Allosteric Modulators

Question 1

Allosteric verses Orthosteric

Answer 1

Orthosteric
 Orthosteric (Greek - “correct”)
 Orthosteric site: the agonist binding site on a receptor
 Orthosteric agonists can exert agonist activity on their own.
 Orthosteric interaction: two ligands compete for the same binding domain on the receptor.

Allosteric
 Allosteric (Greek - “other”)
 Allosteric site: an additional binding site on a receptor that is distinct from the orthosteric binding site
 An allosteric ligand usually does not have an independent action of its own. It rather modulates the action of hormone or neurotransmitter.
 Allosteric interaction: a 2 way interaction between two topographically distinct binding sites on the same protein complex.

Question 2

Allosteric modulation

Answer 2

• Allosteric modulation is the binding of a ligand at a regulatory site other than the orthosteric site (endogenous ligand binding site)
• It normally acts by causing a conformational change in a protein, which results in a change in the binding affinity of the orthosteric (endogenous) ligand/substrate

Question 3

Allosteric modulation of Enzymes

Answer 3

Binding of an allosteric inhibitor changes the shape of the active site reducing the affinity of the substrate for the active site

Binding of an allosteric activator changes the shape of the active site increasing the affinity of the substrate for the active site

Question 4

All superfamilies of receptors can be allosterically modulated

Answer 4

• ligand gated ion channels
• g-protein coupled receptor
• nuclear hormone receptor
• receptor tyrosine kinase

Question 5

Benzodiazepines

Answer 5

Benzodiazepines are used in the treatment of anxiety.
 Benzodiazepines are PAMs. They bind to allosteric site on GABAA receptor, distinct from GABA binding site.
 Benzodiazepines facilitate opening of GABA activated Cl- channels and increase the affinity of GABA for the receptor.
 Thus potentiate inhibitory effects of GABA throughout CNS
 Have a wide therapeutic index and low toxicity, in contrast to direct-acting GABA receptor agonists which can have serious side effects

Question 6

Allosteric modulation of Receptor Tyrosine Kinases

Answer 6

a)The TrkA small-molecule allosteric inhibitor gambogic amide binds to the intracellular juxtamembrane region and inhibits signalling when NGF binds.
(b) the TrkB receptor is inhibited by the peptide inhibitor cyclotraxin-B which binds to the extracellular region of the receptor.
Cyclotraxin-B doesn’t effect the binding of the orthosteric ligand BDNF but does cause a conformational change that inhibits/decreases signalling.

Question 7

G-proteins are allosteric modulators of GPCRs

Answer 7

Agonist have increased affinity for G-protein bound receptor

Question 8

Structure of a GPCR allosteric modulator complex

Answer 8

The allosteric ligand binds to the extracellular vestibule just above the
orthosteric agonist binding site.

Question 9

Advantages of Allosteric Modulators

Answer 9

• The majority of therapeutic drugs on the market today
are drugs that target orthosteric sites.
• Receptor allosteric modulation is currently a hot topic in
drug discovery, because allosteric drugs have :
– Self-Limiting Activity - once all of the allosteric sites are occupied, no further allosteric effect is possible.
Therefore they are safer in overdose.
– Responses more closely resemble physiological signalling
– Greater receptor subtype selectivity (?)

Question 10

An allosteric modulator offers a more physiologic

alternative

Answer 10

“Fine tune” the signal up/down

Orthosteric ligands are at steady state
Allosteric ligands preserves natural rhythm

see graphs

Question 11

Allosteric modulation is saturable

Answer 11

(self-limiting maximal effect)
The “ceiling” effect

To achieve a long-lasting effect with an orthosteric antagonist high doses of an antagonist must be used this produces a large maximal effect which may lead to toxic interactions.
Large doses of allosteric antagonist have self-limiting maximal effect, therefore increasing the dose will not produce overdose, but will rather only prolong the effect in vivo.

