GPCRs

Question 1

GPCR drugs

Answer 1

Losartan - angiotensin receptor blocker (ARB) for hypertension
Semaglutide - GLP1R (Gs) agonist for type 2 diabetes and obesity, providing blood glucose control and satiation signals
Cariprazine - dopamine receptor partial agonist for schizophrenia and bipolar disorders
Suvorexant - orexin receptors (Gq) antagonist OX1R and OX2R for insomnia
Morphine - MOR (Gi) agonist for acute pain
Cetirizine - histamine receptor antagonist for allergies

Question 2

Untapped potential of GPCR drug discovery

Answer 2

GPCRs are the target of 1/3 of all current therapeutic drugs, but there is still a great untapped potential of GPCRs.
Current medicines only target 27% of non-olfactory GPCRs. These include well-established receptors including adrenoceptors, and histamine, 5-HT, ACh, dopamine and opioid receptors.
17% non-olfactory GPCRs (66) have agents that have reached clinical trials
56% non-olfactory GPCRs (224) are yet to be targeted

Question 3

GPCR classification

Answer 3

GPCRs are 7 TM domain proteins. They have an extracellular N-terminus, intracellular C-terminus, 3 ECL and 3 ICL.
There are 6 GPCR families:
- A - rhodopsin-like family
- B - secretin (B1) and adhesion (B2) family
- C - metabotropic glutamate family
- D - non-mammalian - fungal mating pheromone family
- E - non-mammalian - cAMP receptors
- F - frizzled/smoothened
GRAFS classification was commonly used for mammalian GPCRs in the past. This stands for Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin.

Question 4

Mammalian GPCR characteristics

Answer 4

A - Rhodopsin - short N-terminal, the ligand binds at the transmembrane domain. Over 700 members. They respond to a variety of ligands including amines (e.g. adrenaline, noradrenaline, 5-HT, histamine, ACh), nucleosides like ATP, lipids (e.g. endocannabinoids, prostaglandins) and peptides (e.g. oxytocin, substance P, Leu-enkephalin, somatostatin). Binding occurs at the TM domain.
B1- Secretin - their (medium) extracellular domain and 7TM domain contribute to ligand binding. They manly respond to peptide hormones like secretin, glucagon, CGRP, VIP and PTH.
B2 - Adhesion - their large extracellular domain is responsible for binding, consisting of adhesion domains and a GPCR-Autoproteolysis INducing (GAIN) domain with the conserved GPCR proteolysis site (GPS) motif. They respond to themselves via autoproteolytic cleavage at the GPS, leaving a tethered ligand.
C - Glutamate - need to form heterodimers, have a large venus flytrap binding domain. They also have a cysteine-rich domain (CRD). Respond to glutamate and GABA, and also the tastes sweet and umami.
F - Frizzled - contain a CRD, respond to Wnt ligands. They regulate cell polarity, embryonic development, cell proliferation, and neural synapse formation.

Question 5

G𝛼 and G𝛽𝛾 subunits

Answer 5

G𝛼 is membrane associated, mediated by a post-translational modification. It has GTPase activity and it is active when GTP bound. There are 4 subfamilies G𝛼s, G𝛼i, G𝛼q/11, G𝛼12/13, which have different isoforms for a total of 21 members.
G𝛽𝛾 is an obligate heterodimer that is also membrane associated. There are 6 G𝛽 members and 12 G𝛾 members, but these do not show as much functional difference as G𝛼 subunits.

Question 6

GPCR Signalling

Answer 6

Binding of an agonist induces a conformational change in the receptor, activating it. There is a conformational change in the G𝛼 subunit, allowing the exchange of GDP for GTP. G𝛼-GTP dissociates from the G𝛽𝛾. Both activate effector proteins independently, then rebind once the GTPase activity of the G𝛼 hydrolyses GTP.
There are different G𝛼 subunits, coupled to different signalling pathways.
Gs - adenylyl cyclase activation and increase in intracellular cAMP → protein phosphorylation via PKA
Gi/o/z - adenylyl cyclase inhibition → G𝛼s signalling downregulation
Gq/11 - phospholipase C activation → breakdown of PIP2 into IP3 (Ca2+ release) and DAG (PKC activation) → protein phosphorylation and activation of Ca2+-binding proteins.
G12/13 - RhoA (GTPase) activation → chemotaxis, cytoskeleton changes (cell shape change)
Some receptors are uniquely coupled to one G-protein family, but most show coupling to multiple families. This can be dependent on the expression of different G-proteins in different tissues.

Question 7

Desensitisation and downregulation

Answer 7

Upon persistent receptor activation, G𝛽𝛾 subunits interact with G-protein receptor kinases (GRKs), which phosphorylate the intracellular side of the receptor. Other kinases can also phosphorylate GPCRs.
The phosphorylated receptor has higher affinity for a cytosolic protein, 𝛽-arrestin.
Binding of 𝛽-arrestin prevents the G protein from binding and continuing signalling. This is desensitisation.
𝛽-arrestin scaffolds the formation of a multiprotein complex, including AP2 and clathrin, that drives receptor internalisation.
In the endosome, the ligand and receptor dissociate and the receptor is dephosphorylated.
The GPCR can now be recycled to the cell surface or degraded in the lysosomes, which is downregulation.

Question 8

Measuring signalling

Answer 8

Can measure GPCR signalling through detection of G-protein activation of second messenger production.
We can detect G-protein activation via GTP𝛾S binding. This is a non-hydrolysable form of GTP. It harnesses the G protein cycle to accumulate active G𝛼 proteins.
GTP𝛾S can be labelled with radioactivity, such as 35-S, or fluorescence, such as BODIPY.
Filtration is used to separate bound and unbound GTP𝛾S. Detection and measurement of radioactivity or fluorescence retained in filters is a measure of G protein activation.

Question 9

FRET and BRET

Answer 9

FRET/BRET techniques rely on energy transfer between two fluorophores.
When two fluorophores are in close proximity, the donor emission can be absorbed by the acceptor, causing it to emit light at a different wavelength, which can be detected. Spectral overlap between donor emission and acceptor excitation is required when choosing the fluorophores.
In Fluorescence (Förster) Resonance Energy Transfer (FRET), the donor is a fluorescent molecule, so an external light source is required for excitation.
In Bioluminescence Resonance Energy Transfer (BRET), the donor is an enzyme that causes bioluminescence. A substrate is required.
FRET and BRET approaches are ideal for measuring real-time signalling in live cells.

