunits; 19 GABAA receptor subunits but only 5 are required for a functional receptor.
- Subunit composition confers biophysical properties and pharmacology of the receptor complex
- Diversity of receptors; varying patterns of expression within the nervous system and other tissues
- Attractive targets for new drugs that possess subunit selectivity; i.e. discriminate between receptor isoforms.
What are the three LGIC families?
Give a brief overview of Cys-loop receptors
Cys-loop receptors are a superfamily of LGICs comprising:
- Nicotinic acetylcholine receptors
- GABAA receptors
- 5-HT3 receptors
- Glycine receptors
Pentameric structure; usually 2 alpha-subunits plus 3 others.
Each subunit contains large extracellular N-terminal domain (with Cys-loop).
4 membrane-spanning alpha helices (M1-M4); pore is formed by the M2 helices.
Endogenous ligands bind at interface between subunits in the extracellular domain.
What was the first ligand-gated ion channel to be purified and cloned?
The Nicotinic Aceylcholine (ACh) receptor.
Where is the Nicotinic Acetylcholine expressed?
The Nicotinic acetyulcholin (ACh) recptor is expressed at the neuromuscular junction, autonomic ganglia and in the CNS.
How many binding sites are there for ACh?
There are two binding sites for ACh; at interface between α-subunits and their neighbours. Both sites must bind acetylcholine molecules for receptor activation
What happens as a result of ACh receptor activation?
Activation results in fast synaptic transmission.
Nicotinic ACh receptor - a non-selective cation channel?
- Resting membrane potential of post-synaptic cell is ~-75mV
- High Na+ concentration outside cell, high K+ concentration inside cell
- Pore of the nicotinic receptor is equally permeable to all monovalent cations (e.g. Na+, K+ , Li+); non-selective
- Opening of pore allows ions to flow down concentration gradient; Na+ in, K+ out
Describe Cholinergic transmission at the NMJ
- Acetylcholine concentration in synaptic cleft; 10nM to 500µM in 1 millisecond
- Depolarisation of cell membrane; opening of voltage-gated sodium channels, release of calcium from intracellular stores → muscle contraction
Here’s a hard one… Describe the steps in the Gating of nAChR.
- Gating is highly dependent on presence of specific amino acid residues in pore
- Glutamate and threonine residues that line the pore attract cations and repel anions
- Each subunit has a leucine residue in M2 helix; protrude into pore to form the gate
- In the resting state, leucine residues block the pore – gate closed.
- Two ACh molecules bind to extracellular domain of α-subunits
- Conformational change – twisting of α-subunits, M2 helices swivel out of the way.
- Gate opens – channel activated
- Cations pass down concentration gradient into cell (25,000 Na+ ions per millisecond).
- K+ ions move in opposite direction; 3 Na+ ions enter for every 2 K+ ions leaving
- Net increase in intracellular positive charge; leads to depolarisation of membrane
- One millisecond later, helices move back – gate closes
- ACh dissociates back into synaptic cleft where it is broken down by the enzyme acetylcholinesterase
- Receptor returns to resting state
Table showing subunits of nicoinic nAChR
What is the GABAA receptor?
- Another member of Cys-loop superfamily
- Predominant expression in CNS; GABA is the major inhibitory neurotransmitter in the brain
- Two receptor types; GABAA (LGIC) and GABAB (GPCR)
- GABAA receptor similar to nicotinic ACh receptor; forms heteromeric pentamer
- Nineteen GABAA receptor subunits identified; alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, pi, and rho1-3
- GABAA receptors in CNS typically comprise two alpha-, two beta-, and one gamma-subunit
Describe the mechanisms in GABAA receptor.
- GABAA receptor binds two molecules of GABA; interface between α and β subunits
- GABAA receptor is permeable to chloride and bicarbonate ions
- Chloride influx into post-synaptic neuron causes hyperpolarisation & inhibition
- Activation of GABAA receptors reduces excitability throughout CNS
- GABAA antagonists (e.g. bicuculline, picrotoxin) used experimentally to cause seizures.
Give a brief overview of Ionotropic glutamate receptors
- Three types of ionotropic glutamate receptor: AMPA, kainate and NMDA
- Distinct class of LGIC; form heteromeric tetramers (i.e. 4 subunits) rather than pentamers
- Shortened M2 region, intracellular C-terminus
- Distinct subunits for individual receptor types:
- AMPA: four subunits (GluA1 to GluA4)
- Kainate: five subunits (GluK1 toGluK5)
- NMDA: seven subunits (GluN1, GluN2A to GluN2D, GluN3A, GluN3B)
Give facts about AMPA and NMDA receptors.
