Physiological insights of GPCRs Flashcards
Factors to consider when identifying a receptor as a drug target
Is it a good drug target (e.g., drugability)
What is its role in that cell/tissue, and what is its expression elsewhere/role elsewhere (side effects)
What signalling pathways mediate its effects.
Agonist, antagonist, or allosteric drug? Selective or non-selective, biased or unbiased?
Approaches to understanding GPCR roles pharmacologically and limitations + alternatives
GPCR targeting drug can also have off target effects. Furthermore, the on target effects can occur in other tissue.
Genetic approaches such as siRNA can eliminate the expression. Can identify the role that way.
Can also use knockout animals, however adaptations and developmental changes in the animals mean this isn’t entirely accurate.
Signalling inhibitors can identify the individual roles of different pathways, e.g., pertussis toxin which inhibits Gi/osignalling. Only really applicable in vitro.
DREADDs overview and uses
Designer Receptors Exclusively Activated by Designer Drugs.
It is used to identify the circuitry and cellular signalling that specifies behaviour, perception, emotions and motor functions.
DREADDs example and methodology of creating the designer receptors
CNO (clozapine-N-oxide) is an inert metabolite of clozapine (which also has effects at m3AChR). Very bioavailable.
Pheromone signalling in yeast utilises Ste2 (a GPCR). Genetic engineering can replace that with a human GPCR. The yeast G-proteins are also replaced by chimeric human/yeast G-proteins.
This utilisation of the yeast signalling means that yeast growth only occurs when the ligand for the engineered GPCR is present.
A library of randomly mutated m3AChRs transfected into the yeast was given CNO, and the yeast cultures that grew the most were chosen. Plasmid DNA was isolated from the yeast, and the m3AChRs were sequenced and remutagenized repeatedly to create designer receptors highly selective for the designer drug (CNO).
Further modification of the receptors in DREADDs to enable its application
Once the receptors that have the best profile has been collected, the mutations that create these desired profiles are identified.
Mammalian cell lines are exogenously expressed with the mutant GPCRs. Looking at different mutations and combinations of mutations, the receptor with the highest selectivity is chosen, e.g., Y149C + A239G in m3AChR, where ACh has 40,000-fold lower affinity and CNO has high affinity.
Multiple of the designer receptors can be engineered to couple with different signalling pathways, e.g., hM3Dq (Gq-coupled), hM4Di (Gi coupled), GsD (Gs-coupled using a chimera of the intracellular loops of the B2-AR and an m3AChR), and R165L (a B-arrestin signalling specific GPCR)
Methodology of expressing DREADDs experimentally
The gene for the DREADD can be packaged into a virus (attenuated adenovirus) so that it is expressed in the cells that the virus infects. This normally has a local effect. Wherever it is injected tends to be where they’ll be expressed.
Viral Cre-dependent approach is more common. A single DREADD vector with 2 Lox sties is injected into the target area of a Cre-driver mouse and this restricts the expression to Cre-expressing neurones. DREADDs will only be expressed in cells that contain Cre. This occurs as Cre identifies 2 specific Lox sites in the DNA, inverts the sequence between these two sites. This prevents the stop codon present in the vector. As such, the DREADD is expressed in a Cre-dependent manner.
Ways DREADDs can interrogate physiology and overview of information attained from CNO DREADD
Enables us to understand the role of particular signalling in a specific cell during a behaviour or physiological event.
Glucose stimulated insulin secretion (GSIS) in B-cells occurs in a bimodal fashion. GPCRs play a large role in B-cells in controlling insulin release.
DREADDs were incorporated into the B-cells. One was Gq-coupled the other was Gs-coupled. It was identified that activation of both by CNO increased insulin release. Identifies that Gq and Gs signalling in B-cells plays a role in insulin release.
This methodology can be utilised in a wide range of other cells and physiological functions to identify the specific roles of signalling cascades.