Question 10

Using FRET and BRET to measure GPCR signalling

Answer 10

FRET/BRET can be used to measure GPCR signalling as many signalling processes involve protein-protein interactions, changes in the distance between 2 proteins (e.g. when recruitment occurs), or conformational changes.
We can measure:
1. Recruitment of miniaturised G protein (miniG) - uses a truncated, soluble G𝛼 subunit with no GTPase activity. The miniG is recruited to the agonist-bound GPCR → proximity produces a detectable signal.
2. Dissociation of G𝛼-GTP from G𝛽𝛾 - a signal is identified when the subunits are in close proximity and diminishes as the GPCR is activated and these dissociate.
3. Detection of released G𝛽𝛾 - a membrane-tethered donor with a high affinity for the G𝛽𝛾 subunit is used. An increase in BRET is seen following activation and dissociation as G𝛽𝛾 is free to move and can come in contact with the donor.
4. Recruitment of 𝛽-arrestin to phosphorylated GPCR - the donor is attached to the cytoplasmic face of the GPCR and signalling increases as labelled 𝛽-arrestin is recruited following receptor phosphorylation. This however only occurs with prolonged receptor activation.

Question 11

FRET/BRET cAMP sensors

Answer 11

Binding of cAMP induces a conformational change in the protein that changes the distance between the donor and acceptor, which are attached to different ends of the same protein.
Can be used to measure Gs and Gi activity.
cAMP binding proteins are used to measure cAMP changes in live cells. These proteins undergo a conformational change when cAMP binds, causing two fluorophores to move further apart, detected as a decrease in signalling.

Question 12

FRET/BRET calcium sensors

Answer 12

Gq-coupled receptors mobilise Ca2+ stores from the endoplasmic reticulum.
Calmodulin (CaM) and a calmodulin-binding protein are used to detect changes in Gq signalling. These can be on separate donor and acceptor fluorophores or two subunits on the same probe.
The presence of calcium allows these to bind, allowing the energy transfer to occur.

Question 13

FRET/BRET use for concentration-response graphs

Answer 13

FRET/BRET techniques can be used to detect GPCR activity, and the signal intensity obtained can be used to plot log concentration-response graphs.
This allows us to obtain the Emax and log EC50. However, these determinations are different depending on the assay used. The same ligand can create different concentration-response curves depending on how upstream/downstream of the receptor the measurement is and therefore how amplified this is.
For example, cAMP detection is more amplified than G𝛼-G𝛽𝛾 dissociation - many cAMP molecules are produced as the result of the dissociation of a single G-protein trimeric complex.
Assays that detect proximal effects to the receptor show little amplification and are therefore less sensitive
Further downstream, signals are amplified. These assays are more sensitive.
The assay used can produce different graphs for the same ligand. This can cause partial agonists to appear as full agonists when assays with more amplified endpoints are used.
We can however still tell the difference between a full vs partial agonist using multiple FRET/BRET assays measuring different endpoints if we know how to interpret the graphs.
- With a full agonist, the potency increases with amplification → left shift in the curve, reduction in log EC50.
- With a partial agonist, there is first an increase in the maximal effect. Potency only increases after this has reached 100%.
Receptor reserve can affect Emax and EC50 values in a similar way.

Question 14

Two-state model

Answer 14

In a simple linear two-state model, the receptor exists in either an inactive or active state.
Agonists drive the equilibrium towards the active state by stabilising this, while inverse agonists drive it towards the inactive state.
Antagonists prevent agonist binding without affecting equilibrium.

Question 15

Ternary complex model

Answer 15

Experimental observations did not fit the two-state model.
It was found that guanine nucleotides affect the affinity of agonists but not antagonists. The G protein plays an important role in the binding affinity of agonists.
The ternary complex model takes this into account. We only get a response when the receptor is in its active state, bound to the agonist, AND the G-protein is bound.

Question 16

GPCRs as allosteric proteins

Answer 16

Allosterism refers to a change in shape and activity of a protein that results from combination with another substance at a point other than the chemically active site.
The binding of ligands at allosteric sites affects the shape of the orthosteric site, therefore affecting the binding of the orthosteric ligand. This interaction can be positive, increasing affinity, or negative, decreasing it.
The interaction of GPCRs with G-proteins changes agonist affinity.
AND
The interaction of GPCRs with agonists affects G-protein binding.
Guanine nucleotides affect the binding affinity of agonists but not antagonists.
- We would expect all receptors to be either in the high affinity R* or the low affinity R state.
- This would mean that we can fit a single IC50
- However, what actually happens is that you have an equilibrium between the two states
- This does NOT fit the ternary complex model, as it suggests the presence of two affinities and therefore TWO receptor states that can lead to a response.
- We can force the low affinity state curve with high GTP concentrations, which causes G-protein uncoupling by activating said G-protein and allowing it to dissociate. If GTP concentrations are high enough, we will observe a single affinity which will correspond to the low-affinity R state. GTP affects receptor affinity states by affecting G-protein binding to the receptor; it creates a shift in the affinity of the receptor.
- Antagonists are not affected by G-protein binding.
- Intracellular GTP is normally high, so in (fluorescent) whole-cell assays, receptors are normally in the R state and the low affinity curve is obtained. In membrane assays, commonly done with radioligands, we can control GTP levels.

Question 17

Detecting ligand binding

Answer 17

Labelled ligands are required to detect drug binding to a GPCR. We can use radioisotopes for this, often used in saturation and competition binding.
Limitations of radioligands include:
- require separation by filtration
- not sensitive so require a high amount of material, e.g.cells or membranes
- radioactivity is a health hazard
- environmental issues with disposal
- endpoint assays, so no information about receptor kinetics is gained.
Fluorescent ligands have the following advantages:
- easier separation with washes or due to the probe sensitivity to the environment (may only fluoresce when close to the membrane and not in solution)
- single cell resolution
- safe
- real time information allows kinetic studies
- can be visualised with microscopy
Combination of fluorescent ligands with FRET/BRET approaches improves sensitivity and temporal resolution, as FRET/BRET will only occur for bound ligands. By harnessing resonance energy transfers, we improve the sensitivity and temporal resolution of these assays.