- Both permeable to Na+ and K+ ions; NMDA also permeable to Ca2+
- AMPA receptor carries fast excitatory signals in CNS
- NMDA receptor blocked by Mg2+ at resting membrane potential
- Prolonged depolarisation removes Mg2+ block
- Glycine is co-agonist at NMDA receptor; requires glutamate & glycine to bind for activation
What are the 6 Subdivisions of GPCRs and what are they associated with?
GPCRs can be subdivided into 6 main families, class A to F. Class D is associated with pheromone signaling within Fungi and class E is restricted to cAMP signaling in amoeba. Class F, also known as the Frizzled receptors, were originally identified in drosophila but are now associated with embryonic development and postnatal tissue regeneration. In addition, this class is associated with a number of cancers and is, therefore, a clinically relevant oncogenic target.
However, in terms of pharmacological targets for human disease, the majority of these targets are within classes A to C.
By far the largest family is the class A receptors, also known as the rhodopsin-like GPCRs. Class B is the secretin-like GPCR family and Class C is the metabotropic glutamate GPCR. We will now explore these three classes in a bit more depth, with particular emphasis on receptor structure and ligand binding
Describe the 2D structure of GPCR.
The typical 2D structure of a GPCR consists of a common core of 7 transmembrane-spanning alpha-helices, an extracellular N-terminus, and an intracellular C-terminus. The helices are connected by 3 intracellular and 3 extracellular loops. This structure is common amongst GPCRs but there are distinct differences amongst the classes of GPCRs, particularly in relation to the length of the N-terminus and location of the agonist binding domain.
Describe the 3D structure of Class A GPCRs.
In class A GPCRs, the receptor structure is characterized by a short extracellular N-terminal.
In the 3D structure of GPCRs, the 7 helices form a cavity or pocket. In class A GPCRs, the ligand-binding site is typically found in this pocket.
The Cavity is largely non-polar with a few critical polar residues responsible for specific high-affinity interactions with the ligand
Give some structural features common to Class A GPCRs.
Firstly, cysteine residues in extracellular loops 1 and 2, as shown in green, form a disulfide link. This provides stability and is important for the packing of the helices in the 3D structure.
Secondly, proline residues in transmembrane helices 6 and 7, as shown in yellow, introduce kinks into the alpha-helices. This facilitates a conformational change in the receptor where a small change induced by the agonist is translated to a larger internal change to activate signaling. We will look at this in a bit more detail later on in the lecture.
Thirdly, an asparagine residue in transmembrane 2 and a series of 3 residues (asparagine, arginine, and tyrosine residues) within the 2nd intracellular loop, referred to as the DRY motif, are important for receptor activation of the G-protein.
What is the unique type of Class A receptor?
A unique member of the class A GPCR family is the protease-activated receptors (PAR1-4).
The unique feature of this receptor is that the ligand is contained within the receptor itself. More precisely, it is contained within the N-terminus. A protease activates the receptor by cleaving the N-terminus and exposing 5/6 N-terminal residues that bind to receptor domains in the extracellular loop and activate the receptors. These residues are referred to as tethered agonists.
This cleavage cannot be reversed and eventually, the receptor is desensitized and broken down by the cell. The cell then needs to resynthesize the new receptor protein.
An important example of a protease is thrombin.
Describe features of Class B GPCRs.
There is little sequence homology with class A and class C GPCRs. The structure is stabilized by 3 disulfide bonds within 6 highly conserved cysteine residues.
One of the most striking features of class B GPCRs, as demonstrated in the diagram, is the longer N-terminal tail, consisting of 100-160 residues. This N-terminus incorporates the ligand-binding domain.
What are Class C GPCRs activated by?
Class C GPCRs are activated by glycoprotein hormones and important examples include the metabotropic glutamate receptors, GABAB receptors, and calcium-sensing receptors.
If we look at the schematic of a typical class C GPCR, you can see a clear difference in the N-terminus. This large N-terminus contains a ligand-binding domain called a venus flytrap module. The binding of the agonist to the VFT leads to a conformational change in the receptor and activates the G-protein.
Additionally, allosteric binding sites are usually present within the receptors, influencing the activity of the receptor.
Class C GPCRs typically form dimers. Where the two receptors are the same subtype, this is an example of a homodimer. For example, if the two receptors are metabotropic glutamate receptors.
