From Pharmacology in Drug Discovery and Development

1. Chapter 5
Allosteric Drug Effects
2018B003
Md. Rasheduzzaman Jony

2. Outline
• Introduction
• New Terminology
• Protein Allosterism
• Allosteric Phenotypes
• Unique Effects of Allosteric
• Modulators
• Detecting Allosterism
• Quantifying Allosteric Effect

3. Introduction
• Allosteric molecules can bind to virtually any site on the target protein and show it’s activity.
• If a molecule is bound to the protein at any site the conformational movement of that protein may be affected.
• These effects are allosteric and have become a very important part of new drug discovery.

4. New Terminology
• α = Quantifying the effect of an allosteric modulator on the affinity of a protein for another molecule.
• β = Quantifying the effect of an allosteric modulator on the efficacy of an agonist binding to a receptor protein.
• Cooperativity = The effective interaction between the two cobinding allosteric molecules on the protein.
• Ensemble = A collection of protein molecules will have a selection of different conformations of similar free energy.
• Allosteric Modulator = Helps the protein to bind the cell with the ligand by binding with the protein.
• Negative allosteric modulator = Reduces the affinity or the efficacy of an agonist for a receptor.
• Positive allosteric modulator = increases the affinity and/or the efficacy of an agonist for a receptor.
• Probe dependence = Variation of activity of an allosteric modulator as it modifies the protein interaction with various probes

5. Protein Allosterism
• Allosteric interactions on proteins such as receptors and ion channels occur through the binding of a molecule onto the protein to affect its free
energy of conformation.
• Thus there is a conformational change occurs.
• The energy of the protein depends on the binding but not on the size of the ligand.
• Binding of a ligand with an enzyme with conformational change of the enzyme is referred to as “induced fit” by Daniel Koshland.
• The binding of a ligand initiates a process of conformational selection within the ensemble.
The process of conformational
selection by allosteric ligands

6. Dose-response curve for the binding of a ligand to a single subunit protein and to a protein made up of three identical
cooperatively linked subunits (black curve labeled “trimeric cooperativity”). For the trimer the binding of the
ligand to one subunit promotes the binding to the second and the third, causing the binding curve to be steeper
than that for a monomer.
• The binding of a molecule to one subunit alters the subsequent binding
of molecules to other subunits.

7. COOPERATIVE BINDING OF OXYGEN TO HEMOGLOBIN
• Hemoglobin is a tetrameric protein that binds and transports four oxygen molecules per unit and then releases them to myoglobin.
• The binding of oxygen to hemoglobin is allosterically cooperative.
• Oxygen readily binds to hemoglobin at the high pO2 values in the lung.
• Tetrameric cooperativity of oxygen binding to hemoglobin optimizes the delivery of oxygen to tissues

8. The product of enzyme II (compound C; isoleucine) is structurally different from substrate A (threonine), yet inhibits enzyme I through an
allosteric site. Panel on right shows the inhibition of the utilization of threonine in threonine less mutants of Escherichia coli by isoleucine in the
presence of 10 mM (open circles) and 20 mM L-threonine. This system is optimal for control of output since the overall product then controls the
initial rate of the reaction cascade.
• Proteins such as enzymes can bind molecules at multiple sites to modify their activity.
Negative feedback inhibition by the product of an enzyme cascade

9. ALLOSTERIC PHENOTYPES
• In general there is no way to predict the effects of an allosteric ligand.
• Upon binding an allosteric ligand, a receptor may have altered affinity , efficacy
or be directly stimulated by the modulator to produce response.
Three characteristic parameters of allosteric ligands. Considering a receptor receiving physiological input, the allosteric ligand can alter the
affinity of the endogenous agonist through a cooperativity term α, modify the efficacy of the endogenous agonist through a cooperativity term β or
itself produce direct agonism through a positive allosteric efficacy denoted τB. There are no constraints on the direction or magnitude of any of
these effects for any given allosteric ligand-endogenous agonist pair.

10. The various possible effects of an allosteric modulator on the response to an agonist

11. Phenotypic profiles for allosteric receptor ligands

12. Probe dependence of an allosteric antagonist for three agonists A, B and C.

13. • Allosteric antagonists have the capability of being probe dependent and thus show differential blockade of different probes of the CCR5
receptor.
• TAK779 shows preferential potency for the blockade of CCL3L1-induced receptor internalization over HIV-1 entry.
• TAK652 shows a more favorable profile of greater potency for HIV-1 entry over CCL3L1-induced internalization.

14. UNIQUE EFFECTS OF ALLOSTERIC MODULATORS
The result of an allosterically modulated system can be different for different agonists. This and the property of saturation of effect cause allosteric
modulators to have a unique range of activities. These are;
• The Potential to Alter the Interaction of Very Large Proteins
• The Potential to Modulate but not Completely Activate and/or Inhibit Receptor Function
• Preservation of Physiological Patterns
• Reduction in Side-Effects
• Can Produce Texture in Antagonism
• Can Have Separate Effects on Agonist Affinity and Efficacy
• Allosteric Modulators Exercise “Probe Dependence”

15. The Potential to Alter the Interaction of Very Large Proteins
Disruption of the interaction of two proteins with multiple points of interaction.

18. The Potential to Modulate but not Completely Activate and/or Inhibit Receptor Function
pIC50 curves for allosteric antagonists that produce limite
maximal blockade of functional effects.

19. Reduction in Side-Effects

20. Can Have Separate Effects on Agonist Affinity and Efficacy
Effects of allosteric modulators that have opposite effects on agonist affinity and efficacy

21. Allosteric Modulators Exercise “Probe Dependence”

22. DETECTING ALLOSTERISM
Saturation of allosteric effect

23. QUANTIFYING ALLOSTERIC EFFECT
Alteration of kinetics of receptor dissociation by a PAM and NAM
The following equation defines the response to an agonist (A) in the presence of an allosteric modulator (B)

24. Fitting data to the model of allosteric function; Agonist KA5300 nM, τ 53, Em5100
• Panel A: Antagonism fit to the model for a negative allosteric modulator of KB5100 nM, α50.3, β 50.2. Curves shown in the absence and
presence of 300 nM 2 μM and 10 μM negative modulator.
• Panel B: Data for a PAM of KB5100 nM, α520, β 52. Curves shown in the absence and presence of 3 nM 20 nM and 100 nM positive
modulator

25. Fitting data to the model of allosteric function for modulator with direct agonist action for probe agonist KA5100 nM, τ 53, Em5100
Fitting data to the model of allosteric function for modulator with direct agonist action for control probe agonist KA5100 nM, τ 53, Em5100

26. Thank You for Being Patience
Success is not that what we always see