Question 18

Ligand binding: TR-FRET and nanoBRET

Answer 18

In TR-FRET the receptor is labelled with a SNAP-tag protein able to bind europium (Eu), which can transfer energy to a fluorescent ligand.
This eliminates the non-specific fluorophore binding, as Eu will not be present at these sites to excite the fluorophore.
Eu shows a delay in its energy release following light exposure, so we can temporally resolve emission and only account the one released after a certain period of time. This improves the signal to noise ratio.

In nanoBRET, the SNAP moiety is exchanged for a nano-luciferase. The substrate Furimazine is required for the production of bioluminescence.

Question 19

Extended Ternary Complex Model (ETC)

Answer 19

GTP experiments show that guanine nucleosides affect the binding affinity of agonists but not antagonists. For agonists, there are two affinities and therefore TWO receptor states that can lead to a response.
GPCRs also have constitutive activity. This has been demonstrated by receptor mutants which show an increase in signalling in the absence of an agonist.
The extended ternary complex (ETC) model suggests that GPCRs spontaneously form activated states capable of producing a response through G-proteins in the absence of agonist.

Question 20

Inverse agonists

Answer 20

Constitutive activity explains the mechanism of action of inverse agonists.
Inverse agonists selectively bind to the inactive state of the receptor.
If any receptor happens to be in an active state spontaneously, then an inverse agonist will reverse the resultant constitutive activity.
- Constitutive activity may only be detected in certain assays (e.g. depending on amplification)
- If a receptor has no constitutive activity, the inverse agonist will just act as an antagonist.
Pharmacological effect of inverse agonists is antagonism. The effect on constitutive activity is only relevant if the system is spontaneously active (to a significant degree).
Inverse agonists have negative efficacy.
Constitutive activity may be physiologically important.
- Some viruses (e.g. Herpes virus) express orphan GPCRs that have high constitutive activity and harness cellular machinery to signal toward proliferative signalling (e.g. cancer).
- Some activating mutations that induce high constitutive activity are the cause of disease, e.g. those in CaSR cause hypoparathyroidism.
Some drugs described as antagonists may in fact be inverse agonists for GPCRs of unappreciated or very small constitutive activity (e.g. antihistaminic drugs).
- CA not initially revealed as the assay used was not sensitive enough

Question 21

Receptor reserve and signal amplification

Answer 21

If there’s no receptor reserve, %occupancy = %response. If there is reserve, response > occupancy.
If there is receptor reserve, there is an excess of receptors relative to the cellular signalling machinery. Receptor reserve refers to the condition in a tissue whereby the agonist needs to activate only a small fraction of the existing receptor population to produce the maximal system response.
Cellular systems with receptor reserve are much more sensitive to agonists.
There is a similar relationship with signal amplification.
In the case of signalling amplification, this looks different depending on where we look within the signalling cascade (more upstream vs more downstream).
Both receptor reserve and signalling amplification can happen at the same time.
Different cells will have different receptor expressions and signalling pathways, so the same drug can have different effects in different cells/tissues.
Because EC50 and Emax are determined by system factors as well as drug-receptor factors, these values are dependent on the system used to determine them. Relative agonist effects can depend on receptor reserve and signal amplification.

Question 22

Law of mass action and Operational Models

Answer 22

GPCRs - “Efficacy and biassed agonism” lecture - equation-heavy, so hard to make into flashcards

Question 23

Importance of efficacy

Answer 23

Third generation antipsychotics, such as aripiprazole, are low efficacy dopamine receptor partial agonists rather than antagonists/inverse agonists. This avoids extrapyramidal symptoms.
Salmeterol is a partial agonist selective for the 𝞫2 adrenergic receptor. Partial agonist action may be important for agonist efficacy.
Low efficacy partial agonist opioids might be safer analgesics, avoiding respiratory depression.

Question 24

GPCR biased agonism

Answer 24

Looking at the GLP-1 receptor, the agonist PACAP-27 was more potent than PACAP-38 in the assay measuring cAMP production (Gs), but, when inositol phosphate production (Gq) was measured, PACAP-38 was actually more potent.
This would not be predictable from the operational model, as we would expect the order of potency to be maintained between assays.

Propranolol assays at the beta-2 receptor found that this had an inverse agonist function following isoprenaline activation, as expected. However, a SPAP reporter gene assay found that propranolol is actually an agonist in this pathway.
Propranolol is an agonist in one pathway and antagonist in another.
Propranolol must be stabilising a conformation of the receptor that selectively activates one signalling pathway, but not the other. This is biased agonism.

In the classical model of drug action, agonists, whether partial or full, produce a common active state to induce uniform relative activation of the pathways linked to the receptor.
In biased agonism, different agonists acting at the same receptor stabilise distinct conformations that differentially engage downstream effectors.
If distinct intracellular signalling pathways downstream of a receptor are linked to therapeutic and unwanted side effects, then biassed agonism provides a way to design safer pathway selective drugs that avoid these ‘on target’ side effects.

Question 25

AT1R and heart failure treatment

Answer 25

Angiotensin II acts at AT1 receptors to increase BP.
The RAAS system can be inhibited in the treatment of hypertension. This is often done with ACE inhibitors or AT1 receptor blockers (ARBs), also called sartans, such as losartan.
In chronic heart failure, ARBs inhibit the action of angiotensin II, reducing vascular resistance, improving tissue perfusion and reducing cardiac afterload.
However, ARBs are not used in acute heart failure (AHF) because they decrease cardiac output and cause sustained hypotension.

Question 26

siRNA

Answer 26

Small interfering RNA (siRNA) induces silencing of protein coding genes.
siRNA is a synthetic RNA duplex designed to target a specific mRNA for degradation.
Once the siRNA enters the cell, it’s incorporated into other proteins to form the RNA-Induced Silencing Complex (RISC). It is then unwound to form single-stranded siRNA, which can bind to a complementary mRNA sequence and induce its cleavage.
The cleaved mRNA is recognised as abnormal by the cell and degraded.