If the receptors within the dimer are different receptor subtypes, this is an example of a heterodimer. For example, GABAB1 and GABAB2
Here is a diagram detailing GPCR signaling.
What are the 3 Components of GPCR signaling?
We can break down GPCR signaling into 3 components: receptor; G-protein and effector. The g-protein and the effector protein are responsible for translating an agonist binding event into the generation of a 2nd messenger signaling.
Describe the structure of G-Proteins (Guanine Nucleotide-Binding Regulatory Proteins).
The g-protein is a heterotrimeric g-protein consisting of an alpha, beta and gamma subunit. The g-protein is attached to the membrane by lipid modifications. This includes myristoylation and palmitoylation on the alpha subunit and prenylation of the gamma subunit.
The alpha subunit can bind either GDP or GTP and possesses GTPase activity. We will now fully examine the activation of the G-protein and you will see how these features of the alpha subunit are particularly important.
How do G-proteins cycle between GDP-bound inactive and GTP-bound active states?
Importantly, binding of GDP to the alpha subunit is associated with the inactive state of the G-protein, and binding of GTP is the active state of the G-protein.
The diagram shows the receptor and G-protein at the basal, inactive state, where the G-protein exists as a complete heterotrimer, with GDP bound to the alpha subunit.
Upon agonist binding, the agonist-bound receptor attracts the GDP-bound G-protein.
The receptor-bound G-protein then releases GDP from the alpha subunit.
This allows GTP to bind to the alpha subunit.
G-protein then dissociates into the alpha subunit bound to GTP and a beta-gamma dimer.
Additionally, the agonist dissociates from the receptor upon G-protein activation.
The alpha subunit bound to GTP and the beta-gamma dimer can both bind to effector proteins to modulate downstream signaling.
Remember, the alpha subunit has an intrinsic GTPase activity. This GTPase hydrolyses the bound GTP to GDP.
The GDP bound alpha subunit and the beta-gamma dimer then dissociate from their effectors, leading to the inactivation of the effector proteins.
The G-protein trimer then reforms.
And the G-protein now exists in the basal state, available for another activation cycle
Describe the interaction between G-protein and Receptor.
- Upon ligand binding, transmembrane domains 3 and 6 move away from each other.
- Transmembrane domain 6 also rotates on its own axis by 30 degrees.
- This opens a cleft for the c-terminus of the G-protein alpha subunit to move into. The G alpha subunit is then activated by an interaction with the DRY motif and other sequences.
How Can receptors give specific responses if there is only one pool of G-Proteins?
This can be explained by the existence of a range of G-protein subtypes. There are over 20 G-proteins subtypes identified, with each g-protein subtype classified by the identity of the alpha subunit. Key families include Gs, Gi, Gq, Go, and G12/13.
G-proteins will show selectivity with respect to the receptors and the effectors in which they couple, leading to differential signalling.
Do GPCRs activate one or multiple types of G-Proteins?
Many receptors also have the capacity to activate multiple G-protein subtypes. For example, thrombin activation of the PAR-1 receptor in vascular endothelium can activate Gi, Gq/11, G13 and beta-gamma G-protein signalling, leading to a range of effects associated with angiogenesis, the formation of new blood vessels from pre-existing vessels. In this case, it is essential that multiple signaling pathways are activated to initiate a biological process as complex as angiogenesis.
What is cAMP signaling associated with?
cAMP signaling is associated with a range of biological effects, such as energy metabolism, smooth muscle contraction, cell division, and differentiation. cAMP can be converted to 5-AMP via phosphodiesterases, turning off cAMP signaling.
- cAMP predominantly associated with activation of PKA
- Phosphodiesterases (PDEs) converts cAMP to 5’-AMP
Describe the structure of Protein Kinase A and how it demonstrates how cAMP can activate the enzyme.
The structure of PKA demonstrates how cAMP can activate this enzyme. PKA consists of two regulatory R subunits that have the capacity to bind cAMP and two catalytic C subunits containing the kinase activity of the enzyme. As you can see from the diagram, in the basal state, all 4 subunits are connected and the catalytic subunits are inactive.
Each R unit can bind two cAMP molecules, meaning that each PKA molecule binds 4 cAMP molecules. Once the cAMP binds to the R units, this releases the catalytic subunits, which then become active.
Describe the GPCR-Dependent Activation of Adenylyl Cyclase using Adenosine A2A receptor as an example.
