L12 - Novel avenues for GPCR drug discovery

Question 1

what is biased signalling

Answer 1

concept that different system components stabilize different receptor
conformations, leading to differential activation or inhibition of effectors.

Question 2

Ligand bias signalling

Answer 2

ligand stabilizes a unique conformation that results in differential effector coupling and a biased response - most relevant for drug discovery

Question 3

Receptor bias

Answer 3

modification of a receptor (e.g. through mutations, differential splicing, PTMs), which alters its interaction with ligands and/or effectors

Question 4

system bias

Answer 4

differential expression of effector elements biases specific signalling outputs over others

Question 5

Oliceridine (TRV130) as a biased ligand

Answer 5

Oliceridine in analgesic treatment is used to stabilise the receptor in a conformation that favours the g -protein signalling pathway the causes pain relief.

This is opposed to typical morphine use that activates both g protein and beta arrestin pathways that are associated with negative side effects of opioid use

Question 6

Allosterism

Answer 6

the phenomenon whereby binding of a
ligand other than at the orthosteric site (the site where the endogenous ligand binds) influences receptor function

Question 7

how does allosterism influence receptor function

Answer 7

1. Direct modulation of receptor conformation leading to effector coupling
2. Altering the receptor conformation induced by an orthosteric ligand, thereby altering the orthosteric ligand’s effects (allosteric modulators)

Question 8

4. Advantages of targeting GPCRs allosterically

Answer 8

1. Preservation of physiological signalling with AMs- maintains natural spatio temporal rhythms of endogenous ligands, limiting side effects
2. Effect saturation with AMs - complete occupancy (saturation) means there can be no overdosing
3. Greater selectivity at GPCR subtypes - Greater variance in the amino acid sequence in the allosteric binding pockets
   - selective cooperativity (interaction) between the allosteric and orthosteric site (and/or effector coupling sites)
4. Targeting ‘undruggable’ GPCRs - GPCRs with complex geometry of the orthosteric binding site or the exceptionally high site occupancy by the endogenous ligand may be successfully targeted by allosteric ligands

Question 9

example of undruggable GPCR

Answer 9

Class B GPCRs have large peptidic orthosteric ligands and poorly druggable binding sites.

Generation of small molecule allosteric ligands is a viable approach to therapeutically targeting them.

Question 10

Maraviroc as an allosteric modulator

Answer 10

gp120 is a protein on the HIV virus that allows from the binding and entry of the virus into the host cell.

When treated with maraviroc decreases the affinity of gp120 for CCR5 (receptor) it decreases the binding decreasing entry

Question 11

types of GPCR oligomerization

Answer 11

Obligate (necessary for formation of a functional receptor)

Non-obligate (receptor can function as a monomer, however can also interact with other receptors to form a functional oligomer).

GPCR homo-oligomerization - same receptor

GPCR hetero-oligomerization - two different receptors

Question 12

function of GABAb1 receptor

Answer 12

Responsible for ligand binding, Retained in the ER when expressed
alone

Question 13

function of GABAb2 receptor

Answer 13

Required for G protein coupling, Co-traffics with GABAR1 enabling its localisation at the cell membrane

Question 14

how can homodimerization be used to cause internalisation and resensitisation

Answer 14

the u opiod receptor is a homodimer
- morphine and DAMGO are agonists
for this receptor
- morphine treatment develops
tolerance and addiction because
morphine binding prevents
internalization and therefore
resensitization
- Binding of DAMGO to one monomer
induces μ-OR internalization and
“drags” in morphine bound receptor
-μ-OR system can then be resensitized
ready to be exposed to morphine

Question 15

how can homodimerization be used to treat diminished receptor function due to mutated heterodimerization (benefit of homodimerization)

Answer 15

Mutant GPCRs can interact with wild-type receptors to alter their function

Chemokine CCR5 receptor are co-receptors for HIV binding
- mutant, CCR5Δ32, can dimerize with
the wild-type CCR5 and reduce its cell
surface expression
- This mutation reduces the infective
capacity of HIV and delays the onset of
AIDS

Question 16

what are the benefits of heterodimerization

Answer 16

formation of new complexes with novel
functional properties, Providing an additional level of regulation at which receptor function and cellular response can be modulated