Question 27

Angiotensin II analogues

Answer 27

SII-angiotensin is an angiotensin II analogue where the first 3 residues have been replaced.
In model cell lines, Ang II activates the Gq pathway, but SII does not. Both angiotensin II and SII are however able to stimulate ERK phosphorylation downstream of 𝛃 arrestin.
This led to a model suggesting that angiotensin binding leads to not only Gq activation, but also arrestin recruitment.
Arrestin is not only a regulatory protein, but it is also able to complex its own signalling pathway leading to ERK signalling and cell proliferation.
Both Ang II and SII cause shortening of cardiomyocytes, which is indicative of positive ionotropic effects.
In the case of SII, but not Ang II, shortening of cardiomyocytes can be completely inhibited by genetic knockout of 𝛃-arrestin 2.
Therefore, Gq and 𝛃 arrestin 2 dependent signals downstream of the AT1R control myocyte function, but SII activates only 𝛃 arrestin 2-dependent signals.
This lead to the hypothesis that a 𝛃 arrestin biassed agonist at the AT1R will exert the renal and vascular effects of an ARB (through competitive inhibition of Gq signalling), with the additional benefits of enhanced myocardial contractility and survival provided by 𝛃-arrestin-2 engagement and pro-proliferative ERK signalling.
These biased agonists will NOT cause vasoconstriction and fluid retention like Ang II, but in fact cause vasodilation and reduced Na+/fluid retention.
So, we can use a biassed agonist in the treatment of chronic heart failure rather than an antagonist.

Question 28

TRV120027

Answer 28

TRV120027 is a derivative of SII designed as a 𝛃-arrestin pathway biased agonist at AT1 receptors for the treatment of heart failure.
TRV causes arrestin recruitment but no Gq signalling. It also competes with ang II to block Gq signalling.
TRV causes cardiomyocyte shortening comparable to Ang II.
TRV was able to cause a reduction in mean arterial blood pressure (MAP) and no change in heart rate in live rats, having comparable effects to telmisartan. However, TRV has the advantage of causing an increase in cardiac contractility, while telmisartan causes a decrease, which is undesirable in the treatment of heart failure.
Telmisartan but not TRV120027 decreased stroke volume, making TRV the better agent.
RV120027 therefore seems like a very promising therapeutic agent for heart failure.
TRV moved into phase 2B clinical trials. However, looking at the means of the TRV and placebo, they actually found no significant effect in any of the measured parameters, including shortness of breath, rehospitalisation, worsening of heart failure and mortality. There were no safety issues, but TRV did not confer any benefit over placebo at any dose → the drug was safe, but not efficacious.
This is the result of 95% of all drug developments. This can occur due to incomplete understanding of disease states meaning that our preclinical models are not accurate. This is still the closest a biased agonist has gotten to clinical use.

Question 29

Biased 𝜇 opioid receptor agonists for pain - theory

Answer 29

It was found that knocking out arrestin sustained the analgesic effects of morphine and so decreased tolerance development, AND decreased the degree of respiratory depression.
This led to the theory that the G-protein pathway controlled analgesia, while the arrestin pathway was responsible for side effects.

Question 30

Quantifying agonist bias

Answer 30

This section uses dopamine receptors as an example.
Receptor bias becomes harder to quantify when both drugs activate different pathways, but by different extents.
This is because system and observation bias (relative sensitivity of the assay used) starts to come into play. Graphs are also difficult to interpret.
To quantify this, we apply the operational model to the dose-response curves to obtain a ratio between 𝜏 and Ka.
Each agonist will have its own ratio for each pathway. We then normalise these ratios to a reference agonist, such as dopamine, to remove the influences of system and observation bias.
We can then obtain logBias by taking away the (normlised) ΔLog ratio of pathway B from that of pathway A. This gives us the degree by which the compounds prefer one pathway over another in a quantitative manner.

Question 31

Exploiting biassed agonism for safer MOR analgesics

Answer 31

A group used bias quantification for MOR agonists and they found that some compounds that are very biased for G-protein activation over the arrestin pathway have a larger therapeutic window and are therefore safer.
However, other groups found that if you knock out arrestin, mice still suffer from respiratory depression, and that this may actually be mediated by the G-protein pathway.
Testing MOR agonists at different levels of G-protein activation and arrestin recruitment found that there was no actual ligand bias observed → conflicting evidence.
A different explanation was therefore needed. This was that the weaker the partial agonist, the safer the drug, while no correlation was found with G-protein bias.
A paper actually found that, by analysing opioids that caused overdose, some of the more deadly compounds are very efficacious in the G-protein pathway and only partially efficacious in the arrestin pathway. This again provides conflicting evidence.
The biased agonism hypothesis doesn’t really fit the approach of making these drugs safer. Simpler mechanisms, like optimising the degree of partial agonism, may actually be more useful.

Question 32

Closed and open systems

Answer 32

Closed systems are used to define drug affinity.
In a closed system, the drug and target are present at constant concentrations. This allows an equilibrium to be established between bound drug, free drug and the target.
Closed systems therefore allow determination of parameters of target engagement such as KD as a measure of affinity.
In open systems, such as the human body, the concentration of drug available for interaction with the target in a particular tissue or cell is in constant flux.
This is driven by systemic circulation, distribution into tissue, metabolism, and diffusion within the tissue to cells harbouring the target.
Therefore, equilibrium measures of drug–target interactions may not be predictive of drug action in open systems.
The length of time the drug interacts with the receptor for, i.e. the residence time, determined by kinetic parameters, may be important too.

Question 33

The association constant

Answer 33

kon is a bimolecular or second order rate constant as it describes the rate at which two molecules, the drug and the receptor, bind to each other, estimating the rate of drug-target complex formation.
It is therefore, expressed as M−1 s−1
Forward binding occurs when the drug and receptor collide due to diffusion. The rate is proportional to the concentration of drug [D] and receptor [R].
The rate constant kon reflects that not every collision leads to binding.

Encounter limited binding indicates that before a reaction can occur, the reactants must undergo some degree of reorganisation, e.g. reorientation and/or desolvation.
If the diffusion rate is very slow, diffusion limited binding can occur, where the reactants need only collide with one another for binding to occur.