For example, activation of the adenosine A2A receptor is associated with vascular smooth muscle relaxation. We can now relate this to the regulation of cAMP. In this case, activation of the A2A receptor activates the Gs subtype of G protein. This leads to increased activity of adenylyl cyclase which generates cAMP. This, in turn, leads to the activation of PKA which phosphorylates multiple substrates to relax the vascular muscle. A key example of such a substrate is myosin light chain kinase. The phosphorylation of MLCK by PKA inhibits this enzyme, preventing the phosphorylation of MLC, a key step in smooth muscle contraction.
Why do GPCRs generate a second messenger such as cAMP?
Initially, it might seem strange why a receptor would need to generate a second messenger, such as cAMP. The reason for this is the principle of signal amplification. The activation of a receptor will generate a large number of 2nd messenger molecules by regulating enzyme activity. In this case, the activation of adenylyl cyclase produces a large number of cAMP molecules that can then act on a large number of PKA enzymes.
Hence, this ability to amplify a signal from a ligand binding to a receptor provides a quick, efficient way to transfer that initial message to a biological response.
Here is a table of Gi-coupled receptors and Gs-coupled receptors.
An added level of complexity is associated with the adenylyl cyclase enzyme itself. There are 10 different adenylyl cyclase isoforms, some of which respond selectively to Galpha s or Galpha i
What does the effect of cAMP on muscle contraction largely depend on?
The effect of cAMP on muscle contraction will largely depend on the muscle type and the PKA target substrate. In smooth muscle, cAMP/PKA is associated with muscle relaxation, as explained previously. In contrast, in cardiac muscle, PKA phosphorylates voltage-gated calcium channels, leading to calcium entry into the cell and increasing the force of contraction.
Describe the role of cAMP in the liver.
cAMP also has an important role in the liver, for example, in response to epinephrine or glucagon. The activation of PKA leads to phosphorylation and inactivation of glycogen synthase, inhibiting the production of glycogen. Additionally, PKA phosphorylation and activation of phosphorylase kinase leads to the breakdown of glycogen into glucose, resulting in the release of glucose into the blood.
Describe cAMP signalling in White Adipose Tissue.
In white adipose, stimulation of cAMP leads to phosphorylation and activation of triglyceride lipase, resulting in the breakdown of triglycerides into fatty acids and glycerol.
Describe the structure of Membrane Phospholipids.
These phospholipids contain a hydrophilic phosphate head and 2 hydrophobic fatty acid tails, joined together by a glycerol molecule. They are a major component of the lipid bilayer in cellular membranes.
Describe the 2nd main signaling pathway involving inositol trisphosphate/diacylglycerol.
- We will focus on the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2), an important substrate for cellular signaling.
- The hydrolysis of PIP2 by the enzyme phospholipase C leads to the generation of two distinct second messenger molecules: 1,2 Diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3).
- DAG, the hydrophobic product of hydrolysis that consists of the fatty acid tails, remains in the membrane. IP3, the hydrophilic phosphate product, enters the cytosol.
How do GPCRs regulate the hydrolysis of PIP2 and what cellular signalling is associated with DAG and IP3?
- GPCR-dependent hydrolysis of PIP2 is generally associated with the receptors that couple to the Gq G protein subtype. For example, the alpha1 adrenoceptor. Upon activation of the receptor, this leads to an increase in the activity of the PLC enzyme, generating IP3 and DAG.
- The cytosolic IP3 acts on the IP3 receptor, a ligand-gated calcium channel present on the membrane of the ER. This induces the release of calcium from the ER, leading to an increase in intracellular calcium concentration.
- The emptying of the intracellular calcium stores subsequently triggers the opening of store-operated calcium channels at the plasma membrane, resulting in an influx of calcium from outside the cell.
- Intracellular calcium binds to protein kinase C.
- This results in the translocation of PKC to the plasma membrane where it binds to DAG and becomes active and has the capacity to phosphorylate a range of substrates.
Tabled examples of Gq-Coupled GPCRs.
What Makes Calcium an Ideal Intracellular Messenger?
First, calcium can be rapidly mobilized and removed, providing a signaling communication that responds quickly and efficiently to changes in the physiological environment.
Secondly, calcium binds tightly to target proteins. For example, the binding of calcium to calmodulin. Further study of the calcium-binding site of calmodulin shows that the calcium ion binds to oxygen atoms of three aspartates, glutamate, water, and the backbone carbonyl.
By linking multiple regions of a protein, calcium-binding induces significant conformational changes.
What is Calmodulin and how does its structure relate to its function?
