14th and 17th Oct Flashcards
Outline del Castillo and Katz (1957) model of drug action at GPCRs
A+R AR AR*
For GPCRs: A+R AR+G ARG –> Response
Outline de Lean’s (1980) Ternary complex model
It is cyclic
A+R AR+G ARG A+RG A+R….
Does GTP binding affect the affinity of the receptor?
Yes the agonists GTP presence increases the binding affinity of the receptor with the -GTP curve being slightly sigmoidal indicating heterogeneous binding.
Outline the Extended Tertiary Complex Model by Samama et al., 1993
AR AR- AR-G R-G R- R AR…
This takes into account agonist independent R-R* conformational change
What is the name of Weiss’ model in 1996?
The Cubic ternary complex model
This is the most encompassing model
Does ligand binding induce receptor conformational change?
No, the receptor is a dynamic structure therefore will naturally oscillate between R and R* states. Ligand binding stabilises the R* state.
What is the ratio of R to R* upon full agonist binding?
R*»R
What is the ratio of R to R* upon partial agonist binding?
R*>R
What is the ratio of R to R* upon antagonist binding?
R*=R
What is the ratio of R to R* upon inverse agonist binding?
R*
What is a CAM GPCR?
A constitutively active mutant GPCR
What are the key regions of a GPCR that give rise to CAMs?
D/ERY sequence mutations
Membrane proximal regions of the i3 loop
TM6/e4 interface
What disease is caused by a CAM TSH receptor?
Hyperthyroidism
What is caused by a CAM Luteinising Hormone Receptor?
Male precocious puberty - which drives puberty in men as young as 3 yo
What disease is caused by a CAM Rhodopsin?
Retinitis pigmentosa and night blindness
What disease is caused by a CAM parathyroid hormone receptor?
Short-limb dwarfism
What disease is caused by a CAM Ca2+ sensing receptor?
Hypocalaemia/hypercalcuria
Do neutral antagonists and inverse agonists cause pharmacologically distinct actions?
In a quiescent system neutral antagonists and inverse agonists are functionally indistinguishable
In a constitutively active system neutral antagonists and inverse agonists have very different effects. Only inverse agonists will suppress constitutive activity therefore are the only likely drugs for diseases caused by CAM GPCRs
Why is the choice between neutral antagonists and inverse agonists therapeutically important?
In a constitutively active system neutral antagonists will not suppress activity.
Treatment with inverse agonists can cause effects on receptor expression levels e.g. chronic treatment w/ histamine
Can GPCRs adopt multiple conformational states?
Yes
How can we observe receptor conformational change?
Monitor how FRET changes after agonist application
Crystallographic information on GPCR structure - allows us to look at different activity states of GPCRs e.g. Rhodopsin
What are the differences between bound and unbound GPCR opsins structures?
Differences between bound and unbound:
- In binding pocket part of the space usually occupied by the ligand is filled by Trp265
- The cytoplasmic end of TM6 is moved outward therefore activating the receptor
- The unbound form has two openings of the retinal binding pocket
What is biased agonism?
When one ligand binding promotes one conformation and thus one molecular pathway, and another ligand binding to the same receptor promotes a different conformation and thus a different molecular pathway.
What is the evidence for agonist-directed trafficking of receptor stimuli?
Berg 1998
5HT-2 receptors were targeted by a variety of ligands with a cell background
All ligands stimualted both Gi and Gq
TFMPP and Quipazine have a higher maximal response for Gq (measured through IP3 accumulation)
DOI and LSD have a higher maximal response for Gi (measured through aa release)
–> the different ligands appear to bias the output of the receptor
Outline allosteric modulation of the GABAa receptor
The orthosteric site = alpha beta subunit interfaces
Activation can be modulated by benzodiazepiens binding at the interface between alpha and a gamma subunit altering the ability of GABA to gate the channel
FOR ALLOSTERIC ACTION GABA MUST BE BOUND AT THE ORTHOSTERIC SITE
Positive Allosteric Modulator (PAM) - BZD agonists enhance GABA evoked Cl- conductance at sub maximal [GABA]
Neutral Allosteric Modulator - BZD antagonists can block PAM and NAM action
Negative Allosteric Modulator (NAM) - BZD inverse agonists decrease GABA enabled Cl- conductance
Give an example of a postive allosteric modulator for GABAa receptor
Diazepam
Give an example of a neutral allosteric modulator for the GABAa receptor
Flumanezil
Give an example of a negative allosteric modulator
sapmazenil
What are the three different classes of allosteric binding sites?
Class A
Class B
Class C
What are the three main effects that allosteric modulation can have
Altering orthosteric ligand binding affinity
Altering orthosteric ligand efficacy
Direct allosteric agonism
What are the advantages of targeting allosteric sites when developing drugs?
Orthosteric binding sites are usually fairly conserved therefore specificity is difficult. Allosteric sites are much less conserved
Allosteric modulators can alter the activity of endogenous ligands without altering the pattern of stimulation therefore complex intermittent/phasic patterns of receptor stimulation can be preserved
What is a protean agonist?
a protean agonist would produce an agonist
response in a quiescent receptor system (consisting of receptors the majority of
which are in the inactive state) and be an inverse agonist in a constitutively active
receptor system (one in which the receptors have been induced into the active state
with respect to G-protein coupling)
What did flourescence lifetime spectroscopy of beta2 adrenoceptors labelled at Cys265 show?
Showed a Gaussian distribution for probe environments indicating continuous fluctuations in receptor conformation
What are RAMPs?
Receptor activity modifying proteins
What is homolgous desensitisation of receptors?
Phosphorylation of agonist occupied receptors
What is heterologous desensitisation of receptors?
Phosphorylation of all GPCRs of that type
Who introduced the concept of constitutively active receptors in 1989?
Costa and Herz