Question 12

Saturation of allosteric effect

Answer 12

An allosteric antagonist modulator will have a limited maximal antagonism

• A competitive orthosteric antagonist will produce limitless shifts to the right of the agonist dose response curve
• an orthosteric non-competitive antagonist will depress the maximal response to an agonist to
baseline values
• An allosteric modulator will cease to have an effect when all the allosteric sites are occupied

Question 13

Orthosteric and allosteric binding sites

Answer 13

• The orthosteric and allosteric binding sites vary across the different GPCR families

Conservation of the Orthosteric Site
makes the design of selective drugs difficult

Reduced Conservation of Sequence in the
Extracellular Loops
Targeting this site might facilitate the design of selective drugs

Question 14

Therapeutic Use of Allosteric Modulators

Answer 14

e.g. HIV
Drug - Maraviroc
Action - NAM of CCR5
Status - in clinical use

Question 15

Three modes of action of GPCR allosteric modulators

Answer 15

• Affinity modulation
• Efficacy modulation
• Agonism/inverse agonism

Allosteric modulators exhibit one or more of these properties

Question 16

Allosteric Modulators can effect affinity and/or efficacy

Answer 16

Effects of modulator- induced increases or decreases in agonist affinity. Changes in the affinity of the receptor for agonists result in shifts along the concentration axis of the concentration response curves

Effects of modulator- induced increases or decreases in agonist efficacy . As increased maximal responses to a full agonist is not possible, modulators which increase efficacy result in shifts to the left of the concentration response curve. Modulators which decrease efficacy lead to a decease in maximal response.

Effects of allosteric modulators that have opposite effects on agonist affinity and efficacy.
(A) The allosteric ligand reduces agonist affinity and increases efficacy of a partial agonist; this will shift the concentration response curve to the right but increase maximal response.
(B) A modulator that increases the affinity of the agonist but decreases the efficacy may shift the concentration response curve to the left and will decrease the maximal response

Question 17

Experiments for detecting allosteric modulators of GPCRs

Answer 17

• Radioligand binding assays
– Equilibrium binding assays
– Kinetic binding studies (association & dissociation)
• Receptor function assays

Question 18

Allosteric Modulators:

Dissociation kinetics

Answer 18

Changes in the kinetics of interaction of the protein target with agonists and/or radioligands can only be produced by allosteric ligands.

Question 19

The difference between orthosteric & allosteric antagonism

Answer 19

Allosteric antagonists produce limited antagonism, which is reflected in the curvilinear Schild regression analysis

Question 20

The C9 bisacridine is a negative allosteric modulator of norepinephrine at the α1A-adrenoceptor

Answer 20

On a graph, maximum % response reduces

Question 21

Allosteric Modulators:

Functional Assays

Answer 21

• The modulator, CPCCOEt, reduces mGlu1R signalling without affecting binding or
potency.
• CPCCOEt is a negative allosteric modulator (NAM) at the metabotropic glutamate
receptor subtype 1 (mGlu1R).

• Org27569 decreases efficacy while increasing the potency and binding of CP55940 (CP55940 is a cannabinoid, CB1 receptor agonist).
• Org 27569 is an allosteric enhancer (PAM) of the CB1 cannabinoid receptor, but may also be an inverse agonist.

Question 22

Quantifying allosteric effects

Answer 22

• τ is a measure of efficacy
• K is a measure of affinity
• α effect of an allosteric modulator on the affinity of a protein for another molecule
  (note α is bidirectional)
• β effect of an allosteric modulator on the efficacy of an agonist binding to a receptor protein

Allosteric modulators cause left/rightward shift of the response curve of the orthosteric ligand

• Positive allosteric modulator α > 1
• Negative allosteric modulator α < 1

Allosteric agonist τ ≥ 0 i.e. modulator has direct agonist activity

Question 23

Allosteric Modulation is probe-dependent

Answer 23

Allosteric modulator produces competitive antagonism to agonist A, noncompetitive antagonism to agonist B and no effect to agonist C