Question 34

The dissociation constant

Answer 34

koff describes the dissociation of a single species. Therefore, it is a unimolecular or first order rate constant.
koff is entirely dependent on specific interactions between the drug and its target. In the case of GPCRs this is often a binding pocket.
The rate of dissociation is only proportional to the concentration of the drug-receptor complex [DR].
koff is independent of the local concentration of free drug
It is expressed in units of s−1

Question 35

Measuring dissociation rate

Answer 35

We can measure dissociation by blocking reassociation.
We add a known concentration of a labelled drug and the receptor of interest. These are allowed to reach equilibrium.
The influence of rebinding can then be removed by:
- Isotopic dilution - once equilibrium is reached, a high concentration of a high-affinity unlabelled ligand is added, which immediately binds to any free binding sites.
- Infinite dilution - we dilute our reaction >100-fold, so that when the labelled drug unbinds it is at such a low concentration that it does not bind again.
The reaction can be separated at different timepoints to obtain a curve.
The bound and unbound drug has to be removed by filtration.
Drug dissociation from a GPCR follows a monophasic exponential dissociation.
Y = Span.e^(-KX) + plateau
The dissociation half-life, t1/2, is an experimental measurement of the duration of a drug-target complex, or a drug’s residence time.
t1/2 - 0.693/kodd

Question 36

Measuring association rate

Answer 36

Measuring the association rate is more difficult, as in the observed association phase, dissociation is still playing a role.
We measure the amount of drug bound by stopping the reaction at different time points, but this is influenced by both kon and koff.
We fit a curve which can give us the equation:
Y = Ymax.(1 - e^(-kobs.X))
The value obtained is actually the observed association rate, kobs, but you can use this to obtain the actual association rate, kon.
The observed association rate, kobs (min-1), is dependent on kon but also koff and the drug concentration [D].
To work out kon from kobs we can use the equation:
𝑘𝑜𝑛 = (𝑘𝑜𝑏𝑠 − 𝑘𝑜𝑓𝑓) / [𝐷𝑟𝑢𝑔]

Question 37

Measuring kon if you don’t know koff

Answer 37

𝑘𝑜𝑛 = (𝑘𝑜𝑏𝑠 − 𝑘𝑜𝑓𝑓) / [𝐷𝑟𝑢𝑔]
If you do not know koff you could also perform an association experiment with multiple known concentrations of your drug.
kobs will vary with [D], whereas kon and koff will be constant, as they are intrinsic properties of the drug-receptor interaction.
We can ‘globally’ fit multiple association curves of the same drug at different concentrations to obtain these parameters.
Then solve using simultaneous equations.

Question 38

Measuring unlabelled ligands

Answer 38

Labelling is expensive and impractical to do with a large number of ligands in screening.
Labelling may also alter the drug kinetics.
If we know the kinetics of a labelled tracer ligand we can perform competition experiments to obtain the kinetics of the unlabelled competing ligand.
Competition kinetics assays use a single concentration of labelled tracer ligand and increasing concentrations of our unlabelled drug of interest.
The time taken for a single concentration of the tracer to reach equilibrium will depend on the concentration of the tracer and competing drug, and the koff and kon of these.
We can analyse competition kinetic assays to determine the koff and kon of an unlabelled drug using the Motulsky-Mahan Method.

Note: if the koff of the competing ligand is slower than that of the tracer, the specific curve will ‘overshoot’ and go up before coming back down and plateauing.

Question 39

Resonance energy transfer

Answer 39

Classical methods using radioligands require separation of the bound and free ligand using filtration or centrifugation, so a new reaction tube must be created for each timepoint. This is laborious, time consuming and expensive.
Newer methods make use of resonance energy transfer (RET) based methods
A signal is only observed when the fluorescent ligand and energy source, which is a fluorescent or bioluminescent protein, are in close proximity (< 10 nm), so there is no need for separation. This also offers greater temporal resolution.
Time-resolved fluorescence resonance energy transfer (TR-FRET) utilises proteins, such as SNAP, which are then fused to the receptor of interest to create a tagged receptor. Tagged receptors are labelled with a lanthanide substrate, such as terbium cryptate or europium, which forms a covalent bond.
FRET detection occurs following the transfer of energy from the terbium donor to an acceptor fluorophore attached to a ligand selective for the receptor of interest.
Elements such as terbium (Tb) and europium (Eu) have long-lived fluorescence. This allows for a time delay between excitation and measurement of the resulting fluorescence, which reduces auto-fluorescence and improves the signal to noise ratio.
In bioluminescence resonance energy transfer (BRET), the receptor is tagged with the small, bright luciferase, NanoLuc, which in the presence of its substrate furimazine produces bioluminescent light. If in close enough proximity (< 10 nm), the resonance energy from the NanoLuc can be transferred to a fluorescent ligand bound to the receptor. This results in emission at a different wavelength.

Question 40

Surface plasmon resonance

Answer 40

FRET and BRET require a fluorescent ligand and modification of the receptor, which could affect receptor-ligand interactions.
Surface plasmon resonance (SPR) is a label free approach.
The protein of interest is immobilised on a coated gold chip, which is then exposed to the compound of interest under continuous flow.
Ligand binding to the receptor immobilised on the chip’s surface induces a change in the refractive index on the sensor surface, which can be measured.
A curve is plotted and different parts of this curve will give us the kon and koff values.
Such biophysical techniques have not been extensively used on GPCRs, as these receptors are integral membrane proteins and rapidly deteriorate when taken out of their membrane environment.

Question 41

Drug-target residence time

Answer 41

Drug-target residence time, represented by the dissociation half-life, refers to the duration a drug molecule remains bound to its target. It is a measure of the stability of the drug-target complex.
This metric is increasingly recognised as potentially more crucial than a drug’s affinity for its target, particularly in living organisms where drug concentrations constantly fluctuate due to ADME processes.
Traditionally, drug development primarily focused on equilibrium-derived parameters such as affinity (Ki) or potency (IC50). However, in vivo environments are more dynamic, and RT is a more dynamic parameter.
Longer RT is often associated with more profound and sustained effects.
Optimising RT can contribute to enhanced drug efficacy by prolonging target engagement, leading to longer-lasting therapeutic effects.
Mathematical models have been developed to predict the duration of pharmacological effects based on a drug’s RT, enabling a more comprehensive understanding of drug action in vivo.
Investigating structure-kinetic relationships (SKRs), which explore the connection between a drug’s chemical structure and its binding kinetics, is crucial for optimising drug-target RT during drug development. SKR studies can be combined with traditional SAR studies for a more comprehensive understanding of drug-target interactions.

