Schild regression
Encyclopedia
Schild regression analysis, named for Heinz Otto Schild, is a useful tool for studying the effects of agonist
s and antagonist
s on the cellular response caused by the receptor
or on ligand-receptor binding.
Using a dose-response curve or an equivalent curve with concentration and binding %, it is possible to determine the dose ratio, this is a measure of the potency of a drug; it is obtained by dividing the increased equilibrium constant due to drug inhibition by the equilibrium constant without the drug. A Schild plot is a double logarithmic plot, typically Log(dr-1) as the ordinate and Log[B] as the abscissa. This is because a competitive drug B will have a linear plot with the
.
These experiments must be carried out on a very wide range (therefore the logarithmic scale) as the mechanisms differ over a large scale, such as at high concentration of drug.
binding is used. A ligand A will bind to a receptor
R according to an equilibrium constant :
Kd=k-1/k1
Although the equilibrium constant is more meaningful, texts often mention its inverse, the affinity constant (Kaff = k1/k-1): A better binding means an increase of binding affinity.
The equation for simple ligand binding to a single homogeneous receptor is
This is the Hill-Langmuir equation, which is practically the Hill equation (biochemistry) describen for the agonist binding. In chemistry, this relationship is called the Langmuir equation
, which describes the adsorption of molecules onto sites of a surface (see adsorption
).
[R]total is the total number of binding sites, and when the equation is plotted it is the horizontal asymptote to which the plot tends; more binding sites will be occupied as the ligand concentration increases, but there will never be 100% occupancy. The binding affinity is the concentration needed to occupy 50% of the sites; the lower this value is the easier it is for the ligand to occupy the binding site.
The binding of the ligand to the receptor at equilibrium follows the same kinetics as an enzyme at steady-state (Michaelis-Menten equation) without the conversion of the bound substrate to product.
Agonists and antagonists can have various effects on ligand binding. They can change the maximum number of binding sites, the affinity of the ligand to the receptor, both effects together or even more bizarre effects when the system being studied is more intact, such as in tissue samples. (Tissue absorption, densitization, and other non equilibrium steady-state can be a problem.)
A surmountable drug changes the binding affinity:
A nonsurmountable drug changes the maximum binding:
The Schild regression also can reveal if there are more than one type of receptor and it can show if the experiment was done wrong as the system has not reached equilibrium.
A radio-receptor assay requires the separation of the bound from the free ligand. This is done by filtration
, centrifugation
or dialysis
.
A method that does not require separation is the scintillation proximity assay that relies on the fact that β-rays from 3H travel extremely short distances. The receptors are bound to beads coated with a polyhydroxy scintillator. Only the bound ligands to be detected.
Today, the fluorescence method is preferred to radioactive materials due to a much lower cost, lower hazard, and the possibility of multiplexing the reactions in a high-throughput manner. One problem is that fluorescent-labeled ligands have to bear a bulky fluorophore that may cause it to hinder the ligand binding. Therefore, the fluorophore used, the length of the linker, and its position must be carefully selected.
An example is by using FRET
, where the ligand's fluorophore transfers its energy to the fluorophore of an antibody raised against the receptor.
Other detection methods such as surface plasmon resonance
do not even require fluorophores.
Agonist
An agonist is a chemical that binds to a receptor of a cell and triggers a response by that cell. Agonists often mimic the action of a naturally occurring substance...
s and antagonist
Receptor antagonist
A receptor antagonist is a type of receptor ligand or drug that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses...
s on the cellular response caused by the receptor
Receptor (biochemistry)
In biochemistry, a receptor is a molecule found on the surface of a cell, which receives specific chemical signals from neighbouring cells or the wider environment within an organism...
or on ligand-receptor binding.
Using a dose-response curve or an equivalent curve with concentration and binding %, it is possible to determine the dose ratio, this is a measure of the potency of a drug; it is obtained by dividing the increased equilibrium constant due to drug inhibition by the equilibrium constant without the drug. A Schild plot is a double logarithmic plot, typically Log(dr-1) as the ordinate and Log[B] as the abscissa. This is because a competitive drug B will have a linear plot with the
.These experiments must be carried out on a very wide range (therefore the logarithmic scale) as the mechanisms differ over a large scale, such as at high concentration of drug.