Allows for diversification of receptor function and cellular response, by extending the number of signalling complexes

Question 17

how can heterodimerization affect GPCR pharmacology

Answer 17

Cell surface receptor expression - Altered expression as a result of cotrafficking/sequestration

Ligand interactions with the receptor -
Altered ligand affinity and/or selectivity,
formation of new binding site

Receptor signalling - Altered level of signalling, altered signalling partner/pathway, transactivation

Desensitization - Altered level or
selectivities of interactions with GRKs or arrestins

Internalization and intracellular trafficking - Co-internalization, or trans-inhibition of internalisaton/trafficking

Question 18

clinical relevance of GPCR heterodimerization

Answer 18

Heteromerisation of the AT1 and CCR2 in chronic kidney disease (CKD) mediate inflammation
- in a rat model of CKD, dual receptor blockade significantly reduced pathogenesis of CKD

Question 19

explain compartmentalized GPCR signalling

Answer 19

This signalling is spatially and
temporally distinct from G protein
signalling at the plasma
membrane (PM)

Intracellular GPCRs can signal
through both G protein dependent and G protein independent pathways

Question 20

endosome GPCR signalling

Answer 20

Usually occurs following receptor
internalization.

Question 21

Mitochondrial GPCR signalling

Answer 21

GPCRs are believed to be targeted to the mitochondria using standard targeting motifs and sorting
machinery

Question 22

endoplasmic reticulum GOCR signalling

Answer 22

ER localization can be regulated
by classical RXR motifs, which retain receptors in the biosynthetic
pathway.

Question 23

Golgi body GPCR signalling

Answer 23

GPCRs can sort retrogradley from
endosomes to the Golgi after PM
internalization
or
they can be retained in the Golgi during biosynthetic trafficking en route to the PM after synthesis at the endoplasmic reticulum (ER)

Question 24

nuclear membrane GPCR signalling

Answer 24

GPCRs can localize to nuclear
membranes after synthesis or
after internalizing from the plasma
membrane. Localization to nuclear
membranes can be mediated by a
nuclear localization sequence
(NLS) and/or importins.

Question 25

How does compartmentalized signalling generate different signalling outputs?

Answer 25

Temporal component - Sustained signalling responses are usually associated with GPCR signalling from non-plasma membrane locations Spatial component - Differences in protein (e.g. effector) expression/colocalization, Differences in lipid composition

Question 26

how does lipid composition generate different signalling pathways

Answer 26

lipid composition varies throughout the cell - Cholesterol can allosterically modulate GPCR activity in a variety of ways including receptor dimerization, ligand binding, G protein coupling, and signalling

Question 27

how do GPCRs transmit pain signals

Answer 27

GPCRs at the peripheral terminals of primary sensory neurons detect ligands from inflamed and injured tissues GPCRs control the activity of second-order spinal neurons that transmit pain signals centrally

Question 28

which receptors show a difference in the plasma membrane vs endosomal signalling pathways that may relate to pain

Answer 28

protease activated receptor neurokinin 1 receptor u and delta opioid receptor

Question 29

how does GPCR signalling change acute pain to chronic pain

Answer 29

plasma membrane G proteins are activated by agonists leading to acute pain signals as the GPCR is bound by arrestin and internalised into an endosome it is believed that arrestin mediated signalling occurs and endosomal signalling contribute to neural hyper sensitivity that causes chronic pain

Question 30

how can endocytosis inhibitors prevent chronic pain signalling

Answer 30

these prevent the internalisation of GPCRs but are only useful in research settings because application in humans would causes unwanted side effects

Question 31

how can Tripartite lipidated probes prevent chronic pain signalling

Answer 31

Insert an antagonist drug cargo into the PM via liquidated probe connected by a linker, this will undergo endocytosis, the high internal acidity will break bond between the drug and the linker delivering the antagonist to GPCR

Question 32

how can pH-dependent nanoparticles be used to prevent chirnuc pain signalling

Answer 32

Hydrophilic shell, hydrophobic core –self-assemble in an aqueous environment, encapsulating hydrophobic drug cargo. They undergo endocytosis and in the acidic endosomal environment, protonation of the hydrophobic monomer results nanoparticle disassembly and drug cargo release.