Question 42

Why is it important to understand kinetics?

Answer 42

Equilibrium conditions are not always possible when measuring GPCR activation. For example, calcium mobilisation is pulsatile and transient.
The system may become desensitised before equilibrium is reached.
Different cellular signalling events will have distinct kinetic profiles. For example, calcium mobilisation is a short event, while changes in gene transcription, measured using reporter gene assays, are long events.
If drugs with slow dissociation rates are measured before equilibrium is reached then their potency will be underestimated.
Consideration should be given to the influence of kinetics in observations of biassed agonism, particularly when measuring agonist action at two different signalling endpoints at different times.

Question 43

Assumptions of equilibria for models of competitive antagonists

Answer 43

Gaddum/Schild analysis assume that sufficient time is allowed for equilibrium to be established among the receptors, the agonist, and the antagonist.
The time required to achieve equilibrium with an agonist in the presence of an antagonist may be much longer than the time required for only the agonist if the koff of the antagonist is much slower than that of the agonist.
- The trace goes up before going back down again - the initial phase is because the competitive antagonist with a slow dissociation rate hasn’t had time to reach equilibrium.
For antagonists with a slow dissociation rate, there are implications of drug binding kinetics at hemi-equilibrium conditions.
The antagonist is not irreversible, but it dissociates really slowly, so a long time is required for equilibrium to be reached. At hemi-equilibrium conditions, the Emax appears to be lower, which can lead us to interpret the agonist to act in an irreversible manner if we do not have a better understanding of the drug’s kinetics.

Question 44

The action of LAMAs for the treatment of COPD

Answer 44

Long-acting muscarinic antagonists (LAMAs), such as tiotropium, are used for the treatment of COPD, which involves inflammation, airway remodelling and airflow limitation.
M1 receptors are found in parasympathetic ganglia and their blockade results in reduced reflex bronchoconstriction.
M3 receptors mediate the bronchoconstrictor and mucus secretion action of ACh. The most important effects of anticholinergics in the treatment of COPD are mediated through antagonism of M3 receptors.
M2 receptors are located at cholinergic nerve terminals and act as autoreceptors to inhibit ACh release, so blockade increases ACh and causes bronchoconstriction. Systemic M2 antagonism in the heart would also increase the risk of tachycardia.
We therefore want M1/3 selective antagonists, but the orthosteric site is highly conserved.
All LAMAs have similar affinities for all mAChR subtypes.
Dissociation kinetics of LAMAs are what underlies an improved side effect profile. All LAMAs dissociate more rapidly from M2 autoreceptors than from M3 receptors, so M3 RT is longer, which is proposed to lead to an improved side effect profile.
The better the kinetic selectivity, the shorter the duration and lower the effect to increase heart rate. Aclidinium has a shorter residence time at the M2 receptor than tiotropium, so the increases in heart rate caused by aclidinium are not as large and they are also more transient.
The duration of action of LAMAs is predicted by their relative dissociation rate from M3 receptors.
The drugs have kinetic selectivity for the receptor subtypes.

Question 45

Antipsychotic binding kinetics and side effect profile

Answer 45

The dopamine hypothesis proposes that Schizophrenia is related to an imbalance in dopamine signalling.
Positive symptoms are caused by hyperactive mesolimbic projections to the striatum and NucAcc, resulting in D2 hyperstimulation.
Negative and cognitive symptoms are caused by hypoactive mesocortical projections to the prefrontal cortex, resulting in D1 hypostimulation.
All antipsychotics currently used are D2 receptor antagonists.
On-target side effects include extrapyramidal side effects (EPS) like parkinsonism due to blockade of nigrostriatal D2Rs, and hyperprolactinemia due to blockade of D2Rs in the tuberoinfundibular pathway.
Atypical antipsychotics like clozapine have a lower tendency to cause EPS, but the reason why is unclear.
The difference in side effects seen with atypicals may be explained by the fast off hypothesis. Drugs like clozapine have a really fast dissociation rate compared to drugs like haloperidol.
Fast dissociating antagonists can be displaced by transient increases in dopamine concentrations at the synapse, maintaining some dopaminergic signalling patterns and so avoiding EPS. Drugs like haloperidol stay on the receptor for a very long time, acting almost like irreversible antagonists.
Kinetic analysis was carried out on antipsychotic drugs. They then looked at whether the kinetic of these drugs predicted their likelihood of causing side effects through meta-analysis of patient reports.
Prolactin release is correlated with koff but not kon. There is a decrease in release as logkoff increases.
The slower the dissociation rate (koff), the more likely the drug to cause hyperprolactemia.
The same relationship does NOT apply for EPS, as expected.
Extrapyramidal side effects are correlated with kon but not koff. There is an increase in the EPS odds ratio as logkon increases.
The faster the association rate (kon), the more likely the drug to cause extrapyramidal side effects.
To avoid both types of side effects we therefore want a fast koff and slow kon → short residence time at the receptor.
This phenomenon may be explained by drug rebinding.

Question 46

Drug rebinding

Answer 46

Drug-receptor binding models assume free diffusion of drugs. However, in certain tissue microenvironments, this assumption may not be valid.
The small aqueous compartment within a dopamine synapse is unlikely to mix well with the bulk aqueous environment of plasma.
Rebinding is the process whereby a reversibly bound ligand dissociates from a receptor into the local aqueous environment but then rebinds the same or a nearby receptor before it is able to diffuse from the synaptic cleft. It maintains high drug concentrations near the receptor.
The degree of rebinding is determined by receptor density, the kon, and anatomical and physicochemical factors.
The faster the kon the more likely for rebinding to occur.
In the hypothalamic–pituitary portal system, there is free diffusion of dopamine and the drug as these are carried away in the blood, so rebinding is negligible. Therefore, koff drives duration/level of D2R blockade and so koff is predictive of hyperprolactinemia.
At the striatal DA synapse, there is a small aqueous compartment and diffusion out of this into the bulk aqueous environment is slow. This leads to high drug concentrations in the synapse, so rebinding can occur, controlled by kon. A fast kon causes sustained D2R blockade and therefore EPS, so kon is predictive of EPS.

Haloperidol has a slow koff and fast kon → more side effects
Clozapine has a fast koff and slow kon → less side effects
This hypothesis does not exclude other mechanisms, such as polypharmacology: antipsychotics are termed ‘dirty drugs’ as they act as receptors other than DA receptors - e.g. clozapine acts at ~20 receptors, which may contribute to side effects.