Schild regression for ligand binding
Although most experiments use cellular response as a measure of the effect, the effect is, in essence, a result of the binding kinetics; so, in order to illustrate the mechanism, ligandLigand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
binding is used. A ligand A will bind to a receptor
Receptor (biochemistry)
In biochemistry, a receptor is a molecule found on the surface of a cell, which receives specific chemical signals from neighbouring cells or the wider environment within an organism...
R according to an equilibrium constant :
Kd=k-1/k1
Although the equilibrium constant is more meaningful, texts often mention its inverse, the affinity constant (Kaff = k1/k-1): A better binding means an increase of binding affinity.
The equation for simple ligand binding to a single homogeneous receptor is
This is the Hill-Langmuir equation, which is practically the Hill equation (biochemistry) describen for the agonist binding. In chemistry, this relationship is called the Langmuir equation
Langmuir equation
The Langmuir equation relates the coverage or adsorption of molecules on a solid surface to gas pressure or concentration of a medium above the solid surface at a fixed temperature. The equation was developed by Irving Langmuir in 1916...
, which describes the adsorption of molecules onto sites of a surface (see adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
).
[R]total is the total number of binding sites, and when the equation is plotted it is the horizontal asymptote to which the plot tends; more binding sites will be occupied as the ligand concentration increases, but there will never be 100% occupancy. The binding affinity is the concentration needed to occupy 50% of the sites; the lower this value is the easier it is for the ligand to occupy the binding site.
The binding of the ligand to the receptor at equilibrium follows the same kinetics as an enzyme at steady-state (Michaelis-Menten equation) without the conversion of the bound substrate to product.
Agonists and antagonists can have various effects on ligand binding. They can change the maximum number of binding sites, the affinity of the ligand to the receptor, both effects together or even more bizarre effects when the system being studied is more intact, such as in tissue samples. (Tissue absorption, densitization, and other non equilibrium steady-state can be a problem.)
A surmountable drug changes the binding affinity:
- competitive ligand K'd=Kd (1+[B]/Kb)
- cooperative allosteric ligand K'd=Kd (KB+[B]/(KB+[B]/α))
A nonsurmountable drug changes the maximum binding:
- noncompetitive binding [R]t'=[R]t /(1+[B]/Kb)
- irreversible binding
The Schild regression also can reveal if there are more than one type of receptor and it can show if the experiment was done wrong as the system has not reached equilibrium.
Radioligand binding assays
The first radio-receptor assay (RRA) was done in 1970 by Lefkowitz et al., using a radiolabeled hormone to determine the binding affinity for its receptor.A radio-receptor assay requires the separation of the bound from the free ligand. This is done by filtration
Filtration
Filtration is commonly the mechanical or physical operation which is used for the separation of solids from fluids by interposing a medium through which only the fluid can pass...
, centrifugation
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
or dialysis
Dialysis
In medicine, dialysis is a process for removing waste and excess water from the blood, and is primarily used to provide an artificial replacement for lost kidney function in people with renal failure...
.
A method that does not require separation is the scintillation proximity assay that relies on the fact that β-rays from 3H travel extremely short distances. The receptors are bound to beads coated with a polyhydroxy scintillator. Only the bound ligands to be detected.
Today, the fluorescence method is preferred to radioactive materials due to a much lower cost, lower hazard, and the possibility of multiplexing the reactions in a high-throughput manner. One problem is that fluorescent-labeled ligands have to bear a bulky fluorophore that may cause it to hinder the ligand binding. Therefore, the fluorophore used, the length of the linker, and its position must be carefully selected.
An example is by using FRET
Fret
A fret is a raised portion on the neck of a stringed instrument, that extends generally across the full width of the neck. On most modern western instruments, frets are metal strips inserted into the fingerboard...
, where the ligand's fluorophore transfers its energy to the fluorophore of an antibody raised against the receptor.
Other detection methods such as surface plasmon resonance
Surface plasmon resonance
The excitation of surface plasmons by light is denoted as a surface plasmon resonance for planar surfaces or localized surface plasmon resonance for nanometer-sized metallic structures....
do not even require fluorophores.
External links
- curvefit.com - Dose-response curves in the presence of antagonists, for a clear explanation.