Calmodulin is a small dumbbell-shaped protein. Apo-calmodulin is the form of calmodulin in the absence of calcium. When calmodulin binds 4 calcium atoms, the hydrophobic helix folds around a partner protein. For example, CaM kinase or myosin light chain kinase. The binding of calmodulin then induces a conformational change in the target protein, altering the target protein’s function.
Calcium-binding to calmodulin is positively co-operative. In other words, a small increase in calcium concentration leads to a larger change in calmodulin activity
Diagram of Receptor Tyrosine Kinases
Describe the steps involved in Receptor Tyrosine Kinase activation.
Diagram of Examples of Tyrosine Kinase Activation
How does IGF-1 get phosphorylated?
- Insulin/IGF-I are soluble monomeric polypeptides
- Receptors are expressed as disulphide bond-linked preformed heteromers
- Insulin/IGF-I bind to the alpha subunits to induce a conformational change that is transmitted to the beta subunits which then transphosphorylate each other
Diagram of Tyrosine Kinase Receptor Signalling
Describe the Docking and Adaptor Proteins-intracellular events.
The phosphorylated tyrosine residues act as high-affinity docking sites for intracellular proteins.
One important group is SH2 domain proteins which contain a 100AA recognition site.
Individual SH2 proteins bind selectively- making the response to receptor activation specific.
What is the end result of RTK signaling involving SH2 domain proteins?
- Many SH2 domain proteins are enzymes- kinases or phospholipases
- End result is activation or inhibition of transcription factors that migrate to the nucleus
- This can alter gene transcription.
- An important transcription factor is NFkappaB.
- Normally complexed with an inhibitor in the cytoplasm
- RTKs can phosphorylate the inhibitor to allow the NFkappaB to start moving
- Could be a possible drug target.
Describe with diagrams how Ras activation occurs.
Diagram detailing how Ras activation leads to MAPK activation.
Diagram detailing Ras activation to PI3- kinase activation.
Diagram detailing PI3K activation to PKB/Akt activation.
Diagram detailing PI3K activation to PKB/Akt activation.
Diagram detailing P-Tyr residues bind specific SH2 domain-containing proteins.
Diagram detailing different P-Tyr Residues being able to bind specific SH2 Domain-Containing Proteins
What is the correlation between RTKs and Cancer?
- The HER family member HER2 overexpressed in 20-30% of breast cancers
- The receptor is Constitutively active
- Inappropriate amplification of mitogenic signalling
- Aggressive tumour growth
- Increased risk of metastasis
- A possible therapeutic action would be to suppress the receptor
- HERCEPTIN/Trastuzumab = Monoclonal Ab which binds and inactivates HER2
- Lapatinib = HER-targeted tyrosine kinase inhibitor molecule
Labelled Diagram of a Nuclear Receptor’s structure.
What does the hinge region allow for in a nuclear receptor?
The Hinge region allows for confirmational change upon ligand binding.
Diagram of Type I NR.
Diagram of Class II NRs activation
What are some PPAR agonist drugs?
And whats is the pharmacology behind it?
PPAR gamma agaonists: Thiazolidinediones (Roziglitazone, Pioglitazone).
Pharmacology: Agonist Receptor complex enhances transcription of responsive genes. Glucose entry to muscle and gluconeogenesis suppressed- reversal of insulin resistance
Mechanism of Fibrates in a flow chart thingy
Diagram detailing Glucocorticoids and Inflammation.
How is the potential difference measured in a cell?
Measurable by placing an electrode inside the cell and measuring vs bath electrode, outside the cell. Potential is always quoted as inside vs outside.
How is the membrane potential established?
- Depends on what is inside vs. outside of the cell i.e. charged ions
- The Physiochemical level of the cell.
- Membrane & membrane components are crucial
Diagram of cell membrane which has potential?
Diagram of charges being separated being responsible for potential.
Diagram depicting differing magnitudes of p.d.
Figure depicting concentration and permeability of ions.
How does the membrane potential change?
What is Ion Leakage and what is the cell’s solution to this?
Diagram depicting Synaptic transmission.
What does the magnitude of the Graded Potential depend on?
Diagram of Summation of EPSPs
Describe how an AP occurs in the context of EPSPs.
Diagram detailing an AP.
Diagram detailing AP, TP, RP, and HP.
Diagrams detailing the steps from rest to ap
What is the problem with Ion Exchange?
Diagram detailing AP propagation.
Diagram of Node of Ranvier between Myelin Sheath.