Question 47

Allosteric site location

Answer 47

The allosteric site is a binding site on a receptor that is non overlapping and spatially distinct from, but conformationally linked to, the orthosteric site.
Allosteric sites can be found wherever the orthosteric ligand-binding site is not, so the location depends on the GPCR family.
These can be found:
- Above the orthosteric binding site in family A (rhodopsin)
- At the bottom of the receptor in family B (adhesion/secretin)
- For family C (glutamate), the orthosteric site is in the venus flytrap domain (VFTD), so the allosteric site can be in the helical bundle
These are just examples, and the allosteric site can be found at different locations too.

Question 48

Advantages of allosteric drug discovery

Answer 48

Increased subtype selectivity - less conserved, might not even bind any endogenous ligands and can therefore offer great variety.
Endogenous spatiotemporal rhythms are maintained - e.g. orthosteric antagonists show persistent receptor binding only dependent on drug concentrations and PK/PD, but allosteric modulators still allow for endogenous agonist binding and only modulate the response down.
Saturability of effect/ceiling level effect as the extent of modulation is limited by cooperativity → increased therapeutic window, decreased overdose risk.

Question 49

GPCRs as allosteric proteins

Answer 49

The interaction of GPCRs with G proteins increases agonist affinity
The interaction of GPCRs with ligands increases G protein binding
This is due to allosterism
GPCRs are allosteric proteins, which is how agonist binding at the extracellular surface causes G protein activation at the intracellular surface.
Guanine nucleotides affect the binding affinity of agonists but not antagonists. This is because GTP causes the G protein to dissociate from the receptor, so we lose the high affinity R* state.

Question 50

Allosteric parameters

Answer 50

KA is the affinity of the orthosteric ligand for the orthosteric site
KB is the affinity of the allosteric ligand for the allosteric site
𝜏B is the intrinsic efficacy of the allosteric ligand - allosteric ligands can sometimes activate the receptor on their own.

The binding of the allosteric ligand can change the conformation of the GPCR.
This change in conformation can change the affinity of the orthosteric ligand for the allosteric ligand-bound receptor.
𝛼 is a ‘cooperativity factor’ that indicates how much the binding of the allosteric ligand affects the binding affinity of an orthosteric ligand.
This change in conformation can also change the ability of the orthosteric agonist to activate the allosteric ligand-bound receptor.
𝛽 is a ‘cooperativity factor’ that indicates how much the binding of the allosteric ligand affects the efficacy of an orthosteric ligand.
We can potentially tune allosteric modulators to change their effect on affinity and/or efficacy.
Therefore, the action of an allosteric modulator at a GPCR can be defined in terms of KB, 𝛼, 𝛽 and 𝜏B.

Question 51

Allosteric ternary complex model (ATCM)

Answer 51

See GPCR ‘Allostery’ lecture.
The ternary complex is formed of the receptor, agonist and allosteric ligand: ARB.
The binding cooperativity factor α affects the formation of this complex.
Different α values tell us different things about cooperativity
- α > 1 positive cooperativity
- 0 < α < 1 negative cooperativity
- α = 1 neutral cooperativity

Question 52

Allosteric modulator titration binding assay

Answer 52

Allosteric ligands can either increase or decrease the binding of an orthosteric ligand.
These assays are similar to a competition binding assay.
We add a single concentration of labelled orthosteric ligand with a known affinity for the target and increassing concentrations of our unlabelled allosteric ligand. Measurements are taken at equilibrium.
The allosteric ligand does not compete with the orthosteriv tracer, but it changes its affinity for the receptor. The allosteric sites can become saturated, so a plateau is eventually reached.
Cooperativity determines the amount of orthosteric ligand bound, and we can see an increase, decrease or no change in specific binding.
A PAM with an α of 10 increases the affinity of A 10-fold. This means the same concentration of orthosteric ligand now occupies a higher proportion of receptors, so specific binding increases.
A NAM with an α 0.1 would decrease the affinity of A 10-fold. This causes a decrease in binding, but the effect is saturable as allosteric sites become fully occupied. This distinguishes a NAM from a competitive ligand.
A silent allosteric modulator (SAM), also called a neutral allosteric ligand (NAL), with an α of 1 would cause no change. These are usually used to compete with PAMs or NAMs when we want to reverse their effects.
The affinity of A in the presence of an allosteric ligand is given by KA/α. Remember that KA is the concentration required for 50% receptor occupancy at equilibrium, so the smaller the KA the higher the affinity.

Question 53

Quantification of the ATCM

Answer 53

See GPCR ‘Allostery’ lecture.
The fractional occupancy (Y) of the receptor by orthosteric ligand A in the presence of allosteric modulator B can be fitted to binding data to get parameters of allosteric ligand affinity (KB) and cooperativity (𝛂).

Question 54

Allostery and unlabelled drugs

Answer 54

See GPCR ‘Allostery’ lecture.

We can carry out competition binding experiments using a labelled antagonist and increasing concentrations of an unlabelled agonist to obtain a displacement curve.
We can then carry this out in the presence of increasing concentrations of an allosteric modulator to observe the effects on the dissociation curve.
- Two orthosteric ligands and one allosteric ligand are used.
- The allosteric ligand will have an effect on both orthosteric ligands → we will obtain two 𝛂 values
We can use these displacement curves to obtain the KB and two 𝛂 values of the allosteric ligand.
- 𝛂 is the affinity cooperativity factor for the LABELED antagonist
- 𝛂’ is the affinity cooperativity factor for the UNLABELED agonist
Looking at 15f, this is a NAM of [3H]NMS antagonist affinity but a PAM of ACh agonist affinity.
This is probe dependence - the effect of the modulator is dependent on the orthosteric ligand used.
The allosteric ligand 15f promotes the R* conformation of the receptor, so it prefers to bind the agonist-bound over the antagonist-bound receptor, which is found in the R state.

𝛂 affects the specific binding at the start of the curve - 15f decreased antagonist affinity, so the specific binding at the beginning, before ACh is added, was decreased.
𝛂’ shifts the curve left or right - 15f increases the affinity of ACh, so it causes a (limited) left shift in the curve

Question 55

IC50 and allosterism

Answer 55

For a competitive antagonist, IC50 is related to the concentration of the tracer ligand in a displacement assay. This is a Cheng-Prusoff relationship.
For noncompetitive antagonists, IC50 does not change.
Allosteric ligands can be an amalgam of competitive and non-competitive antagonists in terms of the relationship between IC50 and tracer ligand concentration, as allosteric antagonism is saturable.
Binding kinetics are more appropriate for the quantification of allosteric effects.

In general, the kinetics of most allosteric modulators have been shown to be faster than the kinetics of binding of the tracer ligand.
Therefore, the rate of offset of the tracer ligand in the presence of various concentrations of allosteric ligand can be used to detect allosterism and to quantify both the affinity (1/KB ) and 𝛂 value for the allosteric ligand.
Negative allosteric modulators will generally decrease the rate of association and/or increase the rate of dissociation of the tracer ligand.

Question 56

Probe dependence

Answer 56

An allosteric agonist may stabilise the R* of the receptor, favouring the binding of agonists but not antagonists.
However, probe dependence can also be seen between different agonist molecules.
For example, an allosteric antagonist may block the binding of one agonist but have no effect on the binding of another agonist.
- We saw differences in effects between agonists and antagonists, but there can also be differences with drugs of the same class.
Unlike orthosteric antagonists, which produce a uniform effect on all agonists, allosteric modulators can produce different effects on different agonists. This means that allosterically modified receptors may have different conformations from each other, which could lead to differences in resistance profiles with chronic treatment.

Question 57

Allosteric modulator examples

Answer 57

Two modulators have made it onto the clinic:
Maraviroc, a NAM at the CCR5 chemokine receptor (class A) used to treat HIV.
Cinacalcet, a PAM at the class C calcium sensing receptor (CaSR) used to treat osteoporosis and parathyroidism.

Other allosteric modulators developed include:
Gallamine - NAM of muscarinic receptors. It produces surmountable antagonism of acetylcholine, shifting the concentration-response curve to the right without affecting the maximal response.
Alcuronium - PAM of muscarinic M2 receptors that is not blocked by the classical muscarinic antagonist QNB.

Question 58

Dissociation binding assays and allosteric modulators

Answer 58

Kinetic binding studies can be used to detect an allosteric mechanism of action.
A change in the orthosteric ligand dissociation rate indicates an allosteric interaction.
The binding of an allosteric modulator can change the conformation of the receptor, changing the affinity of the orthosteric ligand for the orthosteric site and therefore the rate of dissociation.
Competitive ligands cannot change the rate of dissociation because competitive ligands will only bind once the endogenous ligand has already dissociated.
Therefore, a change in the koff rate is a signature of an allosteric interaction.
The kon may also change when an allosteric ligand is used
Negative modulators increase (speed up) the dissociation rate, while positive modulators decrease it.
Equilibrium binding assays may lead us to believe that a NAM with a high negative cooperativity is a competitive antagonist as it leads to a decrease in specific binding, seeming to displace the endogenous ligand. However, the modulator is actually just increasing the koff. This is why kinetic studies are needed.

Question 59

Allosteric modulation and receptor function

Answer 59

It’s the effect of a modulator on receptor function, i.e. its action to engage downstream signalling, that will determine its effect, so effects on orthosteric drug affinity are not the whole story.
We must consider how the allosteric modulator affects agonist efficacy and whether it has efficacy in its own right.

Question 60

PAMs

Answer 60

A PAM can have effects on agonist affinity, agonist efficacy and be an agonist in its own right (an allosteric agonist).
A PAM can increase agonist potency, shifting concentration-response curves to the left. The shift is limited as allosteric sites become saturated.
A PAM can also increase efficacy, increasing the maximal response.
When a PAM is an agonist in its own right (𝛕B>0), the baseline response at the beginning of the curve increases (no longer 0). As at the start of concentration-response curves there is not enough orthosteric ligand to cause an effect, we know that this is due to intrinsic efficacy of the allosteric ligand, which activates the receptors.
Information is fed back to chemists so they know how different changes are affecting allosteric ligand function, and fed forward in preclinical models to see how these different modulation mechanisms change disease states.

We can use an updated operational model of allosterism to quantify effects. This is a functional interaction study. → see GPCR ‘Allostery II’

Question 61

The Schild Plot

Answer 61

In concentration-response curves, there is a parallel rightward shift with increasing concentrations of a competitive antagonist.
There is no decrease in Emax
We can use this shift to work out the affinity of our antagonist.
The Gaddum equation is:
CR-1 = [B]/KB
The logarithmic form of this is the Schild equation:
Log(CR-1) = Log[B] - LogKB
This is a linear equation with a slope of 1 if our antagonist is competitive.
The x-intercept gives you LogKB.

Question 61

NAMs

Answer 61

When a NAM is used in a concentration-response curve, there is a limited decrease in agonist potency with increasing antagonist concentrations → the rightward shift reaches a limit.
This occurs as the allosteric antagonist binding sites are saturated, but the agonist is still able to bind, even if affinity might be lowered. This is limited by the 𝛂 effect.
We can fit NAM curves with the operational model to obtain 𝛂.
Plotting this on a Schild plot gives us a curvilinear line, which is characteristic of a NAM.
If we try to plot the Schild plot of a NAM using linear regression, the gradient won’t be 1.
NAMs can also decrease Emax. These compounds would give a 𝛃<1 when fitted with the operational model.
A decrease in Emax can also be a result of:
- The action of irreversible antagonists
- The action of competitive antagonists in hemi-equilibrium conditions
Multiple experiments, including kinetic studies, would be needed to confirm an allosteric mechanism of action - remember NAMs can affect the rate of dissociation of an orthosteric agonist, but orthosteric antagonists cannot.
NAMs can also have negative intrinsic efficacy, acting similarly to inverse agonists at the orthosteric site by switching off basal signalling.

Question 61

Probe dependence

Answer 61

Probe dependence refers to the fact that the allosteric effect can change depending on the orthosteric ligand used.
But why does this happen?
Structurally different agonists could cause slightly different conformational changes in the receptor.
The allosteric drug also stabilises a distinct receptor conformation. The allosteric modulator promotes a conformation of the receptor that is favourable for the binding of one orthosteric ligand but not the other.